Developing Rat Brain Research Articles

Lasting effects of chronic fluoxetine treatment on the late developing rat brain: Age-dependent changes in the serotonergic neurotransmitter system assessed by pharmacological MRI

With the growing prevalence of psychotropic drug prescriptions among children and adolescents, the need for studies on lasting effects of drug exposure on the developing brain rises. Fluoxetine is the only selective serotonin reuptake inhibitor (SSRI) officially registered to treat major depressive disorder in children. Although various (pre)clinical studies have assessed the (long-term) effects of fluoxetine exposure in the perinatal period and in adulthood, limited data is available on its effects on the developing brain later in life, i.e. during adolescence. The present study aimed at investigating the effects of age following chronic SSRI treatment on the central serotonin (5-HT) system. To this end, pharmacological MRI (phMRI) was performed in chronic fluoxetine-treated (5 mg/kg, oral gavage for 3 weeks) juvenile (PND25) and adult rats (PND65) after a 1-week washout period, using an acute fluoxetine challenge (5 mg/kg, i.v.) to trigger the 5-HT system. We observed a diminished brain response to the acute challenge in adult treated animals when compared to control animals, whereas this response was increased in juvenile treated rats. As a result, a significant age by treatment interaction effect was seen in several (subcortical) 5-HT related brain regions. An opposite effect of chronic fluoxetine treatment was seen in the developing brain compared to that in matured brain, as assessed non-invasively using phMRI. These findings most likely reflect neuronal imprinting effects of juvenile SSRI treatment and may underlie emotional disturbances seen in animals and children treated with this drug. Also, our findings suggest that phMRI might be ideally suited to study this important issue in the pediatric population.

Protective effect of pyruvate against ethanol-induced apoptotic neurodegeneration in the developing rat brain

Exposure to alcohol during the early stages of brain development can lead to neurological disorders in the CNS. Apoptotic neurodegeneration due to ethanol exposure is a main feature of alcoholism. Exposure of developing animals to alcohol (during the growth spurt period in particular) elicits apoptotic neuronal death and causes fetal alcohol effects (FAE) or fetal alcohol syndrome (FAS). A single episode of ethanol intoxication (at 5g/kg) in a seven-day-old developing rat can activate the apoptotic cascade, leading to widespread neuronal death in the brain. In the present study, we investigated the potential protective effect of pyruvate against ethanol-induced neuroapoptosis. After 4h, a single dose of ethanol induced upregulation of Bax, release of mitochondrial cytochrome-c into the cytosol, activation of caspase-3 and cleavage of poly (ADP-ribose) polymerase (PARP-1), all of which promote apoptosis. These effects were all reversed by co-treatment with pyruvate at a well-tolerated dosage (1000mg/kg). Histopathology performed at 24 and 48h with Fluoro-Jade-B and cresyl violet stains showed that pyruvate significantly reduced the number of dead cells in the cerebral cortex, hippocampus and thalamus. Immunohistochemical analysis at 24h confirmed that ethanol-induced cell death is both apoptotic and inhibited by pyruvate. These findings suggest that pyruvate treatment attenuates ethanol-induced neuronal cell loss in the developing rat brain and holds promise as a safe therapeutic and neuroprotective agent in the treatment of neurodegenerative disorders in newborns and infants.

Nuclear Clusterin Is Associated with Neuronal Apoptosis in the Developing Rat Brain upon Ethanol Exposure

Fetal alcohol spectrum disorder (FASD) is often accompanied by reduced brain volumes, reflecting brain cell death induced by ethanol, but the molecular mechanisms were less elucidated. This study was set up to investigate whether clusterin (Clu) was involved in neuronal cell death in developing rats. Seven-day-old rats were subcutaneously injected with 20% ethanol in normal saline at 3 g/kg twice. The upregulation of Clu and cell death was detected by immunohistochemistry, immunofluorescence microscopy, and/or Western blotting. Protein-protein interaction was detected by immunoprecipitation and immunoblotting. To identify the isoform interacting with Bcl-XL, HT22 mouse hippocampal cells were transfected with nuclear Clu(nClu)- or secretory Clu-expressing vector, and confocal microscopy was performed. Clu transcripts were knocked down in primary cortical cells using siRNA. We found that Clu increased in the cerebral cortex following acute ethanol treatment. The Clu upregulation was related to increased cell death, which was assessed by activated caspase-3 and TUNEL staining. The upregulated Clu was a nuclear isoform that was nuclear translocated upon ethanol exposure and interacted with Bcl-XL, mediating apoptosis. This study shows that nClu plays a pro-apoptotic role in ethanol-induced cell death in the developing brain, providing new insights for development of FASD.

Chlorpromazine Protects Against Apoptosis Induced by Exogenous Stimuli in the Developing Rat Brain

BackgroundChlorpromazine (CPZ), a commonly used antipsychotic drug, was found to play a neuroprotective role in various models of toxicity. However, whether CPZ has the potential to affect brain apoptosis in vivo is still unknown. The purpose of this study was to investigate the potential effect of CPZ on the apoptosis induced by exogenous stimuli.MethodologyThe ethanol treated infant rat was utilized as a valid apoptotic model, which is commonly used and could trigger robust apoptosis in brain tissue. Prior to the induction of apoptosis by subcutaneous injection of ethanol, 7-day-old rats were treated with CPZ at several doses (5 mg/kg, 10 mg/kg and 20 mg/kg) by intraperitoneal injection. Apoptotic cells in the brain were measured using TUNEL analysis, and the levels of cleaved caspase-3, cytochrome c, the pro-apoptotic factor Bax and the anti-apoptotic factor Bcl-2 were assessed by immunostaining or western blot.FindingsCompared to the group injected with ethanol only, the brains of the CPZ-pretreated rats had fewer apoptotic cells, lower expression of cleaved caspase-3, cytochrome c and Bax, and higher expression of Bcl-2. These results demonstrate that CPZ could prevent apoptosis in the brain by regulating the mitochondrial pathway.ConclusionsCPZ exerts an inhibitory effect on apoptosis induced by ethanol in the rat brain, intimating that it may offer a means of protecting nerve cells from apoptosis induced by exogenous stimuli.

Lipopolysaccharide-Induced Preconditioning Against Ischemic Injury Is Associated With Changes in Toll-Like Receptor 4 Expression in the Rat Developing Brain

Lipopolysaccharide (LPS) preconditioning reduces ischemic injury in adult brain by activating Toll-like receptor 4 (TLR-4). We sought to investigate the effect of brain maturity on the efficacy of LPS preconditioning against hypoxic-ischemic (HI) injury in the developing rat brain. Rat pups at the specified age were randomly assigned to LPS-treated (0.1 mg/kg) or saline-treated groups. HI injury was induced 48 h later by occluding the right common carotid artery followed by transient hypoxia. Brains were removed 1 wk after HI injury, and infarct volumes were compared between the two groups. TLR-4 expression was also compared among different ages. We found that LPS treated P7, P9, and P14 rat pups had significantly smaller infarct volume compared with saline-treated pups (p = 0.006, 0.03, and 0.01, respectively). This significant reduction in infarct volume was not observed in P3 and P5 rats. TLR-4 expression was significantly higher in older rats compared with P3 and P5 rats (p < 0.01). These findings indicate that LPS-induced preconditioning is a robust neuroprotective phenomenon in the ischemic developing brain that is age dependent. Pattern of TLR-4 expression is also affected by brain maturity and likely to be responsible for differences in the efficacy of LPS preconditioning.

Chronological changes in prosaposin in the developing rat brain

Prosaposin is the precursor protein of four glycoproteins, saposins A, B, C, and D, which activate sphingolipid hydrolases in lysosomes. Besides this role, intact prosaposin is also known as a potent neurotrophic factor that prevents neuronal cell death and stimulates neurite outgrowth in in vivo and in vitro experiments. In the present study, we examined chronological changes in prosaposin immunoreactivity in the rat brain using immunofluorescence staining and Diaminobenzidine (DAB) immunohistochemistry. In the hippocampal regions CA1, CA3, and dentate gyrus, the strongest staining of prosaposin was observed on postnatal day 1. The prosaposin immunoreactivity then decreased gradually until postnatal day 28. But in the cerebral cortex, prosaposin staining intensity increased from postnatal day 1 to 14, then decreased until postnatal day 28. The prosaposin immunoreactivity co-localized with the lysosomal granules labeled by an anti-Cathepsin D antibody, indicating that prosaposin mainly localized in the lysosomes of the neurons. We also examined the chronological changes in prosaposin mRNA and its two alternatively spliced variants using in situ hybridization. We found that both the mRNA forms, especially the one without a nine-base insertion, increased significantly from embryonic day 15 to postnatal day 7, then decreased gradually until postnatal day 28. Abundant prosaposin expression in the perinatal stages indicates a potential role of prosaposin in the early development of the rat brain.

The effects of the perinatal treatment with 5-hydroxytryptophan or tranylcypromine on the peripheral and central serotonin homeostasis in adult rats

Serotonin (5HT) is a biologically active amine present in mammals in the brain and the peripheral tissues. Autism is a neurodevelopmental disorder in which 5HT homeostasis is disturbed both centrally and peripherally, but the relationship between the 5HT disturbances in the two compartments is not understood. In an attempt to explore the relationship between the disturbed peripheral 5HT homeostasis and central 5HT functioning, we exposed the developing rat brain to increased 5HT concentrations, by treatment of rats with subcutaneous injections of the immediate 5HT precursor 5-hydroxy-l-tryptophan (5HTP, 25mg/kg), or the non-selective MAO inhibitor tranylcypromine (TCP, 2mg/kg), during the period of the most intensive development of 5HT neurons – from gestational day 13 to post-natal day 21. The effects of the mentioned treatments on peripheral and central 5HT levels were then studied in adult rats. Platelet and plasma 5HT concentrations (measured by ELISA), as well as cortical and midbrain 5HT, tryptophan and 5-hydroxyindoleacetic acid levels (measured by HPLC) were determined in twelve 5HTP treated and eight TCP treated rats, and compared with the values measured in 10 control, saline treated rats. Treatment with 5HTP significantly raised peripheral but not central 5HT concentrations. At adult age, peripheral 5HT homeostasis was re-established, while modest decrease in 5HT concentration was observed in frontal cortex, presumably due to hyperserotonemia-induced loss of 5HT terminals during brain development. Treatment with TCP induced significant 5HT elevations in both compartments. At adult age, permanent changes in 5HT homeostasis were observed, both peripherally (as hyperserotonemia) and centrally (as altered 5HT metabolism with decreased 5HT concentrations). Further studies are planned in order to explore the nature of the different disturbances of 5HT homeostasis induced by the two compounds, and their results are expected to shed some light on the role of hyperserotonemia in autism.

Switching of N-Methyl-d-aspartate (NMDA) Receptor-favorite Intracellular Signal Pathways from ERK1/2 Protein to p38 Mitogen-activated Protein Kinase Leads to Developmental Changes in NMDA Neurotoxicity

Excitotoxicity mediated by overactivation of N-methyl-D-aspartate receptors (NMDARs) has been implicated in a variety of neuropathological conditions in the central nervous system (CNS). It has been suggested that N-methyl-D-aspartate (NMDA) neurotoxicity is developmentally regulated, but the definite pattern of the regulation has been controversial, and the underlying mechanism remains largely unknown. Here, we show that NMDA treatment leads to significant cell death in mature (9 and 12 days in vitro) hippocampal neurons or hippocampi of young postnatal day 12 and adult rats but not in immature (3 and 6 days in vitro) neurons or embryonic day 18 and neonatal rat hippocampi. In contrast, NMDA promotes survival of immature neurons against tropic deprivation. Interestingly, it is found that NMDA preferentially activates p38 MAPK in mature neuron and adult rat hippocampus, but it favors ERK1/2 activation in immature neuron and postnatal day 0 rat hippocampus. Moreover, it is shown that NMDA neurotoxicity in mature neuron is mediated via p38 MAPK activation, and neuroprotection in immature neuron is mediated via ERK1/2 activation, whereas all these effects are NR2B-containing NMDAR-dependent, as well as Ca(2+)-dependent. We also revealed that mature and immature neurons showed no difference in the amplitude of NMDA-induced intracellular calcium ([Ca(2+)](i)) increase. However, the basal level of [Ca(2+)](i) is shown to elevate with the maturation of neuron, and this elevation is attributable to the changes in NMDA neurotoxicity but not to the switch of the NMDAR signaling pathway. Taken together, our results suggest that a switch of NMDA receptor-favorite intracellular signal pathways from ERK1/2 to p38 MAPK and the elevated basal level of [Ca(2+)](i) with age might be critical for the developmental changes in NMDA neurotoxicity in the hippocampal neuron.

The clinically available NMDA receptor antagonist, memantine, exhibits relative safety in the developing rat brain

The N-methyl- d-aspartate glutamate receptor (NMDAR) has been implicated in preterm brain injury (periventricular leukomalacia (PVL)) and represents a potential therapeutic target. However, the antagonist dizocilpine (MK-801) has been reported to increase constitutive neuronal apoptosis in the developing rat brain, limiting its clinical use in the developing brain. Memantine is another use-dependent NMDAR antagonist with shorter binding kinetics and has been demonstrated to be protective in a rat model of PVL, without effects on normal myelination or cortical growth. To further evaluate the safety of memantine in the developing brain, we demonstrate here that, in contrast to MK-801, memantine at neuroprotective doses does not increase neuronal constitutive apoptosis. In addition, there are no long-term alterations in the expression of NMDAR subunits, AMPAR subunits, and two markers of synaptogenesis, Synapsin-1 and PSD95. Evaluating clinically approved drugs in preclinical neonatal animal models of early brain development is an important prerequisite to considering them for clinical trial in preterm infants and early childhood.

CB1 Cannabinoid receptor Regulates Ethanol‐mediated inhibition of Neurogenesis

CB1 cannabinoid receptor (CB1R) activation stimulates neurogenesis in developing rat brain. Chronic ethanol treatment has been shown to inhibit developmental and adult brain neurogenesis. The objective of the present study is to determine if CB1R regulates ethanol‐mediated inhibition of developmental neurogenesis in zebrafish. We have used green fluorescence protein (GFP)‐expressing transgenic zebrafish (ISLET‐1) embryos to study neurogenesis using confocal microscopy. We have shown that zCB1R mRNA transcript is expressed in the different regions of zebrafish brain at 48 and 72 hpf (hour post‐fertilization). We also showed that CB1R agonist CP55940 (1–10 μM) treatment stimulated zCB1R expression and motor neurogenesis in zebrafish brain. We further showed knocking down CB1R using morpholino or antagonist‐mediated blockade of CB1R attenuated CB1R‐mediated increase in neurogenesis. Further we have found that chronic ethanol (0.5%–1.5%)‐treatment during embryonic development (6hpf–36 hpf) dose‐dependently reduced zCB1R expression and motor neurogenesis. Co‐treatment with CB1R agonist CP5540 or FAAH‐inhibitor URB597 attenuated ethanol‐mediated inhibition of developmental neurogenesis. Together, results from this study showed that zCB1R plays a vital role in the regulation developmental neurogenesis as well as ethanol‐mediated deleterious effects on neurogenesis.

Interaction of PFOS and BDE-47 Co-exposure on Thyroid Hormone Levels and TH-Related Gene and Protein Expression in Developing Rat Brains

Perfluorooctane sulfonate (PFOS) and 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) are two persistent environmental contaminants that are toxic to developing nervous systems, particularly via their disruption of thyroid hormone (TH) function. To investigate whether an interaction existed between PFOS and BDE-47 on TH-mediated pathways, adult female Wistar rats were exposed to 3.2 and 32 mg/kg of PFOS or BDE-47 in their diet and co-exposed to a combination of each chemical (3.2 mg/kg) from gestational day 1 to postnatal day (PND) 14. Serum and brain tissues from both male and female neonates were collected on PNDs 1, 7, and 14 to examine TH-regulated gene and protein expression. The results revealed that (1) a significant accumulation difference occurred between the two chemicals; (2) On a equimolar basis, BDE-47 and PFOS affected serum total triiodothyronine and total thyroxine differently in adults and offspring; (3) there were region-specific and exposure- and time-dependent alterations in TH concentrations and tested gene and protein expression levels; and (4) interaction for the combined chemicals was only observed for brain-derived neurotrophic factor (BDNF), which exhibited a synergistic effect on PND 1 in the cortex and an antagonistic effect on PND 14 in the hippocampus. Our results suggest a complex TH-mediated gene and protein response to BDE-47 and/or PFOS exposure that seems little related to TH homeostasis and that little combined interaction of co-exposures was observed except on BDNF. The underlying mechanisms remain uncertain but seem to involve more actions than just TH-regulated pathway.

Effect of perinatal thyroid hormone deficiency on expression of rat hippocampal conventional protein kinase C isozymes

Thyroid hormone (TH) is essential for the proper development of mammalian central nervous system. TH deficiency during critical period of brain development results in permanent cognitive and neurological impairments. Hippocampus is a structure involved in various memory processes that are essential for creating new memories, and lesions to hippocampus result in impaired learning and memory. Protein kinase C (PKC) isoforms play an important role in many types of learning and memory, and deletion of specific PKC genes results in deficits in learning. In the present study, we used real-time PCR and Western blot to investigate the conventional PKC expression in developing rat hippocampus with different thyroid status, trying to establish a correlation between TH deficiency and conventional PKC expression in developing rat hippocampus. We found that PKCβI and PKCγ expression decreased significantly both in mRNA and protein levels in hypothyroid group compared with the normal controls, and thyroxine replacement could restore it. As for PKCα, we did not find any difference between different thyroid status. Though the expression of PKCβII also decreased in the TH deficiency group, the change was not significant. Taken together, our data indicate TH deficiency can cause hippocampal PKCβ1 and PKCγ downregulation during rat brain development. Since there are other PKC isoforms in the rat brain, whether these change is related to impaired learning and memory of perinatal hypothyroid rats requires further researches.

Ketamine-Induced Neurotoxicity and Changes in Gene Expression in the Developing Rat Brain

Ketamine, an N-methyl-D-aspartate (NMDA) receptor antagonist, is widely used for analgesia and anesthesia in obstetric and pediatric practice. Recent reports indicate that ketamine causes neuronal cell death in developing rodents and nonhuman primates. The present study assessed the potential dose- and time-dependent neurotoxic effects and associated changes in gene expression after ketamine administration to postnatal day 7 (PND-7) rat pups. Pups were exposed to ketamine subcutaneously at doses of 5, 10, or 20 mg/kg, in one, three or six injections respectively. Control animals received the same volume of saline at the same time points. The animals were sacrificed 6 h after the last ketamine or saline administration and brain tissues were collected for RNA isolation and histochemical examination. Six injections of 20 mg/kg ketamine significantly increased neuronal cell death in frontal cortex, while lower doses and fewer injections did not show significant effects. The ketamine induced cell death seemed to be apoptotic in nature. In situ hybridization demonstrated that NMDA receptor NR1 subunit expression was dramatically increased in the frontal cortex of ketamine treated rats. Microarray analysis revealed altered expression of apoptotic relevant genes and increased NMDA receptor gene expression in brains from ketamine treated animals. Quantitative RT-PCR confirmed the microarray results. These data suggest that repeated exposures to high doses of ketamine can cause compensatory up-regulation of NMDA receptors and subsequently trigger apoptosis in developing neurons.

Maturation of sleep homeostasis in developing rats: a role for preoptic area neurons

The present study evaluated the hypothesis that developmental changes in hypothalamic sleep-regulatory neuronal circuits contribute to the maturation of sleep homeostasis in rats during the fourth postnatal week. In a longitudinal study, we quantified electrographic measures of sleep during baseline and in response to sleep deprivation (SD) on postnatal days 21/29 (P21/29) and P22/30 (experiment 1). During 24-h baseline recordings on P21, total sleep time (TST) during the light and dark phases did not differ significantly. On P29, TST during the light phase was significantly higher than during the dark phase. Mean duration of non-rapid-eye-movement (NREM) sleep bouts was significantly longer on P29 vs. P21, indicating improved sleep consolidation. On both P22 and P30, rats exhibited increased NREM sleep amounts and NREM electroencephalogram delta power during recovery sleep (RS) compared with baseline. Increased NREM sleep bout length during RS was observed only on P30. In experiment 2, we quantified activity of GABAergic neurons in median preoptic nucleus (MnPN) and ventrolateral preoptic area (VLPO) during SD and RS in separate groups of P22 and P30 rats using c-Fos and glutamic acid decarboxylase (GAD) immunohistochemistry. In P22 rats, numbers of Fos(+)GAD(+) neurons in VLPO did not differ among experimental conditions. In P30 rats, Fos(+)GAD(+) counts in VLPO were elevated during RS. MnPN neuronal activity was state-dependent in P22 rats, but Fos(+)GAD(+) cell counts were higher in P30 rats. These findings support the hypothesis that functional emergence of preoptic sleep-regulatory neurons contributes to the maturation of sleep homeostasis in the developing rat brain.

Expression of sphingosine kinase 1 in amoeboid microglial cells in the corpus callosum of postnatal rats

Sphingosine kinase 1 (SphK1), a key enzyme responsible for phosphorylating sphingosine into sphingosine-1-phosphate (S1P) has been shown to be expressed in monocytes and monocyte-derived peripheral macrophages. This study demonstrates SphK1 immunoexpression in amoeboid microglial cells (AMC), a nascent monocyte-derived brain macrophage in the corpus callosum of developing rat brain. SphK1 immunofluorescence expression, which appeared to be weak in AMC in normal brain, was markedly induced by lipopolysaccharide (LPS) or hypoxia treatment. Western blot analysis also showed increased expression level of SphK1 in the corpus callosum rich in AMC after LPS treatment. Detection of SphK1 mRNA and its upregulation after LPS treatment was confirmed in primary culture AMC by RT-PCR. Administration of N, N-dimethylsphingosine (DMS), a specific inhibitor of SphK1, effectively reduced upregulated SphK1 immunoexpression in AMC both in vivo and in vitro. This was corroborated by western blot which showed a decrease in SphK1 protein level of callosal tissue with DMS pretreatment. Remarkably, LPS-induced upregulation of the transcription factor NFκB was suppressed by DMS. We conclude that SphK1 expression in AMC may be linked to regulation of proinflammatory cytokines via an NFκB signaling pathway.

Post-ischemic hypothermia promotes generation of neural cells and reduces apoptosis by Bcl-2 in the striatum of neonatal rat brain

Hypothermia is a potential therapy for cerebral hypoxic ischemic injury in adults and neonates. However, the mechanism of hypothermia neuroprotection after hypoxic-ischemia (HI) on the developing rat brain remains unclear. In this research, 7-day-old rats were subjected to left carotid artery ligation followed by 8% oxygen for 2 h. They were divided into hypothermia (rectal temperature, 32–33 °C for 24 h) and normothermia (36–37 °C for 24 h) groups immediately after hypoxia-ischemia. All rats were given 50 mg/kg/day 5-bromodeoxyuridine (BrdU) intraperitoneally at 4–6 days and sacrificed at 1 or 2 weeks after HI. There was a significant decrease in infarct volume in the hypothermia group at 7 days after HI compared with that in the normothermia group. The numbers of nestin-labeled cells did not change greatly, but β-tubulin III (Tuj-1) immuno-positive cells increased significantly in the striatum at 1 and 2 weeks after HI in the hypothermia compared to normothermia group. Neurogenesis was assessed by double immunohistochemical/immunofluorescent labeling of BrdU with nestin, Tuj-1 or microtubule-associated protein 2 (Map-2). Newborn neural progenitors (BrdU +-nestin +) did not change dramatically, but newborn immature (BrdU +-Tuj-1 +) and mature (BrdU +-Map-2 +) neurons increased significantly in the hypothermia compared with normothermia group. Meanwhile, the apoptosis rate of neural precursors, immature and mature neurons, assessed by double labeling of active Casp-3 with nestin/Tuj-1/Map-2, decreased noticeably in the hypothermia compared with normothermia group. We also found that hypothermia significantly increased expression of Bcl-2, which coexisted with nestin/Tuj-1/Map-2. Inhibition of Bcl-2 expression reversed the decreased apoptosis rate of neural precursors and neurons in hypothermia animal striatum of neonatal rat brain. These results suggest that neuroprotection effects of hypothermia on injured developing rat brain may associate with enhanced generation of neuronal cells and Bcl-2-mediated reduction of apoptosis of these cells. These observations are noteworthy regarding clinical hypothermia therapy following cerebral HI injury during the perinatal period.

Sur8/Shoc2 Involves Both Inhibition of Differentiation and Maintenance of Self-Renewal of Neural Progenitor Cells via Modulation of Extracellular Signal-Regulated Kinase Signaling

Sur8/Shoc2 is a scaffold protein that regulates the Ras-extracellular signal-regulated kinase (ERK) pathway. However, the roles of Sur8 in cellular physiologies are poorly understood. In this study, Sur8 was severely repressed in the course of neural progenitor cell (NPC) differentiation in the cerebral cortex of developing rat embryos. Similarly, Sur8 was also critically reduced in cultured NPCs, which were induced differentiation by removal of basic fibroblast growth factor (bFGF). Sur8 regulation occurs at the protein level rather than at the mRNA level as revealed by both in situ hybridization and reverse transcriptase polymerase chain reaction analyses. The role of Sur8 in NPC differentiation was confirmed by lentivirus-mediated Sur8 knockdown, which resulted in increased differentiation, whereas exogenous expression of Sur8 inhibited differentiation. Contrastingly, NPC proliferation was promoted by overexpression, but was suppressed by Sur8 knockdown. The role of Sur8 as an antidifferentiation factor in the developing rat brain was confirmed by an ex vivo embryo culture system combined with the lentivirus-mediated Sur8 knockdown. The numbers and sizes of neurospheres were reduced, but neuronal outgrowth was enhanced by the Sur8 knockdown. The Ras-ERK pathway is involved in Sur8-mediated regulations of differentiation, as the treatment of ERK kinase (MEK) inhibitors blocks the effects of Sur8. The regulations of NPCs' differentiation and proliferation by the Ras-ERK pathway were also shown by the rescues of the effects of bFGF depletion, neuronal differentiation, and antiproliferation by epidermal growth factor. In summary, Sur8 is an antidifferentiation factor that stimulates proliferation for maintenance of self-renewal in NPCs via modulation of the Ras-ERK pathway.

Absence of neurotoxicity with medicinal grade terbutaline in the rat model

To evaluate neurological effects of terbutaline, rats were injected with saline, terbutaline (Sigma or American Pharmaceutical Partners (APP™)) at 0.5 mg/kg-d or 10 mg/kg-d between postnatal days (PND) 2–5 or 11–14. Brains collected 24 h after last injection were used to determine corpus-callosum thickness, Purkinje cell and neuronal number in the cerebellum. Ambulation, distance traveled, resting time and time on rotarod were analyzed. Terbutaline (both doses/grades at PND 11–14) decreased corpus-callosum thickness. Ambulation time was significantly decreased in the 10 mg/kg-d (Sigma) and 0.5 mg/kg-d of terbutaline (APP™) (PND 2–5) juvenile-rats and 10 mg/kg-d-Sigma adult-rats, 0.5 mg/kg-d APP™ (PND 11–14) adult-rats. Resting time was increased in both doses of APP™ (PND 2–5) in juvenile-rats, 10 mg/kg-d Sigma adult-rats. 10 mg/kg-d-Sigma (PND 2–5) decreased distance traveled in adult-rats. 0.5 mg/kg-d-Sigma (PND 2–5 and PND 11–14) decreased the time spent on rotarod (30 RPM) in adult-rats. Sigma terbutaline Sigma had 2× as much free base compared to APP™. In conclusion, APP™ terbutaline did not have a deleterious effect on the developing rat brain.

Beta-amyloid increases the expression level of ATBF1 responsible for death in cultured cortical neurons

BackgroundRecently, several lines of evidence have shown the aberrant expression of cell-cycle-related proteins and tumor suppressor proteins in vulnerable neurons of the Alzheimer's disease (AD) brain and transgenic mouse models of AD; these proteins are associated with various paradigms of neuronal death. It has been reported that ATBF1 induces cell cycle arrest associated with neuronal differentiation in the developing rat brain, and that gene is one of the candidate tumor suppressor genes for prostate and breast cancers in whose cells overexpressed ATBF1 induces cell cycle arrest. However, the involvement of ATBF1 in AD pathogenesis is as yet unknown.ResultsWe found that ATBF1 was up-regulated in the brains of 17-month-old Tg2576 mice compared with those of age-matched wild-type mice. Moreover, our in vitro studies showed that Aβ1-42 and DNA-damaging drugs, namely, etoposide and homocysteine, increased the expression ATBF1 level in primary rat cortical neurons, whereas the knockdown of ATBF1 in these neurons protected against neuronal death induced by Aβ1-42, etoposide, and homocysteine, indicating that ATBF1 mediates neuronal death in response to these substances. In addition, we found that ATBF1-mediated neuronal death is dependent on ataxia-telangiectasia mutated (ATM) because the blockage of ATM activity by treatment with ATM inhibitors, caffeine and KU55933, abolished ATBF1 function in neuronal death. Furthermore, Aβ1-42 phosphorylates ATM, and ATBF1 interacts with phosphorylated ATM.ConclusionsTo the best of our knowledge, this is the first report that Aβ1-42 and DNA-damaging drugs increased the ATBF1 expression level in primary rat cortical neurons; this increase, in turn, may activate ATM signaling responsible for neuronal death through the binding of ATBF1 to phosphorylated ATM. ATBF1 may therefore be a suitable target for therapeutic intervention of AD.

The Neuroprotective Effects of 6-cyano-7-nitroquinoxalin-2,3-dione (CNQX) Via Mediation of Nitric Oxide Synthase on Hypoxic-ischemic Brain Injury in Neonatal Rats

Purpose: Current studies have demonstrated the neuroprotective effects of 6-cyano-7-nitroquinoxalin-2,3-dione (CNQX) in many animal models of brain injury, including hypoxic-ischemic (HI) encephlopathy, trauma and excitotoxicity, but limited data are available for those during the neonatal periods. Here we investigated whether CNQX can protect the developing rat brain from HI injury via mediation of nitric oxide synthase. Methods: In an in vivo model, left carotid artery ligation was done in 7-day-old Sprague-Dawley (SD) rat pups. The animals were divided into six groups; normoxia (N), hypoxia (H), hypoxia with sham-operation (HS), hypoxia with operation (HO), HO treated with vehicle (HV), and HO treated with CNQX at a dose of 10 mg/kg (HC). Hypoxia was made by exposure to a 2 hr period in the hypoxic chamber (92% N2, 8% O2). In an in vitro model, embryonic cortical neuronal cell culture of SD rats at 18-day gestation was done. The cultured cells were divided into three groups: normoxia (N), hypoxia (H), and hypoxia treated with CNQX (HC). The N group was prepared in 5% CO2 incubators and the other groups were placed in 1% O2 incubators (94% N2, 5% CO2) for 16 hr. Results: In the in vitvo and in vivo models, the expressions of iNOS and eNOS were reduced in the hypoxia group when compared to the normoxia group, whereas they were increased in the CNQX-treated group compared to the hypoxia group. In contrast, the expression of nNOS was showed reversely. Conclusion: CNQX has neuroprotective property over perinatal HI brain injury via mediation of nitric oxide synthase.