Activation Of Neural Stem Cells Research Articles

Hes1 oscillation frequency correlates with activation of neural stem cells

Quiescent neural stem cells (NSCs) are occasionally activated to undergo proliferation and subsequent neuronal differentiation. It was previously shown that the transcriptional repressor Hes1 is involved in both active and quiescent states of NSCs: when Hes1 expression oscillates, it periodically represses the proneural gene Ascl1, thereby driving Ascl1 oscillations, which regulate the active state, while sustained Hes1 expression continuously suppresses Ascl1, promoting quiescence. However, it remains to be analyzed how the transition from quiescent to active states of NSCs is controlled. Here, we found that overexpression of the active form of Notch1 significantly activates NSCs in both in-vitro and in-vivo conditions and that its levels are proportional to NSC activation. The active form of Notch1 induces a burst of Hes1 oscillations in quiescent NSCs, and the frequency of Hes1 oscillations, rather than the Hes1 peak levels, correlates with the efficiency of NSC activation. These results raised the possibility that bursting Hes1 oscillations could increase the chance of Ascl1 oscillations in quiescent NSCs, suggesting that Notch1-induced Hes1 oscillation is a cue for a transition from quiescent to active states of NSCs.

Detrimental effects of adolescent escalating low-dose Δ9 -tetrahydrocannabinol leads to a specific bio-behavioural profile in adult male rats.

Adolescent cannabis use is associated with adult psychopathology. When Δ9 -tetrahydrocannabinol (THC), mainly in high doses, is administered to adolescence rats there are also long lasting effects in adults. This study aims to determine the specific adult bio-behavioural profile after adolescent low-dose THC, which better mirrors adolescent recreational cannabis use. Adolescent male Sprague-Dawley rats were treated with escalating low-dose of THC. In adulthood, they were evaluated for their spontaneous locomotion, sensorimotor gating, higher order and spatial cognitive functions. Dopaminergic activity and cannabinoid receptor expression were measured in distinct brain regions. Hippocampal neurogenic activity of neural stem cells was determined and protein levels of neuroplasticity-related biomarkers were quantified. Adolescent low-dose THC exposure increased spontaneous open-field activity, without affecting prepulse inhibition and attentional set-shifting performance. Region-specific dopaminergic alterations and CB1 receptor up-regulation in the prefrontal cortex were observed. Impaired spatial memory, as assessed with the object location task and Morris water maze test, was associated with significantly decreased proliferative activity (SOX2-positive cells), neurogenic potential (decreased doublecortin-positive cells) in the adult hippocampus and defective neuroplasticity, including reduced BDNF expression in the hippocampus and prefrontal cortex. Our findings reveal the adverse impact of adolescent low-dose THC on the psychomotor profile, dopaminergic neurotransmission, compensatory cannabinoid receptor response, cognition-related neurobiological and behavioural functions. Our adolescent low-dose THC animal model does not induce tangible psychotic-like effects, such as those reported in high-dose THC studies, but it impairs cognitive functions and points to hippocampal vulnerability and disrupted neurogenesis.

Traditional Chinese Medicine Monomers: Novel Strategy for Endogenous Neural Stem Cells Activation After Stroke.

Stem cell therapy, which has become a potential regenerative medical treatment and a promising approach for treating brain injuries induced by different types of cerebrovascular disease, has various application methods. Activation of endogenous neural stem cells (NSCs) can enable infarcted neuron replacement and promote neural networks’ regeneration without the technical and ethical issues associated with the transplantation of exogenous stem cells. Thus, NSC activation can be a feasible strategy to treat central nervous system (CNS) injury. The potential molecular mechanisms of drug therapy for the activation of endogenous NSCs have gradually been revealed by researchers. Traditional Chinese medicine monomers (TCMs) are active components extracted from Chinese herbs, and some of them have demonstrated the potential to activate proliferation and neurogenesis of NSCs in CNS diseases. Ginsenoside Rg1, astragaloside IV (AST), icariin (ICA), salvianolic acid B (Sal B), resveratrol (RES), curcumin, artesunate (ART), and ginkgolide B (GB) have positive effects on NSCs via different signaling pathways and molecules, such as the Wingless/integrated/β-catenin (Wnt/β-catenin) signaling pathway, the sonic hedgehog (Shh) signaling pathway, brain-derived neurotrophic factor (BDNF), nuclear factor erythroid 2-related factor 2 (Nrf2), and heme oxygenase 1 (HO-1). This article may provide further motivation for researchers to take advantage of TCMs in studies on CNS injury and stem cell therapy.

Declining lamin B1 expression mediates age-dependent decreases of hippocampal stem cell activity.

Neural stem cells (NSCs) generate neurons throughout life in the hippocampal dentate gyrus. With advancing age, levels of neurogenesis sharply drop, which has been associated with a decline in hippocampal memory function. However, cell-intrinsic mechanisms mediating age-related changes in NSC activity remain largely unknown. Here, we show that the nuclear lamina protein lamin B1 (LB1) is downregulated with age in mouse hippocampal NSCs, whereas protein levels of SUN-domain containing protein 1 (SUN1), previously implicated in Hutchinson-Gilford progeria syndrome (HGPS), increase. Balancing the levels of LB1 and SUN1 in aged NSCs restores the strength of the endoplasmic reticulum diffusion barrier that is associated with segregation of aging factors in proliferating NSCs. Virus-based restoration of LB1 expression in aged NSCs enhances stem cell activity invitro and increases progenitor cell proliferation and neurogenesis invivo. Thus, we here identify a mechanism that mediates age-related decline of neurogenesis in the mammalian hippocampus.

Indoxyl sulfate caused behavioral abnormality and neurodegeneration in mice with unilateral nephrectomy.

Chronic Kidney Disease (CKD) and neurodegenerative diseases are aging-related diseases. CKD with declined renal function is associated with an elevation of circulating indoxyl sulfate, a metabolite synthesized by gut microbes. We explored the roles of gut microbial metabolites in linking with Central Nervous System (CNS) diseases by administrating indoxyl sulfate intraperitoneally to male C57BL/6 mice with unilateral nephrectomy. Upon exposure, the accumulation of indoxyl sulfate was noted in the blood, prefrontal cortical tissues, and cerebrospinal fluid. Mice showed behavioral signs of mood disorders and neurodegeneration such as anxiety, depression, and cognitive impairment. Those behavioral changes were accompanied by disturbed neuronal survival, neural stem cell activity, expression of Brain-Derived Neurotrophic Factor, serotonin, corticosterone, and Repressor Element-1 Silencing Transcription Factor, and post-receptor intracellular signaling, as well as upregulated oxidative stress and neuroinflammation. Uremic toxin adsorbent AST-120 improved the above mentioned changes. Intriguingly, intracerebroventricular indoxyl sulfate administration only caused limited alterations in the normal mice and the alterations were reversed by aryl hydrocarbon receptor antagonism. The findings suggest pathogenic roles of indoxyl sulfate in the development of CNS diseases, and highlight gut microbiota as alternative targets for intervention with the aim of slowing down the progression of CKD and decreasing CNS complications.

In vitro investigation of growth factors including MGF and IGF-1 in neural stem cell activation, proliferation, and migration

Neurogenesis is mainly activated after damage in adult tissues. This destruction activates the neural stem cells (NSCs) by exiting from a quiescent state and initiating proliferation, differentiation, and migration towards the damaged area. Although studies have investigated to clarify the process of NSC biology and neurogenesis, there are still significant artifacts in understanding the primary mechanism. It is known that only a small percentage of NSC become neurons and integrate into the brain tissue after this process. The significant proportion differentiates to become either astrocytes or oligodendrocytes. Furthermore, the quiescent stem cells in the niche are mainly activated by the stimuli affect. In recent years, many studies have been conducted with varying hormones, some of which might provide neuro-stimulation effect and/or involved in the regeneration of the brain tissue and/or neuroprotection from traumatic or ischemic pathologies, including Insulin-like growth factor 1 (IGF-1), Mechano Growth Factor (MGF), Basic Fibroblast Growth Factor (FGF-2), Erythropoietin (EPO), Epidermal Growth Factor (EGF), Nerve Growth Factor (NGF) and Brain-Derived Neurotrophic Factor (BDNF).In this study, we examined the effects of FGF-2, MGF, IGF-1, EPO, EGF, NGF, and BDNF alone or with various combinations on rat hippocampal NSC by tracking the changes in the expression of Nestin, GFAP, TUBB3, and DCX genes during 24 h (h), 72 h and 168 h time frame. The apoptosis analysis revealed that FGF-2 and FGF-2 coupled growth factors effectively protect NSCs against apoptosis, whereas MGF coupled growth factors failed in this protection. The cell cycle analysis demonstrated that these growth factors had accumulated the NSCs exit from the quiescent phase to the Mitosis phase, mostly without being long in the Synthesis Phase. Neurosphere sizes were increased with MGF, signifying MGF being effective in neural progenitor cells. The combined use of MGF with FGF-2 was more effective in postmitotic neurons than MGF alone.We have comparatively demonstrated the effect of cytokines alone and combined administration on activation, proliferation, and migration of NSCs. Although many issues are still waiting to be investigated in adult neurogenesis, neural regeneration, and adult neural stem cell biology, the results provide vital resources to the researchers that are interested in the varying effect of growth factor on NSC.

NSCs: Sentinel Cells of the Brain.

Adult neural stem cells (NSCs) have the ability to oscillate between activated and dormant states in response to extrinsic regulators. In this issue of Cell Stem Cell, Belenguer etal. (2020) identify a direct role for systemic TNF-α, which acts through its receptors TNFRII and TNFRI as a regulator of NSC activation and a return to quiescence, respectively.

Mechanical Brain Injury Increases Cells' Production of Cystathionine β-Synthase and Glutamine Synthetase, but Reduces Pax2 Expression in the Telencephalon of Juvenile Chum Salmon, Oncorhynchus keta.

The considerable post-traumatic brain recovery in fishes makes them a useful model for studying the mechanisms that provide reparative neurogenesis, which is poorly represented in mammals. After a mechanical injury to the telencephalon in adult fish, lost neurons are actively replaced due to the proliferative activity of neuroepithelial cells and radial glia in the neurogenic periventricular zone. However, it is not enough clear which signaling mechanisms are involved in the activation of adult neural stem cells (aNSC) after the injury (reactive proliferation) and in the production of new neurons (regenerative neurogenesis) from progenitor cells (NPC). In juvenile Pacific salmon, the predominant type of NSCs in the telencephalon are neuroepithelial cells corresponding to embryonic NSCs. Expression of glutamine synthetase (GS), a NSC molecular marker, was detected in the neuroepithelial cells of the pallium and subpallium of juvenile chum salmon, Oncorhynchus keta. At 3 days after a traumatic brain injury (TBI) in juvenile chum salmon, the GS expression was detected in the radial glia corresponding to aNSC in the pallium and subpallium. The maximum density of distribution of GS+ radial glia was found in the dorsal pallial region. Hydrogen sulfide (H2S) is a proneurogenic factor that reduces oxidative stress and excitotoxicity effects, along with the increased GS production in the brain cells of juvenile chum salmon. In the fish brain, H2S producing by cystathionine β-synthase in neurogenic zones may be involved in maintaining the microenvironment that provides optimal conditions for the functioning of neurogenic niches during constitutive neurogenesis. After injury, H2S can determine cell survivability, providing a neuroprotective effect in the area of injury and reducing the process of glutamate excitotoxicity, acting as a signaling molecule involved in changing the neurogenic environment, which leads to the reactivation of neurogenic niches and cell regeneration programs. The results of studies on the control of the expression of regulatory Sonic Hedgehog genes (Shh) and the transcription factors Paired Box2 (Pax2) regulated by them are still insufficient. A comparative analysis of Pax2 expression in the telencephalon of intact chum salmon showed the presence of constitutive patterns of Pax2 expression in neurogenic areas and non-neurogenic parenchymal zones of the pallium and subpallium. After mechanical injury, the patterns of Pax2 expression changed, and the amount of Pax2+ decreased (p < 0.05) in lateral (Dl), medial (Dm) zones of the pallium, and the lateral zone (Vl) of the subpallium compared to the control. We believe that the decrease in the expression of Pax2 may be caused by the inhibitory effect of the Pax6 transcription factor, whose expression in the juvenile salmon brain increases upon injury.

Adult neural stem cell activation in mice is regulated by the day/night cycle and intracellular calcium dynamics

Neural stem cells (NSCs) in the adult brain transit from the quiescent state to proliferation to produce new neurons. The mechanisms regulating this transition in freely behaving animals are, however, poorly understood. We customized invivo imaging protocols to follow NSCs for several days up to months, observing their activation kinetics in freely behaving mice. Strikingly, NSC division is more frequent during daylight and is inhibited by darkness-induced melatonin signaling. The inhibition of melatonin receptors affected intracellular Ca2+ dynamics and promoted NSC activation. We further discovered a Ca2+ signature of quiescent versus activated NSCs and showed that several microenvironmental signals converge on intracellular Ca2+ pathways to regulate NSC quiescence and activation. Invivo NSC-specific optogenetic modulation of Ca2+ fluxes to mimic quiescent-state-like Ca2+ dynamics in freely behaving mice blocked NSC activation and maintained their quiescence, pointing to the regulatory mechanisms mediating NSC activation in freely behaving animals.

A multi-channel collagen scaffold loaded with neural stem cells for the repair of spinal cord injury.

Collagen scaffolds possess a three-dimensional porous structure that provides sufficient space for cell growth and proliferation, the passage of nutrients and oxygen, and the discharge of metabolites. In this study, a porous collagen scaffold with axially-aligned luminal conduits was prepared. In vitro biocompatibility analysis of the collagen scaffold revealed that it enhances the activity of neural stem cells and promotes cell extension, without affecting cell differentiation. The collagen scaffold loaded with neural stem cells improved the hindlimb motor function in the rat model of T8 complete transection and promoted nerve regeneration. The collagen scaffold was completely degraded in vivo within 5 weeks of implantation, exhibiting good biodegradability. Rectal temperature, C-reactive protein expression and CD68 staining demonstrated that rats with spinal cord injury that underwent implantation of the collagen scaffold had no notable inflammatory reaction. These findings suggest that this novel collagen scaffold is a good carrier for neural stem cell transplantation, thereby enhancing spinal cord repair following injury. This study was approved by the Animal Ethics Committee of Nanjing Drum Tower Hospital (the Affiliated Hospital of Nanjing University Medical School), China (approval No. 2019AE02005) on June 15, 2019.

Glia-Like Cells from Human Mesenchymal Stem Cells Protect Neural Stem Cells in an In Vitro Model of Alzheimer's Disease by Reducing NLRP-3 Inflammasome.

Background and PurposeNeural stem cells (NSCs) have the ability to regenerate, proliferate, and differentiate, enabling them to play important roles in the recovery of the damaged nervous system. However, in neurodegenerative diseases such as Alzheimer's disease (AD), the NSCs are damaged as well. Glia-like cells from human mesenchymal stem cells (ghMSCs) are functionally enhanced adult stem cells. In the present study, we investigated whether ghMSCs could protect NSCs from amyloid beta (Aβ)-mediated toxicity.MethodsRat NSCs were obtained from E13–14 fetal rat cortices. NSCs were seeded in pre-coated plates, and the next day, cells were simultaneously treated with 20 μM Aβ and 0.4 μm pore insert well-seeded ghMSCs. After 48 hours of co-treatment, cell viability and proliferation were evaluated. After 2 hours of co-treatment, western blotting was performed to measure inflammasome-related factors, such as NOD-like receptor family pyrin domain containing 3, caspase-1, and interleukin-1β.ResultsThe results showed that ghMSCs increased viability and proliferation and reduced the toxicity of NSCs injured by Aβ by reducing the NRLP3 inflammasome activation of NSCs induced by Aβ.ConclusionsIn this study, we confirmed that ghMSCs could protect NSCs in an in vitro model of AD through the regulation of inflammatory response.

Electroacupuncture on the Scalp over the Motor Cortex Ameliorates Behavioral Deficits Following Neonatal Hypoxia-Ischemia in Rats via the Activation of Neural Stem Cells.

Electroacupuncture (EA) therapy via alternating current stimulation on the scalp over the motor cortex is used for the treatment of brain disorders. Perinatal hypoxia-ischemia (HI), a brain injury in newborns, leads to long-term neurologic complications. Here, we investigated whether EA could promote functional improvements and neurogenesis in a neonatal HI rat model. A neonatal HI rat model was induced by permanent ligation of the left carotid artery in postnatal day 7 pups. EA for neonatal HI rats was performed at 2 Hz (1, 3, or 5 mA; 20 min) from 4–6 weeks after birth. HI rats undergoing EA had improved motor and memory function, with the greatest improvement after 3 mA EA. The corpus callosum was significantly thicker and showed a significant increase in proliferating astrocytes in the 3 mA EA group. We observed proliferating cells and a greater number of newly developed neurons and astrocytes in the subventricular zone and dentate gyrus of the 3 mA EA group than in those of the HI group. These results suggest that EA promotes functional improvements following neonatal HI assault via the proliferation and differentiation of neural stem cells. This effect was the strongest after 3 mA EA, suggesting that this is the optimal treatment dose.

The Role of NF-κВ in the Realization of Functions of Various Types of Regeneration-Competent Cells of the Nervous Tissue in Ethanol-Induced Neurodegeneration.

The role of NF-κВ in the realization of the growth potential of neural progenitor cells from the subventricular area of cerebral hemispheres and secretion of neurotrophins by glial elements was studied under conditions of in vitro and in vivo modeled ethanol-induced neurodegeneration. It was found that this transcription factor does not participate in the regulation of mitotic activity of neural stem cells and neuronal-committed progenitors under optimal conditions and under the influence of ethanol in vitro. At the same time, NF-κВ suppresses differentiation/maturation of neural progenitor cells. Long-term peroral administration of ethanol to mice was accompanied by the inhibitory influence of NF-κВ on proliferation of progenitor cells. Blockade of NF-κВ in neural stem cells and committed neuronal precursors in animals with neurodegeneration induced cell cycle progression in these elements. The involvement of NF-κВ in the secretory function of astrocytes and oligodendrogliocytes was established. Inactivation of the nuclear transcription factor reduced the production of neurotrophins, in particular, in the case of ethanol exposure. At the same time, no changes in the function of microglia were noted.

Adult and endemic neurogenesis in the vestibular nuclei after unilateral vestibular neurectomy.

We previously revealed adult reactive neurogenesis in deafferented vestibular nuclei following unilateral vestibular neurectomy (UVN) in the feline model. We recently replicated the same surgery in a rodent model and aimed to elucidate the origin and fate of newly generated cells following UVN. We used specific markers of cell proliferation, glial reaction, and cell differentiation in the medial vestibular nucleus (MVN) of adult rats. UVN induced an intense cell proliferation and glial reaction with an increase of GFAP-Immunoreactive (Ir), IBA1-Ir and Olig2-Ir cells 3 days after the lesion in the deafferented MVN. Most of the newly generated cells survived after UVN and differentiated into oligodendrocytes, astrocytes, microglial cells and GABAergic neurons. Interestingly, UVN induced a significant increase in a population of cells colocalizing SOX2 and GFAP 3 days after lesion in the deafferented MVN indicating the probable presence of multipotent cells in the vestibular nuclei. The concomitant increase in BrdU- and SOX2-Ir cells with the presence of SOX2 and GFAP colocalization 3 days after UVN in the deafferented MVN may support local mitotic activity of endemic quiescent neural stem cells in the parenchyma of vestibular nuclei.

CD8+ T cells are increased in the subventricular zone with physiological and pathological aging.

Age‐related cognitive decline and neurodegenerative diseases are associated with less functional neurogenic niches. It has been recently shown that aged subventricular zone (SVZ) suffers an infiltration of T cells, which affects neural stem cell activity in mice. Whether this occurs in human neurogenic niches or to which extent T‐cell infiltration is also taking place in neurodegenerative diseases remains unknown. In this work, we studied the presence of T cells in both human neurogenic niches in young and old individuals. There was a significant increase in the number of CD3+ and CD8+ T cells in the SVZ of elderly individuals, which was not detected in the dentate gyrus. Moreover, we also found CD3+ and CD8+ T cells in the SVZ of individuals with neurodegenerative diseases. However, T‐cell count was similar when compared non‐neuropathological elderly with disease diagnosed patients. Our study reveals the infiltration of T cells in old human brains, particularly in the SVZ under non‐pathological conditions and also in neurodegenerative contexts.

Prospects for using mitogen-activated protein kinases ERK1/2 and p38 of nerve tissue progenitors as pharmacological targets for the treatment of neurodegeneration caused by alcohol

We studied the possibility of using ERK1/2 inhibitor and p38 inhibitor to modulate the functions of nerve tissue precursor cells in ethanol-induced neurodegeneration. It has been found that in chronic alcoholization of mice both these pharmacological substances are able to increase proliferative activity of neural stem cells (NSC). In addition, the blockade of ERK1/2 initiates the progression of the cell cycle of the neuronal-committed progenitors (NCP, clonogenic PSA-NCAM + cells). This reduces the differentiation intensity of NSC and clonogenic PSA-NCAM + cells under the influence of the ERK1/2 inhibitor and such in NSC under the influence of p38 inhibitor. The fundamental difference between the functions of ERK1/2 and p38 in the regulation of the NSC and NCP cell cycle in optimal living conditions and in ethanol-induced neurodegeneration does not allow to draw unambiguous conclusions about the prospect of using these protein kinases as pharmacological targets of drugs for the therapy of alcoholic encephalopathy.

Conserved and Divergent Features of Adult Neurogenesis in Zebrafish.

Adult neurogenesis, i.e., the generation of neurons from neural stem cells (NSCs) in the adult brain, contributes to brain plasticity in all vertebrates. It varies, however, greatly in extent, location and physiological characteristics between species. During the last decade, the teleost zebrafish (D. rerio) was increasingly used to study the molecular and cellular properties of adult NSCs, in particular as a prominent NSC population was discovered at the ventricular surface of the dorsal telencephalon (pallium), in territories homologous to the adult neurogenic niches of rodents. This model, for its specific features (large NSC population, amenability to intravital imaging, high regenerative capacity) allowed rapid progress in the characterization of basic adult NSC features. We review here these findings, with specific comparisons with the situation in rodents. We specifically discuss the cellular nature of NSCs (astroglial or neuroepithelial cells), their heterogeneities and their neurogenic lineages, and the mechanisms controlling NSC quiescence and fate choices, which all impact the neurogenic output. We further discuss the regulation of NSC activity in response to physiological triggers and non-physiological conditions such as regenerative contexts.

Single-Cell Profiling and SCOPE-Seq Reveal Lineage Dynamics of Adult Ventricular-Subventricular Zone Neurogenesis and NOTUM as a Key Regulator

SUMMARYIn the adult ventricular-subventricular zone (V-SVZ), neural stem cells (NSCs) generate new olfactory bulb (OB) neurons and glia throughout life. To map adult neuronal lineage progression, we profiled >56,000 V-SVZ and OB cells by single-cell RNA sequencing (scRNA-seq). Our analyses reveal the molecular diversity of OB neurons, including fate-mapped neurons, lineage progression dynamics, and an NSC intermediate enriched for Notum, which encodes a secreted WNT antagonist. SCOPE-seq technology, which links live-cell imaging with scRNA-seq, uncovers cell-size transitions during NSC differentiation and preferential NOTUM binding to proliferating neuronal precursors. Consistently, application of NOTUM protein in slice cultures and pharmacological inhibition of NOTUM in slice cultures and in vivo demonstrated that NOTUM negatively regulates V-SVZ proliferation. Timely, context-dependent neurogenesis demands adaptive signaling among neighboring progenitors. Our findings highlight a critical regulatory state during NSC activation marked by NOTUM, which attenuates WNT-stimulated proliferation in NSC progeny.

Waking up quiescent neural stem cells: Molecular mechanisms and implications in neurodevelopmental disorders.

Neural stem cells (NSCs) are crucial for development, regeneration, and repair of the nervous system. Most NSCs in mammalian adult brains are quiescent, but in response to extrinsic stimuli, they can exit from quiescence and become reactivated to give rise to new neurons. The delicate balance between NSC quiescence and activation is important for adult neurogenesis and NSC maintenance. However, how NSCs transit between quiescence and activation remains largely elusive. Here, we discuss our current understanding of the molecular mechanisms underlying the reactivation of quiescent NSCs. We review recent advances on signaling pathways originated from the NSC niche and their crosstalk in regulating NSC reactivation. We also highlight new intrinsic paradigms that control NSC reactivation in Drosophila and mammalian systems. We also discuss emerging evidence on modeling human neurodevelopmental disorders using NSCs.

Transcriptional Heterogeneity of Neural Stem Cells during Axolotl Salamander Spinal Cord Regeneration

Unlike humans, axolotl salamanders are capable of restoring sensory and motor function after spinal cord injury. Upon tail amputation or spinal cord transection, endogenous neural stem cells (NSCs) increase proliferation and differentiate into multiple neural and glial cell types of the central and peripheral nervous system. How NSCs transition from a resting state to a regenerative state and coordinate proper neural differentiation is poorly understood. Here, we study the global changes that occur in NSCs after spinal cord injury as well as specific changes in gene transcription as the NSC regenerative program unfolds. We first studied global changes in NSCs after injury by measuring global RNA synthesis temporally and spatially through incorporation of 5‐ethynyl‐uridine into nascent RNA. Substantial upward shifts in overall transcriptional output occurred in specific NSC populations during regeneration, which was correlated with loss of facultative repressive chromatin marks, and was dependent on Hippo‐YAP signaling. We also found an evolutionarily conserved upregulation of nascent RNA production in active neural progenitors in axolotls and mice. In order to dig deeper into neural stem cell dynamics during regeneration, we adopted multiplexed fluorescence in situ hybridization (FISH) in tissue section and whole mount tissues. We produced a user‐friendly, sharable, Docker Container for automated high‐throughput design of Third Generation Hybridization Chain Reaction (HCR) FISH probe sets. An advantage of HCR‐FISH is that it utilizes non‐enzymatic signal amplification, while maintaining low background signal, leading to high signal‐to‐noise detection of mRNAs. We demonstrate that the HCR‐FISH method is amenable to multiplexing through five sequential rounds of hybridization (SeqFISH). Using our probe design pipeline and HCR‐FISH, we imaged NSC activation and differentiation in tissue sections collected after injury using 15 simultaneously‐detected probe sets indicative to CNS and PNS cell types. Whole mount HCR‐FISH for three neural markers were also imaged simultaneously to assess NSC and neuron responses after injury. Overall, the genetic neuroanatomical mapping performed here using molecular classification of NSCs provides novel information on how NSCs respond to injury to repair a CNS injury.Support or Funding InformationThis project was funded by grants NSF 1656429, NSF 1558017, and NIH OD024909