Immature Neuronal Marker Research Articles

A Non-Linear Relation between Levels of Adult Hippocampal Neurogenesis and Expression of the Immature Neuron Marker Doublecortin

We investigated the mechanisms underlying the effects of the antidepressant fluoxetine on behavior and adult hippocampal neurogenesis (AHN). After confirming our earlier report that the signaling molecule β2-arrestin is required for the antidepressant-like effects of fluoxetine, we found that the effects of fluoxetine on proliferation of neural progenitors and on survival of adult-born granule cells are absent in the β2-arrestin knockout (β2-Arr KO) mice. To our surprise fluoxetine induced a dramatic upregulation of doublecortin (DCX) in the β2-Arr KO mice, indicating that DCX expression can be increased even though AHN is not. We discovered two other conditions where DCX expression is regulated non linearly compared to levels of AHN: a chronic stress model where DCX is upregulated and an inflammation model where DCX is down regulated. We conclude that assessing DCX expression alone to quantify levels of AHN can be misleading and that caution should be applied when label retention techniques are not available.

Probiotic treatment (Bifidobacterium longum subsp. longum 35624™) affects stress responsivity in male rats after chronic corticosterone exposure

Mounting evidence suggests that gut microbiota do not only regulate intestinal function and health, but that they also play a role in mental health via the gut-brain axis. Previous research further suggests that probiotics may have beneficial health effects, but more research is needed to confirm these beneficial effects and better understand the underlying mechanisms and potential sex differences in the response to probiotics. Therefore, the current study investigates the effects of chronic administration of the commercially available probiotic Bifidobacterium longum subsp. longum 35624™(B. 35624) to male and female rats under control or “stressed” conditions. For this, 24 male and 24 female Sprague-Dawley rats were either given daily corticosterone injections (40 mg/kg; to induce depressive-like behavior and a “stressed” condition) or oil injections (controls) together with oral administration of B.35624 or vehicle for 21 days (n = 5–7/group). Animals performed the Open Field Test (OFT) and Forced Swim Test (FST) and several blood samples were collected to investigate basal as well as stress-induced corticosterone levels. Rats were sacrificed on day 22 and their brains sliced and stained with doublecortin, a marker of immature neurons. Results showed that B.35624 was not able to rescue depressive-like behavior or induce changes in neurogenesis in males or females, but the probiotic impacted hypothalamic-pituitary-adrenal axis functioning in male animals and tended to reduce anxiolytic behavior in the OFT. More research is needed to further elucidate the potential health effects of probiotics especially in regard to possible sex differences.

Superior Synaptogenic Effect of Electrospun PLGA-PEG Nanofibers Versus PLGA Nanofibers on Human Neural SH-SY5Y Cells in a Three-Dimensional Culture System.

Synapses are touted as the main structural and functional components of neural cells within in the nervous system, providing tissue connectivity and integration via the formation of perineuronal nets. In the present study, we evaluated the synaptogenic activity of electrospun PLGA and PLGA-PEG nanofibers on human SH-SY5Y cells after 14days in vitro. Electrospun PLGA and PLGA-PEG nanofibers were fabricated and physicochemical properties were examined using the HNMR technique. The cells were classified into three random groups, i.e., control (laminin-coated surface), PLGA, and PLGA-PEG. Scaffolds' features, cell morphology, attachment, and alignment were monitored by SEM imaging. We performed MTT assay to measure cell survival rate. To evaluate neurite formation and axonal outgrowth, cells were stained with an antibody against β-tubulin III using immunofluorescence imaging. Antibodies against synapsin-1 and synaptophysin were used to explore the impact of PLGA and PLGA-PEG scaffolds on synaptogenesis and functional activity of synapses. According to SEM analysis, the PLGA-PEG scaffold had less thick nanofibers compared with the PLGA scaffold. Cell attachment, expansion, neurite outgrowth, and orientation were promoted in the PLGA-PEG group in comparison with the PLGA substrate (p < 0.05). MTT assay revealed that both scaffolds did not exert any neurotoxic effects on cell viability. Notably, PLGA-PEG surface increased cell viability compared to PLGA by time (p < 0.05). Immunofluorescence staining indicated an increased β-tubulin III level in the PLGA-PEG group days coincided with axonal outgrowth and immature neuron marker after seven compared with the PLGA and control groups (p < 0.05). Based on our data, both synaptogenesis and functional connectivity were induced in cells plated on the PLGA-PEG surface that coincide with the increase of synapsin-1 and synaptophysin in comparsion with the PLGA and control groups (p < 0.05). Taken together, our results imply that the PLGA-PEG nanofibers could provide the desirable microenvironment to develop perineuronal net formation, contributing to efficient synaptogenesis and neuron-to-neuron crosstalk.

Nwd1 Regulates Neuronal Differentiation and Migration through Purinosome Formation in the Developing Cerebral Cortex.

SummaryEngagement of neural stem/progenitor cells (NSPCs) into proper neuronal differentiation requires the spatiotemporally regulated generation of metabolites. Purines are essential building blocks for many signaling molecules. Enzymes that catalyze de novo purine synthesis are assembled as a huge multienzyme complex called “purinosome.” However, there is no evidence of the formation or physiological function of the purinosome in the brain. Here, we showed that a signal transduction ATPases with numerous domains (STAND) protein, NACHT and WD repeat domain-containing 1 (Nwd1), interacted with Paics, a purine-synthesizing enzyme, to regulate purinosome assembly in NSPCs. Altered Nwd1 expression affected purinosome formation and induced the mitotic exit and premature differentiation of NSPCs, repressing neuronal migration and periventricular heterotopia. Overexpression/knockdown of Paics or Fgams, other purinosome enzymes, in the developing brain resulted in a phenocopy of Nwd1 defects. These findings indicate that strict regulation of purinosome assembly/disassembly is crucial for maintaining NSPCs and corticogenesis.

Insulin‐like Growth Factor‐1 Treatment of Experimental Traumatic Brain Injury Increases Newborn Neuron Migration Through the Granular Cell Layer

Traumatic brain injuries (TBI) are a tragic yet surprisingly common problem throughout the world. In the United States alone, 2.8 million people a year are diagnosed with a TBI. TBI causes cellular death and dysfunction within the brain that is connected with cognitive and behavioral dysfunction both clinically and experimentally.The controlled cortical impact (CCI) model is a well characterized contusion model of TBI that emulates both motor and cognitive deficits in addition to cell loss within the dentate gyrus of the hippocampus. Following CCI there is an early loss of the immature neuron population in the subgranular zone of the dentate gyrus followed by a compensatory increase in newborn neurons. Additionally, there is increased glial reactivity in response to injury. Of these newborn neurons, only a small portion survive and incorporate into the granular cell layer (GCL) of the dentate gyrus. However, the surviving trauma‐born neurons display stunted growth and reduced arborization, and not many make it to a fully functional form.Previous research shows that treatment with insulin‐like growth factor‐1 (IGF‐1) conveys behavioral improvements and increases the survival of doublecortin (DCX) positive trauma‐born neurons with improved dendritic arborization. In the current study, C57BL/6J mice underwent a CCI or a sham injury and were administered IGF‐1 (3mg/mg) or vehicle by intracerebroventricular injection (nCCI&amp;IGF‐1 = 7 | nCCI&amp;Veh = 8 | nSham&amp;IGF‐1 = 4 | nSham&amp;Veh = 4) for 7 days following injury. Animals were euthanized 7 days after injury, and the brain was collected, processed, and sectioned at a thickness of 40mm for immunohistochemistry. Tissue was then immuno‐labeled for DCX, a marker of immature neurons, and glial fibrillary acidic protein (GFAP), a marker of astrocytes in the brain, and imaged.We observed that the trauma‐born neurons of CCI injured mice treated with IGF‐1 migrate from the subgranular zone and into the outer 2/3rds of the GCL more frequently than do those observed in animals treated with vehicle. During development, GFAP positive cells send processes out that serve as a scaffolding by which immature neurons can migrate. As such, we hypothesized that after TBI, immature neurons migrate through the GCL by utilizing GFAP positive processes, and that IGF‐1 would increase the number of processes projecting through the GCL compared to vehicle treated animals.We assessed the number of GFAP positive processes projecting into the GCL by counting the number of processes per millimeter in three brain sections collected from the epicenter of injury. Our findings suggest that there is an increase in GFAP processes following CCI, but treatment with IGF‐1 did not increase the number of these processes. Our findings suggest that increased migration seen with IGF‐1 treatment does not occur along glial processes, or that this is not the only means by which DCX positive cells are able to migrate after injury.Support or Funding InformationFunded through American Physiological Society 2019 UGSRF Fellowship

Alteration of the neurogenesis and long term potential of olfactory bulb in an animal model of PTSD

Failure of extinction of fear-conditioned traumatic memory is the main pathology behind post-traumatic stress disorder (PTSD). Functional and structural dysfunctions in the olfactory system are implicated by studies in PTSD patients. However, little is known regarding the neurobiological networks of trauma‑related odor sensitivity in PTSD. Male Wistar rats were exposed with a female cat for 10 min and long-term stress was evaluated by behavioral tests, containing open field (OF) and elevated plus maze (EPM). To prove the PTSD model, the serum level of cortisol was evaluated and compared with the control group. Local field potential (LFP) was applied to compare the electrophysiology of the OB in two groups. To assess neurogenesis, the expression of nestin, and doublecortin were evaluated. Data from EPM revealed a significant increase in spent time in the closed arms in PTSD group. We observed a significant reduction in OF parameters in terms of the total distance traveled, the time spent in the central zone, and the number of crossing the central zone in PTSD group compared to the control group. The mean serum cortisol level was significantly higher in the PTSD group than the control group. In LFP recording, the slope and the amplitude of field excitatory postsynaptic potential (fEPSP) in the PTSD group were significantly higher than that of the control group. Our results also showed that the mRNA expression level of nestin as a neural progenitor marker and doublecortin, as an immature neuron marker, significantly decreased in the PTSD group compared to the control group. This study has shown that PTSD can disrupt the OB function through decreasing neurogenesis. More information on PTSD and OB would help us to establish a greater degree of accuracy on this matter.

Reproducible differentiation and characterization of neurons from mouse embryonic stem cells

Investigation on the effects of disease-associated mutations on neurodevelopment is an essential approach to understand the molecular basis of neurological disorders and can be achieved by generating suitable animal models. However, some of the mutations preclude development of animal models, leaving cell-based models as the only options. Mouse embryonic stem cells (mESCs) are attractive because of the well-established technologies for introducing disease-associated mutations and the feasibility of investigating the abnormalities during different stages of neurogenesis. Importantly, such transgenic mESCs enable large-scale screening and identification of the most promising small molecules and/or drug candidates before undertaking expensive animal studies. Although neuronal differentiation from mESCs is one of the earliest methods to be developed, we observed that the published as well as publicly available methods did not yield neurons consistently. Here, we describe a 16-day differentiation protocol that consistently induced differentiation of mESCs into neurons. This step-wise protocol enables monitoring of the neuronal differentiation process at different stages as well as characterization using the markers for immature and mature neurons by using immunocytochemistry and quantitative real-time PCRs.•Development of a method for differentiating mouse ES cells into neurons.•Differentiating the mouse ES cells into embryoid bodies prior to induction of neuronal differentiation results in better neuron formation.

Korean red ginseng promotes hippocampal neurogenesis in mice.

Neurogenesis in the adult hippocampus plays a major role in cognitive ability of animals including learning and memory. Korean red ginseng (KRG) has long been known as a medicinal herb with the potential to improve learning and memory; however, the mechanisms are still elusive. Therefore, we evaluated whether KRG can promote cognitive function and enhance neurogenesis in the hippocampus. Eight-week-old male C57BL/6 mice received 50 mg/kg of 5-bromo-2′-deoxyuridine (BrdU) intraperitoneally and 100 mg/kg of KRG or vehicle orally once a day for 14 days. Pole, Rotarod and Morris water maze tests were performed and the brains were collected after the last behavioral test. Changes in the numbers of BrdU- and BrdU/doublecortin (DCX; a marker for neuronal precursor cells and immature neurons)-positive cells in the dentate gyrus and the gene expression of proliferating cell nuclear antigen (a marker for cell differentiation), cerebral dopamine neurotrophic factor and ciliary neurotrophic factor in the hippocampus were then investigated. KRG-treated mice came down the pole significantly faster and stood on the rotarod longer than vehicle-treated mice. The Morris water maze test showed that KRG administration enhanced the learning and memory abilities significantly. KRG also significantly increased BrdU- and BrdU/DCX-positive cells in the dentate gyrus as well as the proliferating cell nuclear antigen, cerebral dopamine neurotrophic factor and ciliary neurotrophic factor mRNA expression levels in the hippocampus compared to vehicle. Administration of KRG promotes learning and memory abilities, possibly by enhancing hippocampal neurogenesis. This study was approved by the Pusan National University Institutional Animal Care and Use Committee (approval No. PNU-2016-1071) on January 19, 2016.

Effects of neurogenesis in the dentate gyrus (DG) of hippocampus on depression-like behaviors in WKY rats

To observe the effects of the neurogenesis in the dentate gyrus (DG) of the hippocampus on depression-like behaviors in adult Wistar Kyoto (WKY) rats. There were three groups in total (n = 10): ① the control (Wistar) group: 9-week-old Wistar rats were treated with saline for 3 weeks (10 mg/kg, intragastric administration); ② the depression model (WKY) group: WKY rats of the same age, tested for depression-like behaviors, were as a rat model of depression, and were treated with saline for 3 weeks (10 mg/kg, intragastric administration); ③ the positive control (AMI+WKY) group: WKY rats of the same age were treated with amitriptyline for 3 weeks (10 mg/kg, intragastric administration). The neurogenesis in hippocampus was detected by immunofluorescence staining for Ki67 (a neuronal proliferation marker) and DCX (an immature neuronal marker). The depression-like behaviors were assessed by sucrose preference test (SPT), open field test (OFT) and forced swimming test (FST). ① When compared with Wistar rats, the number of Ki67+ cells and DCX+ cells of the DG in WKY rats were decreased by 33.0% (P＜0.01) and 39.2% (P＜0.01), respectively; amitriptyline treatment significantly increased the number of Ki67+cells and DCX+ cells in the DG by 43.8% (P＜0.01) and 46.7% (P＜0.01), respectively, as compared with WKY rats. ② When compared with control group, WKY rats showed a significant decrease in sucrose preference (P＜0.01), less total horizontal distance (P＜0.01) and less time entered the center field (P＜0.01) in the OFT, the immobility time in the FST was increased significantly (P＜0.01). Amitriptyline treatment significantly improved the depression-like behaviors in WKY rats. ① The proliferation and differentiation of hippocampal neural stem cells in adult WKY rats are significantly lower than those of Wistar rats, suggesting that the neurogenesis in adult WKY rats is impaired. ②Amelioration of impaired hippocampal neurogenesis can partially reverse the depression-like behaviors in adult WKY rats.

Differential effects of fluoxetine and venlafaxine in the neural embryonic stem cell test (ESTn) revealed by a cell lineage map

There is a need for in vitro tests for the evaluation of chemicals and pharmaceuticals that may cause developmental neurotoxicity (DNT) in humans. The neural embryonic stem cell test (ESTn) is such an in vitro test that mimics early neural differentiation. The aim of this study was to define the biological domain of ESTn based on the expression of selective markers for certain cell types, and to investigate the effects of two antidepressants, fluoxetine (FLX) and venlafaxine (VNX), on neural differentiation. A cell lineage map was made to track neural differentiation and the effects of FLX and VNX in ESTn. Whole transcriptome analysis revealed differentiation from an embryonic stem cell population to a mixed culture of neural progenitors, neurons and neural crest cells 7 days into differentiation. Maturing neurons, astrocytes and oligodendrocytes were present after 13 days. Exposure to FLX or VNX led to different expression patterns between compounds at both time points. On day 7, both compounds upregulated most of the stem cell- and immature neuron markers, but had distinct effects on neural subtype markers. FLX downregulated glycinergic markers and upregulated cholinergic markers, while VNX had the opposite effect. On day 13, FLX and VNX affected their specific therapeutic targets, represented by mainly serotonergic markers by FLX- and dopaminergic and noradrenergic markers in VNX-exposed cultures, as well as oligodendrocyte and glycinergic neuron markers. This proof of concept study shows the added value of assessing DNT in ESTn through a cell lineage map and gives mechanistic insight in the potential neurodevelopmental effects of FLX and VNX. More compounds should be tested to further evaluate the use of the cell lineage map.

Neural cell adhesion molecule (NCAM) and polysialic acid–NCAM expression in developing ICR mice

Abstract Background Coexpression of polysialic acid (PSA)–neuronal cell adhesion molecule (NCAM) with immature neuronal markers is used to indicate the developmental state of neurons generated in the subgranular zone (SGZ) of adult hippocampus. PSA–NCAM is highly expressed throughout the embryonic and juvenile mammalian brain, but heavily downregulated in adult brain. Objective To visualize the expression profiles of NCAM/PSA–NCAM in the dentate SGZ of the hippocampus in developing ICR mice. Methods Cellular distribution, expression, and developmental changes of NCAM/PSA–NCAM were studied in ICR mice at embryonic age 17 days (E17); and similarly at postnatal ages P3, P5, and P7. The SGZ was studied using NCAM and PSA–NCAM immunoreactive staining with or without hematoxylin counterstaining. Western blotting was used to confirm protein expression levels. Results NCAM expression was localized to the surface of neurons and glia and was higher in postnatal mice than it was in embryonic mice. PSA–NCAM was found in cytoplasm and membrane of neural cells, more densely staining in the dentate SGZ at P7, but no staining found at E17. Western blotting of brain tissues also showed expression of both PSA–NCAM and NCAM increased significantly at P5 and P7 compared with expression at P3. Conclusions Progressive increase in NCAM expression occurs in the SGZ during embryogenic and postnatal development. PSA–NCAM was not expressed in embryonic ICR mice, but was increased after birth and highly localized in the SGZ at P7. This NCAM expression pattern in the developing brain indicating structural plasticity and neurogenesis may be useful for study of brain repair.

A positive allosteric modulator of mGluR5 promotes neuroprotective effects in mouse models of Alzheimer's disease

Alzheimer's Disease (AD) is the most prevalent neurodegenerative disorder. Despite advances in the understanding of its pathophysiology, none of the available therapies prevents disease progression. Excess glutamate plays an important role in excitotoxicity by activating ionotropic receptors. However, the mechanisms modulating neuronal cell survival/death via metabotropic glutamate receptors (mGluRs) are not completely understood. Recent data indicates that CDPPB, a positive allosteric modulator of mGluR5, has neuroprotective effects. Thus, this work aimed to investigate CDPPB treatment effects on amyloid-β (Aβ) induced pathological alterations in vitro and in vivo and in a transgenic mouse model of AD (T41 mice). Aβ induced cell death in primary cultures of hippocampal neurons, which was prevented by CDPPB. Male C57BL/6 mice underwent stereotaxic surgery for unilateral intra-hippocampal Aβ injection, which induced memory deficits, neurodegeneration, neuronal viability reduction and decrease of doublecortin-positive cells, a marker of immature neurons and neuronal proliferation. Treatment with CDPPB for 8 days reversed neurodegeneration and doublecortin-positive cells loss and recovered memory function. Fourteen months old T41 mice presented cognitive deficits, neuronal viability reduction, gliosis and Aβ accumulation. Treatment with CDPPB for 28 days increased neuronal viability (32.2% increase in NeuN+ cells) and reduced gliosis in CA1 region (Iba-1+ area by 31.3% and GFAP+ area by 37.5%) in transgenic animals, without inducing hepatotoxicity. However, it did not reverse cognitive deficit. Despite a four-week treatment did not prevent memory loss in aged transgenic mice, CDPPB is protective against Aβ stimulus. Therefore, this drug represents a potential candidate for further investigations as AD treatment.

Genetic Ablation of Neural Progenitor Cells Impairs Acquisition of Trace Eyeblink Conditioning.

Adult-born neurons are believed to play a role in memory formation by providing enhanced plasticity to the hippocampus. Past studies have demonstrated that reduction of neurogenesis impairs associative learning, but these experiments used irradiation or neurotoxic substances, which may have had unintended off-target effects. Therefore, to investigate the role of these adult-born neurons more precisely, we used nestin-HSV-TK transgenic mice (Nes-TK) to selectively ablate newborn neurons. Nes-TK mice were fed a chow infused with valganciclovir to induce the ablation of neural progenitor cells. After being on this diet for 4 weeks, mice were trained on trace eyeblink conditioning, a hippocampus-dependent temporal associative memory task. Following the completion of training, brain sections from these animals were stained for doublecortin, a marker for immature neurons, to quantify levels of neurogenesis. We found that male transgenic mice on valganciclovir had significantly decreased amounts of doublecortin relative to male control animals, indicating a successful reduction in levels of neurogenesis. In conjunction with this reduction in neurogenesis, the male transgenic mice on valganciclovir learned at a significantly slower rate than male control mice. The female Nes-TK mice on valganciclovir showed no significant decrease in neurogenesis and no behavioral impairment relative to female control mice. Ultimately, the results are consistent with, and expand upon, prior studies that demonstrated that adult-born neurons are involved in the formation of associative memories. This study also provides a foundation to continue to explore the physiological role of newborn neurons with in vivo recordings during behavioral training.

Early-life exposure to low levels of permethrin exerts impairments in learning and memory with the effects on neuronal and glial population in adult male mice.

Permethrin, a pyrethroid chemical, is widely used as a pesticide because of its rapid insecticidal activity. Although permethrin is considered to exert very low toxicity in mammals, the effects of early, low-level, chronic exposure on the adult central nervous system are unclear. In this study, we investigated the effects of low-level, chronic permethrin exposure in early life on the brain functions of adult mice, using environmentally relevant concentrations. We exposed mice to the acceptable daily intake level of permethrin (0.3ppm) in drinking water during the prenatal and postnatal periods. We then examined the effects on the central nervous system in adult male offspring. In the permethrin group, we detected behavior that displayed incomplete adaptation to a novel environment, as well as an impairment in learning and memory. In addition, immunohistochemical analysis revealed an increase in doublecortin- (an immature neuron marker) positive cells in the hippocampal dentate gyrus in the permethrin exposure group compared with the control group. Additionally, in the permethrin exposure group there was a decrease in astrocyte number in the hilus of the dentate gyrus, and remaining astrocytes were often irregularly shaped. These results suggest that exposure to permethrin at low levels in early life affects the formation of the neural circuit base and behavior after maturation. Therefore, in the central nervous system of male mice, low-level, chronic permethrin exposure during the prenatal and postnatal periods has effects that were not expected based on the known effects of permethrin exposure in mature animals.

Fluoxetine-induced dematuration of hippocampal neurons and adult cortical neurogenesis in the common marmoset

The selective serotonin reuptake inhibitor fluoxetine (FLX) is widely used to treat depression and anxiety disorders. Chronic FLX treatment reportedly induces cellular responses in the brain, including increased adult hippocampal and cortical neurogenesis and reversal of neuron maturation in the hippocampus, amygdala, and cortex. However, because most previous studies have used rodent models, it remains unclear whether these FLX-induced changes occur in the primate brain. To evaluate the effects of FLX in the primate brain, we used immunohistological methods to assess neurogenesis and the expression of neuronal maturity markers following chronic FLX treatment (3 mg/kg/day for 4 weeks) in adult marmosets (n = 3 per group). We found increased expression of doublecortin and calretinin, markers of immature neurons, in the hippocampal dentate gyrus of FLX-treated marmosets. Further, FLX treatment reduced parvalbumin expression and the number of neurons with perineuronal nets, which indicate mature fast-spiking interneurons, in the hippocampus, but not in the amygdala or cerebral cortex. We also found that FLX treatment increased the generation of cortical interneurons; however, significant up-regulation of adult hippocampal neurogenesis was not observed in FLX-treated marmosets. These results suggest that dematuration of hippocampal neurons and increased cortical neurogenesis may play roles in FLX-induced effects and/or side effects. Our results are consistent with those of previous studies showing hippocampal dematuration and increased cortical neurogenesis in FLX-treated rodents. In contrast, FLX did not affect hippocampal neurogenesis or dematuration of interneurons in the amygdala and cerebral cortex.

Stable Adult Hippocampal Neurogenesis in Cannabinoid Receptor CB2 Deficient Mice

The G-protein coupled cannabinoid receptor 2 (CB2) has been implicated in the regulation of adult neurogenesis in the hippocampus. The contribution of CB2 towards basal levels of proliferation and the number of neural progenitors in the subgranular zone (SGZ) of the dentate gyrus, however, remain unclear. We stained hippocampal brain sections of 16- to 17-week-old wildtype and CB2-deficient mice, for neural progenitor and immature neuron markers doublecortin (DCX) and calretinin (CR) and for the proliferation marker Ki67 and quantified the number of positive cells in the SGZ. The quantification revealed that CB2 deficiency neither altered overall cell proliferation nor the size of the DCX+ or DCX and CR double-positive populations in the SGZ compared to control animals. The results indicate that CB2 might not contribute to basal levels of adult neurogenesis in four-month-old healthy mice. CB2 signaling might be more relevant in conditions where adult neurogenesis is dynamically regulated, such as neuroinflammation.

Aluminum affects neural phenotype determination of embryonic neural progenitor cells.

Aluminum (Al) is a neurotoxin and is associated with the etiology of neurodegenerative diseases, such as Alzheimer's disease (AD). The Al-free ion (Al3+) is the biologically reactive and toxic form. However, the underlying mechanisms of Al toxicity in the brain remain unclear. Here, we evaluated the effects of Al3+ (in the chloride form-AlCl3) at different concentrations (0.1-100µM) on the morphology, proliferation, apoptosis, migration and differentiation of neural progenitor cells (NPCs) isolated from embryonic telencephalons, cultured as neurospheres. Our results reveal that Al3+ at 100µM reduced the number and diameter of neurospheres. Cell cycle analysis showed that Al3+ had a decisive function in proliferation inhibition of NPCs during neural differentiation and induced apoptosis on neurospheres. In addition, 1µM Al3+ resulted in deleterious effects on neural phenotype determination. Flow cytometry and immunocytochemistry analysis showed that Al3+ promoted a decrease in immature neuronal marker β3-tubulin expression and an increase in co-expression of the NPC marker nestin and glial fibrillary acidic protein. Thus, our findings indicate that Al3+ caused cellular damage and reduced proliferation and migration, resulting in global inhibition of NPC differentiation and neurogenesis.

Defining the Vulnerability Window of Anesthesia-Induced Neuroapoptosis in Developing Dentate Gyrus Granule Cells — A Transgenic Approach Utilizing POMC-EGFP Mice

Exposure to commonly used anesthetics is associated with widespread neuroapoptosis in neonatal animals. Vulnerability of developing hippocampal dentate gyrus granule cells to anesthetic neurotoxicity peaks approximately 2 weeks after cell birth, as measured by bromodeoxyuridine birth dating, regardless of the age of the animal. The present study examined whether the vulnerable window can be further characterized by utilizing a transgenic approach. Proopiomelanocortin enhanced green fluorescent protein (POMC-EGFP) mice (postnatal day 21) were exposed to 3% sevoflurane for 6 h. Following exposure, cleaved caspase 3, expression of EGFP and differential maturational markers were quantified and compared with unanesthetized littermates. Electrophysiological properties of EGFP+ and EGFP− cells in the subgranular zone and the inner half of the granule cell layer were recorded by whole-cell patch-clamp. We found that sevoflurane significantly increased apoptosis of POMC-EGFP+ granule cells that accounted for approximate 1/3 of all apoptotic cells in dentate gyrus. Apoptotic EGFP− granule cells more frequently expressed the immature neuronal marker calretinin (75.4% vs 45.0%, P < 0.001) and less frequently the late progenitor marker NeuroD1 (21.9% vs 87.9%, P < 0.001) than EGFP+ granule cells. Although EGFP− granule cells were more mature in immunostaining than EGFP+ granule cells, their electrophysiological properties partially overlapped in terms of input resistance, resting membrane potential and action potential amplitude. Our results revealed the POMC stage, when GABA acts as an excitatory neurotransmitter, only partly captures susceptibility to anesthetic neurotoxicity, suggesting the vulnerable window of anesthesia-induced neuroapoptosis extends from the end of POMC+ stage to the post-POMC+ stage when depolarizing glutamatergic inputs emerge.

Abstract 5224: Conversion of human glioblastoma cells into neurons by neuronal transcription factors

Abstract Background: Glioblastoma is one of the most severe primary cancer types in the central nervous system. Because of genomic and epigenetic heterogeneity, GBM is infiltrative and resistant to conventional treatments such as radiation, chemotherapy and molecular targeting drugs. Glioblastoma often arises from astrocytes, which can be directly converted into neurons according to our earlier work, so we hypothesize that glioblastoma cells might also be converted into non-proliferating neurons. This trans-differentiation therapy might provide a unique approach for glioblastoma treatment. Methods: NeuroD1 was chosen as one of the candidate factors in this study because we have shown its critical roles in astrocyte-to-neuron conversion. Neurog2 and Ascl1 were also tested to understand possible different conversion mechanisms. Single transcription factor or GFP was overexpressed via retrovirus in human glioblastoma cells. Twelve hours after virus infection, culture medium was changed into differentiation medium containing neurotropic factors for neuronal maturation. Immuostainng and other tests were conducted at different days post infection. Results: Retrovirus yielded high infection efficiency in fast-proliferating glioblastoma cells. All three factors tested were capable of converting glioblastoma cells into neuron-like cells efficiently. Besides morphological change, robust pan-neuronal markers were expressed during the conversion, such as immature neuronal markers DCX, Tuj1 and mature neuronal makers MAP2, NeuN. Majority of the converted cells from glioma were immunopositive for glutamatergic neuron marker vGluT1 and hippocampal neuron marker Prox1. Reactive astroglial marker GFAP decreased after conversion, but cancer marker EGFR and IL13Ra2 remained during conversion. Cell proliferation was inhibited during conversion indicated by Ki67 and BrdU. Robust synaptic puncta along dendrites were found in glioma-converted cells, indicated by SV2 immunostaining. Patch-clamp recordings revealed that most of the converted neurons could fire multiple action potentials or single action potential. Conclusion: Our data suggest that several neuronal transcription factors are capable to convert human glioblastoma cells into neuron-like cells efficiently. The converted cells obtained a variety of neuron-specific markers with functional synaptic networks and active electrophysiological properties. This neuronal conversion was also confirmed by reduction of reactive astroglial marker GFAP. Although some cancer markers remain in the converted neurons, glioblastoma cells stopped proliferating once being converted. In summary, our study suggests that converting human glioblastoma cells into neurons could be a potential therapeutic approach for glioblastoma treatment to at least control cancer cell proliferation and inhibit tumor progression. Citation Format: Xin Wang, Zifei Pei, Aasma Hossain, Tania T. Barnatan, Yuting Bai, Gong Chen. Conversion of human glioblastoma cells into neurons by neuronal transcription factors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5224.

A single injection of imipramine affected proliferation in the hippocampus of adult Swiss mice depending on the route of administration, doses, survival time and lodging conditions

Swiss mice may be valuable for the screening of antidepressants in preclinical trials. Acute treatment with antidepressants may affect the behaviour of Swiss mice, but the effects on their hippocampal neurogenesis remain unknown. The present work aims to assess the influence of acute treatment with antidepressants on cell proliferation in the dentate gyrus of the hippocampus of adult Swiss mice. Cell proliferation was estimated by ex vivo counting of Ki-67 immunoreactive nuclei (Ki-67-ir) in the dentate gyrus of Swiss mice housed in standard or enriched environments, at survival-times 2 or 24 h after imipramine injection Independent of the experimental group, intraperitoneal imipramine (0 or 30 mg/kg) failed to change the number of Ki-67-ir in the hippocampus of mice. Through intracerebroventricular route, imipramine reduced the number of Ki-67-ir in the hippocampus of Swiss mice at the dose of 0.06 nmol and increased it at the dose 0.2 nmol. At the dose 0.2 nmol, not 0.06 nmol, imipramine increased the immunoreactivity to doublecortin (a marker for immature neurons) in the hippocampus of mice. The effects of intracerebroventricular injection of imipramine on neurogenesis markers were seen 24 h after the injection in mice housed in standard conditions. The effects of intracerebroventricular injection of imipramine on neurogenesis markers were absent in mice housed in enrichment or 2 h after the injection. These data suggest that acute treatment with imipramine may affect proliferation in the hippocampus of adult Swiss mice depending on the route of administration, doses, survival time and lodging conditions.