Effects of nitric oxide on the prefrontal cortex in stressed rats

BACKGROUND:Nitric oxide(NO)exhibits both protective and detrimental effects in the central nervous system.OBJECTIVE:To investigate the effect of NO on the prefrontal codex in neonatal stressed rats.DESIGN,TIME AND SETTING:A randomized,controlled,animal study was performed at the Anatomical Department of Iran University of Medical Sciences from May 2007 to August 2008.MATERIALS:Forty-eight male,Wistar rats were obtained from Pasteur's Institute,Tehran,Iran.METHODS:Rat stress models were established by immobilization and randomly received intraperitoneal injection of 2 mL physiological saline,L-arginine(200 mg/kg)as a NO precursor,N(G)-nitro-L-arginine methyl ester(20 mg/kg),or subcutaneous injection of 7-nitroindazole(25 mg/kg)as a NO synthase inhibitor.MAIN OUTCOME MEASURES:After the rats were treated for 4 weeks,the frontal codex was harvested for histological observation and NO detection.RESULTS:Subcutaneous administration of N(G)-nitro-L-arginine methyl ester or 7-nitroindazole resulted in significantly lower prefrontal cortex thickness and NO production compared with subcutaneous administration of L-arginine(P < 0.05).Prefrontal cortex thickness significantly increased in rats following L-arginine treatment,compared with physiological saline intervention(P< 0.05).CONCLUSION:NO exhibited protective effects on the prefrontal codex of stressed rats.     

Response of the sensorimotor cortex of cerebral palsy rats receiving transplantation of vascular endothelial growth factor 165-transfected neural stem cells

Neural stem cells are characterized by the ability to differentiate and stably express exogenous ge- nes. Vascular endothelial growth factor plays a role in protecting local blood vessels and neurons of newborn rats with hypoxic-ischemic encephalopathy. Transplantation of vascular endothelial growth factor-transfected neural stem cells may be neuroprotective in rats with cerebral palsy. In this study, 7-day-old Sprague-Dawley rats were divided into five groups： （1） sham operation （control）, （2） cerebral palsy model alone or with （3） phosphate-buffered saline, （4） vascular en- dothelial growth factor 165 ＋ neural stem cells, or （5） neural stem cells alone. The cerebral palsy model was established by ligating the left common carotid artery followed by exposure to hypox- ia. Phosphate-buffered saline, vascular endothelial growth factor ＋ neural stem cells, and neural stem cells alone were administered into the sensorimotor cortex using the stereotaxic instrument and microsyringe. After transplantation, the radial-arm water maze test and holding test were performed. Immunohistochemistry for vascular endothelial growth factor and histology using hematoxylin-eosin were performed on cerebral cortex. Results revealed that the number of vas- cular endothelial growth factor-positive cells in cerebral palsy rats transplanted with vascular endothelial growth factor-transfected neural stem cells was increased, the time for finding water and the finding repetitions were reduced, the holding time was prolonged, and the degree of cell degeneration or necrosis was reduced. These findings indicate that the transplantation of vascu- lar endothelial growth factor-transfected neural stem cells alleviates brain damage and cognitive deficits, and is neuroprotective in neonatal rats with hypoxia ischemic-mediated cerebral palsy.     

miR-21 promotes the differentiation of hair follicle-derived neural crest stem cells into Schwann cells

Hair follicle-derived neural crest stem cells can be induced to differentiate into Schwann cells in vivo and in vitro. However, the underlying regulatory mechanism during cell differentiation remains poorly understood. This study isolated neural crest stem cells from human hair follicles and induced them to differentiate into Schwann cells. Quantitative RT-PCR showed that microRNA(miR)-21 expression was gradually increased during the differentiation of neural crest stem cells into Schwann cells. After transfection with the miR-21 agonist(agomir-21), the differentiation capacity of neural crest stem cells was enhanced. By contrast, after transfection with the miR-21 antagonist(antagomir-21), the differentiation capacity was attenuated. Further study results showed that SOX-2 was an effective target of miR-21. Without compromising SOX2 mRNA expression, miR-21 can down-regulate SOX protein expression by binding to the 3′-UTR of miR-21 mRNA. Knocking out the SOX2 gene from the neural crest stem cells significantly reversed the antagomir-21 inhibition of neural crest stem cells differentiating into Schwann cells. The results suggest that miR-21 expression was increased during the differentiation of neural crest stem cells into Schwann cells and miR-21 promoted the differentiation through down-regulating SOX protein expression by binding to the 3′-UTR of SOX2 mRNA.     

Effects of prenatal exposure to amphetamine in the medial prefrontal cortex of the rat

This study was designed to investigate the effects of prenatal exposure to amphetamine in the organization of the medial prefrontal cortex of the rat, by an evaluation of growth, morphometric and neurochemical parameters. Pregnant Wistar rats were given 10 mg/kg body weight/day of D-amphetamine sulfate, subcutaneously, from gestational days 8 to 22. Control groups of pregnant rats were injected with saline, pair-fed or non-manipulated; litters were culled to eight pups (four males and four females), weighed every other day until postnatal day 30 and every week until day 90. The Gompertz model was used to study body weight evolution and the estimated growth parameters were not significantly different in the experimental groups. At postnatal days 14 and 30, the volumes of the prefrontal cortex, the fraction of neuropile occupied by neurons and the number of neurons per unit surface area were determined. The number of neurons per unit volume of reference area was calculated using the stereological technique of the dissector. For neurochemical analysis, the medial prefrontal cortex was dissected to measure the concentration of dopamine, serotonin and their metabolites. The allometric relationship of forebrain/body growth pointed to a mechanism of sparing and compensatory growth in the amphetamine exposed group. The changes found in the number of neurons per unit volume at postnatal day 14 show a catch-up at postnatal day 30. A decrease in serotonin levels was found in the amphetamine group compared with the pair-fed control, which was reflected in the ratio of serotonin to its metabolite, 5-hydroxyindolacetic acid. These changes, whether permanent or transitory, raise the possibility that some of the effects of prenatal exposure to amphetamine may be due to modifications in the neurotransmitter levels of serotonin.     

Upregulation of Nogo receptor expression induces apoptosis of retinal ganglion cells in diabetic rats

The Nogo receptor is an essential factor for neuronal apoptosis, but the changes in Nogo receptor expression in the retina and the effects of the Nogo receptor on retinal ganglion cell apoptosis in diabetes mellitus remain unclear. We found that Nogo receptor expression was mainly visible in retinal ganglion cells of a rat model of diabetes mellitus induced by streptozotocin. At 12 weeks after onset of diabetes mellitus, Nogo receptor and Rho kinase expression signiifcantly increased in the retina, and retinal ganglion cell apoptosis was apparent. When RNA interference was used to suppress Nogo receptor expression in rat retina, Rho kinase expression was obviously inhibit-ed, and retinal ganglion cell apoptosis was evidently reduced in rats with diabetes mellitus. These results indicate that upregulation of Nogo receptor expression is an important mechanism of retinal ganglion cell apoptosis in rats with diabetes mellitus.     

Lack of experience‐dependent intrinsic plasticity in the adolescent infralimbic medial prefrontal cortex

Fear extinction, an inhibitory learning that suppresses a previously learned fear memory, is diminished during adolescence. Earlier studies have shown that this suppressed fear extinction during adolescence involves an altered glutamatergic plasticity in infralimbic medial prefrontal cortical (IL‐mPFC) pyramidal neurons. However, it is unclear whether the excitability of IL‐mPFC pyramidal neurons plays a role in this development‐dependent suppression of fear extinction. Therefore, we examined whether fear conditioning and extinction affect the active and passive membrane properties of IL‐mPFC layer 5 pyramidal neurons in preadolescent, adolescent and adult mice. Both preadolescent and adult mice exhibited a bidirectional modulation of the excitability of IL‐mPFC layer 5 pyramidal neurons following fear conditioning and extinction, i.e., fear conditioning reduced membrane excitability, whereas fear extinction reversed this effect. However, the fear conditioning‐induced suppression of excitability was not reversed in adolescent mice following fear extinction training. Neither fear conditioning nor extinction affected GABAergic transmission in IL‐mPFC layer 5 pyramidal neurons, suggesting that GABAergic transmission did not play a role in experience‐dependent modulation of neuronal excitability. Our results suggest that the extinction‐specific modulation of excitability is impaired during adolescence.     

Comparison of deficits in electrical self-stimulation after ibotenic acid lesion of the lateral hypothalamus and the medial prefrontal cortex

The aim of the present study was to compare the self-stimulation deficit produced by a unilateral injection of the neurotoxin, ibotenic acid, in the lateral hypothalamus (LH) to the deficit produced by the same unilateral injection in the medial prefrontal cortex (MPC). Four groups of adult male Sprague-Dawley rats were used: in two control groups, electrodes were bilaterally implanted in the LH (5 rats) or in the MPC (6 rats) and self-stimulation (ICSS) was obtained separately with the right and left electrodes. In the two experimental groups the intrinsic neurons of the LH (8 rats) or of the MPC (10 rats) were destroyed unilaterally by local injection of ibotenic acid (4 micrograms in 0.5 microliter); the other side served as the sham-lesioned control. Ten days later ICSS electrodes were implanted bilaterally, one in the lesioned area, the other in the contralateral region. As in the case of the control rats, ICSS was determined separately for each electrode, first by a rate dependent test (nose-poke) then by a 'rate-free' test (shuttle-box). In the LH and MPC control rats, ICSS responses were the same with stimulation on either side. In the LH-lesioned rats, the ICSS rates measured with the nose-poke test were significantly decreased with stimulation on the lesioned side, whereas rates with stimulation of the non-lesioned LH were normal. Likewise, while shuttle responses with stimulation of the non-lesioned LH were normal, the OFF-time was increased and the ON-time was decreased with stimulation of the lesioned LH. In the MPC-lesioned rats, ICSS (nose-poke) was totally suppressed and the shuttle responses were disorganized since neither the ON- nor the OFF-times changed in response to increasing current intensities. Nose-poke responses with stimulation of the non-lesioned MPC were just about normal. These results show that in the two brain regions studied local neurons are involved in ICSS. The difference in the magnitude of the deficit observed suggests, that the neuronal circuits involved in MPC self-stimulation are poorly represented whereas in the LH many neuronal circuits involved in these mechanisms overlap.     

Overexpression of microRNA-124 promotes the neuronal differentiation of bone marrow-derived mesenchymal stem cells

microRNAs(miRNAs) play an important regulatory role in the self-renewal and differentiation of stem cells. In this study, we examined the effects of miRNA-124(miR-124) overexpression in bone marrow-derived mesenchymal stem cells. In particular, we focused on the effect of overexpression on the differentiation of bone marrow-derived mesenchymal stem cells into neurons. First, we used GeneChip technology to analyze the expression of miRNAs in bone marrow-derived mesenchymal stem cells, neural stem cells and neurons. miR-124 expression was substantially reduced in bone marrow-derived mesenchymal stem cells compared with the other cell types. We constructed a lentiviral vector overexpressing miR-124 and transfected it into bone marrow-derived mesenchymal stem cells. Intracellular expression levels of the neuronal early markers β-III tubulin and microtubule-associated protein-2 were significantly increased, and apoptosis induced by oxygen and glucose deprivation was reduced in transfected cells. After miR-124-transfected bone marrow-derived mesenchymal stem cells were transplanted into the injured rat spinal cord, a large number of cells positive for the neuronal marker neurofilament-200 were observed in the transplanted region. The Basso-Beattie-Bresnahan locomotion scores showed that the motor function of the hind limb of rats with spinal cord injury was substantially improved. These results suggest that miR-124 plays an important role in the differentiation of bone marrow-derived mesenchymal stem cells into neurons. Our findings should facilitate the development of novel strategies for enhancing the therapeutic efficacy of bone marrow-derived mesenchymal stem cell transplantation for spinal cord injury.     

Role of apoptosis and autophagy in the control of cell proliferation in dentate gyrus after hippocampal lesion

研究背景：很多病理情况下都可以引起神经发生（新的神经元产生）,新生细胞的命运和这些疾病的预后有着直接的关系。实验证明脑损伤往往导致海马齿状回神经发生区细胞增殖的增加,新生细胞的数量随着时间的推移逐渐减少,但是新生细胞减少的原因尚不知。阐明这些新生细胞的命运将增加对脑损伤病理过程和治疗手段的理解。 目的：本文研究凋亡和自噬这两种程序性细胞死亡在海马损伤诱导的海马齿状回细胞增殖后新生细胞减少中的作用。 设计、时间和设定：海马齿状回细胞增殖的动物实验于2008年2月至2009年3月在苏州大学医学部神经生物学系进行。 材料：成年雄性SD大鼠通过微量注射海人酸(购自美国Sigma公司)建立海马损伤模型。 方法：5-溴脱氧嘧啶核苷（BrdU）用于标记增殖细胞,海马损伤后齿状回的不同时间点BrdU阳性细胞用免疫组织化学方法鉴定,阳性细胞的数量采用无偏差体视学(Stereology)方法进行定量。凋亡相关蛋白caspase-3、Bcl-2、p53和自噬相关蛋白LC3、Beclin1通过免疫荧光和蛋白质印迹(Western blot）的方法进行检测。 主要结果检测：凋亡和自噬的检测,是在海马损伤后不同时间点,在激光共聚焦显微镜下观察BrdU阳性细胞共表达BrdU/caspase-3、BrdU/LC3和BrdU/Beclin 1。同时, 还用Western blot的方法检测齿状回凋亡相关蛋白和自噬相关蛋白的改变。 结果：定量分析显示海马损伤组在连续3天注射BrdU后的第1天有最多的BrdU阳性细胞,BrdU阳性细胞随着时间的推移逐渐减少。海马颗粒细胞层和门区发现BrdU/cleaved caspase-3双标细胞,海马损伤后的各个时间点未发现BrdU/LC3、BrdU/Beclin 1双标细胞。但Western blot结果显示海马损伤后LC3Ⅱ、Beclin 1、p53的表达上调,pro-caspase-3和Bcl-2的表达下调。 结论：成年大鼠海马损伤后能导致齿状回显著的细胞增殖,这些增殖细胞随着时间的推移逐渐减少,凋亡和自噬可能参与海马损伤后增殖细胞的减少。     

Long-term administration of scopolamine interferes with nerve cell proliferation,differentiation and migration in adult mouse hippocampal dentate gyrus,but it does not induce cell death

Long-term administration of scopolamine, a muscarinic receptor antagonist, can inhibit the survival of newly generated cells, but its effect on the proliferation, differentiation and migration of nerve cells in the adult mouse hippocampal dentate gyrus remain poorly understood. In this study, we used immunohistochemistry and western blot methods to weekly detect the biological behaviors of nerve cells in the hippocampal dentate gyrus of adult mice that received intraperitoneal administration of scopolamine for 4 weeks. Expression of neuronal nuclear antigen(Neu N; a neuronal marker) and Fluoro-Jade B(a marker for the localization of neuronal degeneration) was also detected. After scopolamine treatment, mouse hippocampal neurons did not die, and Ki-67(a marker for proliferating cells)-immunoreactive cells were reduced in number and reac hed the lowest level at 4 weeks. Doublecortin(DCX; a marker for newly generated neurons)-immunoreactive cells were gradually shortened in length and reduced in number with time. After scopolamine treatment for 4 weeks, nearly all of the 5-bromo-2′-deoxyuridine(Brd U)-labeled newly generated cells were located in the subgranular zone of the dentate gyrus, but they did not migrate into the granule cell layer. Few mature Brd U/Neu N double-labeled cells were seen in the subgranular zone of the dentate gyrus. These findings suggest that long-term administration of scopolamine interferes with the proliferation, differentiation and migration of nerve cells in the adult mouse hippocampal dentate gyrus, but it does not induce cell death.     

dcc orchestrates the development of the prefrontal cortex during adolescence and is altered in psychiatric patients

Adolescence is a period of heightened susceptibility to psychiatric disorders of medial prefrontal cortex (mPFC) dysfunction and cognitive impairment. mPFC dopamine (DA) projections reach maturity only in early adulthood, when their control over cognition becomes fully functional. The mechanisms governing this protracted and unique development are unknown. Here we identify dcc as the first DA neuron gene to regulate mPFC connectivity during adolescence and dissect the mechanisms involved. Reduction or loss of dcc from DA neurons by Cre-lox recombination increased mPFC DA innervation. Underlying this was the presence of ectopic DA fibers that normally innervate non-cortical targets. Altered DA input changed the anatomy and electrophysiology of mPFC circuits, leading to enhanced cognitive flexibility. All phenotypes only emerged in adulthood. Using viral Cre, we demonstrated that dcc organizes mPFC wiring specifically during adolescence. Variations in DCC may determine differential predisposition to mPFC disorders in humans. Indeed, DCC expression is elevated in brains of antidepressant-free subjects who committed suicide.     

Environmental enrichment reduces the function of D1 dopamine receptors in the prefrontal cortex of the rat

Summary. Environmental enrichment produces changes in spontaneous and psychostimulant-induced motor activity. Dopamine in the prefrontal cortex (PFC), through the activation of D1 receptors, has been suggested to play a role in modulating motor activity. The present study investigated the effects of environmental enrichment on spontaneous motor activity, prefrontal acetylcholine release following local D1 receptor stimulation and D1 receptor expression in the PFC. Male wistar rats (3 months of age) were housed in enriched or isolated conditions during 90 days. Animals were then implanted with guide cannulae to perform microdialysis experiments in the PFC. Spontaneous motor activity and acetylcholine extracellular concentrations were monitored simultaneously. Also spontaneous motor activity was measured in an open field. On completion of the experiments, the density of D1 receptors in the PFC was studied by immunocytochemistry. Rats housed in an enriched environment showed significantly lower spontaneous motor activity in the open field compared to isolated animals. Perfusion of the D1 agonist SKF38393 (50 μM; 40 min) in the PFC produced long lasting increases of spontaneous motor activity and of local dialysate concentrations of acetylcholine in both groups of rats. However, increases of both motor activity and acetylcholine concentrations were significantly lower in enriched compared to isolated animals. Moreover, the density of D1 receptors in the PFC was significantly reduced in animals housed in an enriched environment. These results are the first evidence suggesting that environmental enrichment during adult life changes the function of D1 dopamine receptors in the PFC.     

Apoptosis and autophagy control cell proliferation in the dentate gyrus following hippocampal lesion

Brain injuries often result in the promotion of cell proliferation in the hippocampal dentate gyrus(DG),but the number of newborn cells declines with time.However,the cause of this decline remains poorly understood.Elucidation of the fate of these newborn cells will further the understanding of the pathological process and treatment of brain injury.In the present study,the number of newborn cells was quantitatively analyzed using an unbiased stereological method following hippocampal lesion by kainic acid,i...     

Dopamine depletion in the medial prefrontal cortex induces sensitized-like behavioral and neurochemical responses to cocaine

It has been postulated that behavioral sensitization to cocaine is associated with an attenuation of cocaine-induced dopamine (DA) transmission in the medial prefrontal cortex (mPFC). Hence, experiments were designed to examine the effects of chemically-induced cortical DA depletion on the acute behavioral and neurochemical responses to cocaine. One week following two bilateral 6-hydroxydopamine (6-OHDA) injections into the mPFC, animals received injections of cocaine (7.5, 15 or 30 mg/kg, i.p.) or saline (1 ml/kg, i.p.) in a randomized fashion with a minimum 3 day intertrial interval. Cocaine produced a dose-dependent increase in motor activity which was significantly enhanced in animals depleted (mean of 76%) of dopamine in the mPFC. Likewise, 6-OHDA lesions of the mPFC produced a significant enhancement of cocaine-induced DA transmission in the nucleus accumbens (NAC) as estimated by in vivo microdialysis. These data indicate a permissive involvement of cortical DA in mediating behavioral and neurochemical responses to cocaine, as well as confirm the ability of the mPFC to influence subcortical structures in response to an acute injection of cocaine. Collectively, the present findings suggest that alterations in cortical DA transmission may be a neural substrate mediating the development of sensitization to cocaine, and thus, may contribute to the addictive properties of cocaine.     

Transient axonal glycoprotein-1 induces apoptosis- related gene expression without triggering apoptosis in U251 glioma cells

Previous studies show that transient axonal glycoprotein-1, a ligand of amyloid precursor pro- tein, increases the secretion of amyloid precursor protein intracellular domain and is involved in apoptosis in Alzheimer＇s disease. In this study, we examined the effects of transient axonal glyco- protein-1 on U251 glioma cells. 3-（4,5-Dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide assay showed that transient axonal glycoprotein-1 did not inhibit the proliferation of U251 cells, but promoted cell viability. The terminal deoxynucleotidyl transferase dUTP nick end labeling assay showed that transient axonal glycoprotein-1 did not induce U251 cell apoptosis. Real-time PCR revealed that transient axonal glycoprotein-1 substantially upregulated levels of amyloid precursor protein intracellular C-terminal domain, and p53 and epidermal growth factor recep- tor mRNA expression. Thus, transient axonal glycoprotein-1 increased apoptosis-related gene expression in U251 cells without inducing apoptosis. Instead, transient axonal glycoprotein-1 promoted the proliferation of these glioma cells.     

Nerve growth factor promotes in vitro proliferation of neural stem cells from tree shrews

Neural stem cells promote neuronal regeneration and repair of brain tissue after injury,but have limited resources and proliferative ability in vivo.We hypothesized that nerve growth factor would promote in vitro proliferation of neural stem cells derived from the tree shrews,a primate-like mammal that has been proposed as an alternative to primates in biomedical translational research.We cultured neural stem cells from the hippocampus of tree shrews at embryonic day 38,and added nerve growth factor(100 μg/L) to the culture medium.Neural stem cells from the hippocampus of tree shrews cultured without nerve growth factor were used as controls.After 3 days,fluorescence microscopy after DAPI and nestin staining revealed that the number of neurospheres and DAPI/nestin-positive cells was markedly greater in the nerve growth factor-treated cells than in control cells.These findings demonstrate that nerve growth factor promotes the proliferation of neural stem cells derived from tree shrews.     

Properties and distribution of auditory neurons in the dorsolateral prefrontal cortex of the alert monkey

In alert monkeys, some prefrontal neurons located in the superior dorsolateral area were activated to acoustic stimuli delivered in a restricted range of directions with respect to the animal's head. The effective direction was usually contralateral to the animal. The function of the auditory neurons in connection to that of the visual ones, which are commonly found in the prefrontal cortex, is discussed.     

Regional concentrations of noradrenaline and dopamine in the frontal cortex of the rat: dopaminergic innervation of the prefrontal subareas and lateralization of prefrontal dopamine

Catecholamine levels in the two subareas of the prefrontal cortex and in one non-prefrontal region of the rat frontal lobe were measured radioenzymatically. In contrast with noradrenaline (NA), the distribution of dopamine (DA) in the frontal lobe is markedly heterogeneous. DA levels of the orbitofrontal and medial prefrontal subarea are, respectively, 3 and 4 times higher than those of a non-prefrontal region of the frontal lobe, confirming the expectation of neuroanatomical findings. Furthermore, it appears that at the population level, DA levels of the medial prefrontal subarea are lateralized, the left hemisphere being significantly higher than the right hemisphere.     

Inflammatory response and neuronal necrosis in rats with cerebral ischemia

In the middle cerebral artery occlusion model of ischemic injury, inflammation primarily occurs in the infarct and peripheral zones. In the ischemic zone, neurons undergo necrosis and apoptosis, and a large number of reactive microglia are present. In the present study, we investigated the pathological changes in a rat model of middle cerebral artery occlusion. Neuronal necrosis appeared 12 hours after middle cerebral artery occlusion, and the peak of neuronal apoptosis ap- peared 4 to 6 days after middle cerebral artery occlusion. Inflammatory cytokines and microglia play a role in damage and repair after middle cerebral artery occlusion. Serum intercellular cell adhesion molecule-1 levels were positively correlated with the permeability of the blood-brain barrier. These findings indicate that intercellular cell adhesion molecule-1 may be involved in blood-brain barrier injury, microglial activation, and neuronal apoptosis. Inhibiting blood-brain barrier leakage may alleviate neuronal injury following ischemia,