Comparative sensitivity of human and rat neural cultures to chemical-induced inhibition of neurite outgrowth

There is a need for rapid, efficient and cost-effective alternatives to traditional in vivo developmental neurotoxicity testing. In vitro cell culture models can recapitulate many of the key cellular processes of nervous system development, including neurite outgrowth, and may be used as screening tools to identify potential developmental neurotoxicants. The present study compared primary rat cortical cultures and human embryonic stem cell-derived neural cultures in terms of: 1) reproducibility of high content image analysis based neurite outgrowth measurements, 2) dynamic range of neurite outgrowth measurements and 3) sensitivity to chemicals which have been shown to inhibit neurite outgrowth. There was a large increase in neurite outgrowth between 2 and 24 h in both rat and human cultures. Image analysis data collected across multiple cultures demonstrated that neurite outgrowth measurements in rat cortical cultures were more reproducible and had higher dynamic range as compared to human neural cultures. Human neural cultures were more sensitive than rat cortical cultures to chemicals previously shown to inhibit neurite outgrowth. Parallel analysis of morphological (neurite count, neurite length) and cytotoxicity (neurons per field) measurements were used to detect selective effects on neurite outgrowth. All chemicals which inhibited neurite outgrowth in rat cortical cultures did so at concentrations which did not concurrently affect the number of neurons per field, indicating selective effects on neurite outgrowth. In contrast, more than half the chemicals which inhibited neurite outgrowth in human neural cultures did so at concentrations which concurrently decreased the number of neurons per field, indicating that effects on neurite outgrowth were secondary to cytotoxicity. Overall, these data demonstrate that the culture models performed differently in terms of reproducibility, dynamic range and sensitivity to neurite outgrowth inhibitors. While human neural cultures were more sensitive to neurite outgrowth inhibitors, they also had a lower dynamic range for detecting chemical-induced neurite outgrowth inhibition and greater variability from culture-to-culture as compared to rat primary cortical cultures.     

In vitro and in vivo regulation of synaptogenesis by the novel antidepressant spadin

Background and Purpose

We have described a novel antidepressant peptide, spadin, that acts by blocking the TWIK-related-potassium channel, type 1 (TREK-1). Here, we examined possible mechanisms of action of spadin at both molecular and cellular levels.

Experimental Approaches

Effects of spadin were measured in primary cultures of neurons or tissues from mice injected i.v. with spadin. Western blots, qPCR, histochemical and electrophysiological techniques were used.

Key Results

In vitro, spadin increased neuronal membrane potential and activated both the MAPK and PI3K signalling pathways, in a time- and concentration-dependent manner. The latter pathway was involved in the protective effect of spadin against staurosporine-induced apoptosis. Also, spadin enhanced both mRNA expression and protein of two markers of synaptogenesis, the post-synaptic density protein of 95 kDalton (PSD-95) and synapsin. We confirmed these effects on synaptogenesis by the observation that spadin treatment significantly increased the proportion of mature spines in cortical neurons. Finally, in vivo injections of spadin led to a rapid increase in both mRNA expression and protein level of brain-derived neurotrophic factor (BDNF) in the hippocampus, confirming the antidepressant action of the peptide. We argue for a new role of spadin in synaptogenesis as both PSD-95 and synapsin mRNA expression and protein levels were further enhanced in the hippocampus, following treatment in vivo with the peptide.

Conclusions and Implications

These findings provide new mechanisms of action for the rapidly acting antidepressant peptide spadin by stimulating expression of BDNF and synaptic proteins, both in vitro and in vivo.     

Toluene disrupts synaptogenesis in cultured hippocampal neurons

Prenatal toluene exposure may lead to significant developmental neurotoxicity known as fetal solvent syndrome. Emerging evidence suggests that toluene embryopathy may arise from an elusive deviation of the neurogenesis process. One key event during neural development is synaptogenesis, which is essential for the progression of neuronal differentiation and the establishment of neuronal network. We therefore aim to test the hypothesis that toluene may interfere with synaptogenesis by applying toluene to cultured hippocampal neurons dissected from embryonic rat brains. In the presence of toluene, hippocampal neurons displayed a significant loss of the immunostaining of synapsin and densin-180 punctas. Notably, a dramatic reduction was also discerned for the colocalization of the two synaptic markers. Moreover, Western blotting analyses revealed that toluene exposure resulted in considerable down-regulation of the expression of synapse-specific proteins. None of the preceding observations can be attributed to toluene-induced cell death effects, since toluene treatments failed to affect the viability of hippocampal neurons. Overall, our data are consistent with the idea that toluene may alter the expression and localization of essential synaptic proteins, thereby leading to a disruption of synapse formation and maintenance.     

Oroxylin A Induces BDNF Expression on Cortical Neurons through Adenosine A2A Receptor Stimulation: A Possible Role in Neuroprotection

Oroxylin A is a flavone isolated from a medicinal herb reported to be effective in reducing the inflammatory and oxidative stresses. It also modulates the production of brain derived neurotrophic factor (BDNF) in cortical neurons by the transactivation of cAMP response element-binding protein (CREB). As a neurotrophin, BDNF plays roles in neuronal development, differentiation, synaptogenesis, and neural protection from the harmful stimuli. Adenosine A2_A receptor colocalized with BDNF in brain and the functional interaction between A2_A receptor stimulation and BDNF action has been suggested. In this study, we investigated the possibility that oroxylin A modulates BDNF production in cortical neuron through the regulation of A2_A receptor system. As ex-pected, {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 (A2_A receptor agonist) induced BDNF expression and release, however, an antagonist, ZM241385, prevented oroxylin A-induced increase in BDNF production. Oroxylin A activated the PI3K-Akt-GSK-3β signaling pathway, which is inhibited by ZM241385 and the blockade of the signaling pathway abolished the increase in BDNF production. The physiological roles of oroxylin A-induced BDNF production were demonstrated by the increased neurite extension as well as synapse formation from neurons. Overall, oroxylin A might regulate BDNF production in cortical neuron through A2_A receptor stimulation, which promotes cellular survival, synapse formation and neurite extension.     

Studies with neuronal cells: From basic studies of mechanisms of neurotoxicity to the prediction of chemical toxicity.

Neurotoxicology considers that chemicals perturb neurological functions by interfering with the structure or function of neural pathways, circuits and systems. Using in vitro methods for neurotoxicity studies should include evaluation of specific targets for the functionalism of the nervous system and general cellular targets. In this review we present the neuronal characteristics of primary cultures of cortical neurons and of cerebellar granule cells and their use in neurotoxicity studies. Primary cultures of cortical neurons are constituted by around 40% of GABAergic neurons, whereas primary cultures of cerebellar granule cells are mainly constituted by glutamatergic neurons. Both cultures express functional GABAA and ionotropic glutamate receptors. We present neurotoxicity studies performed in these cell cultures, where specific neural targets related to GABA and glutamate neurotransmission are evaluated. The effects of convulsant polychlorocycloalkane pesticides on the GABAA, glycine and NMDA receptors points to the GABAA receptor as the neural target that accounts for their in vivo acute toxicity, whereas NMDA disturbance might be relevant for long-term toxicity. Several compounds from a list of reference compounds, whose severe human poisoning result in convulsions, inhibited the GABAA receptor. We also present cell proteomic studies showing that the neurotoxic contaminant methylmercury affect mitochondrial proteins. We conclude that the in vitro assays that have been developed can be useful for their inclusion in an in vitro test battery to predict human toxicity.     

Mechanisms for dendritic morphogenesis of cerebellar purkinje cells: role of receptor-type protein tyrosine phosphatase zeta]

Cerebellar Purkinje cells have the most elaborate dendritic trees among neurons in the central nervous system, which are formed into a characteristic morphology during postnatal cerebellar development. PTPzeta is a receptor-type protein tyrosine phosphatase that is expressed predominantly in the central nervous system and synthesized as a chondroitin sulfate proteoglycan. PTPzeta and pleiotrophin, a ligand of PTPzeta, are distributed around Purkinje cell dendrites during postnatal cerebellar development. Our study using an organotypic slice culture system demonstrated that pleiotrophin-PTPzeta signaling is involved in the morphogenesis of Purkinje cell dendrites. An aberrant morphology of Purkinje cell dendrites such as multiple and disoriented primary dendrites was induced by disturbing pleiotrophin-PTPzeta signaling in slice cultures. Pleiotrophin-PTPzeta signaling appears to act on Bergmann glia and control formation and/or maintenance of GLAST-positive lamellate processes of Bergmann glia, which regulate the morphogenesis of Purkinje cell dendrites.     

胍丁胺对慢性应激大鼠海马神经元和星形胶质细胞的影响

目的在海马神经元和星形胶质细胞水平探讨胍丁胺抗抑郁作用机制。方法采用多种应激方式建立大鼠慢性应激抑郁模型,通过开场实验和蔗糖饮水实验观察模型组、阳性对照药氟西汀组(10 mg.kg-1,ig)和胍丁胺组(20 mg.kg-1,ig)大鼠的行为学变化;应用神经元标志物——微管相关蛋白2(microtubule-associated protein 2,MAP2)和星形胶质细胞标志物——胶质原纤维酸性蛋白(glial fibrillary acidicprotein,GFAP)免疫组织化学染色,观察各组大鼠海马神经元和星形胶质细胞形态改变。结果慢性应激模型组大鼠开场实验水平运动得分和垂直运动得分明显降低,蔗糖偏嗜度明显降低,氟西汀和胍丁胺可逆转此变化;免疫组化结果显示,慢性应激模型组MAP2和GFAP表达明显减弱,表现为海马神经元突起长度和数目以及星形胶质细胞的数量和突起数量明显降低,氟西汀和胍丁胺可改善此病理变化。结论海马神经元和星形胶质细胞共同参与抑郁症发生,胍丁胺可逆转慢性应激引起的细胞形态结构改变,发挥抗抑郁作用。     

Estrogen and environmental estrogenic chemicals exert developmental effects on rat hypothalamic neurons and glias.

We investigated effects of 17beta-estradiol (E(2)) and endocrine disrupters, nonylphenol (NP) and bisphenol-A (BPA), focusing on the neuronal development in cultures of fetal rat hypothalamic cells. We applied different concentrations of E(2), NP or BPA to the cultured hypothalamic cells and observed their effects on dendritic and synaptic development by immunocytochemistry using anti-microtubule associated protein-2 (MAP2) and anti-synapsin I antibodies, respectively. Administration of E(2) for 7 days affected MAP2-positive area as well as synapsin I-positive area. NP and BPA also influenced neuronal developments. The significant increase both in MAP2- and synapsin I-positive areas was observed at 10 and/or 100 nM of them, while 1 microM of them reduced the positive areas. Synaptic densities calculated from synapsin I-positive area/MAP2-positive area were not constant among different doses of three chemicals, but increased at 10 and/or 100 nM and decreased at 1 microM. Furthermore, immunostaining of NP-treated cells with the antibody against glial fibrillary acidic protein (GFAP) revealed that glial development was similarly influenced by NP. Therefore, the present results demonstrated that not only E(2) but also the environmental estrogenic chemicals, NP and BPA, affect development of fetal rat hypothalamic cells in vitro.     

The potential of caffeine for functional modification from cortical synapses to neuron networks in the brain

Structure and function of the brain are use-dependent variables based on "synapse plasticity". Since synapses are driven by chemical transmitters, synaptic functions are liable to be modified by extrinsic chemicals displaying affinities for synaptic receptors or modulators. Caffeine is a widely used chemical substance that can invade synapses, and has several biochemical and metabolic actions on synaptic activities. This review focuses on the actions of caffeine on changes in structure and function in the region of the hippocampal formation and neocortex, which exhibit high synapse plasticity. At the synapse level, various synaptic receptors and channel activities are modulated by caffeine via mobilization of intracellular calcium, inhibition of phosphodiesterase, antagonism of adenosine receptors and GABA receptors. These actions of caffeine enable neurons to induce plastic changes in the properties of synaptic activities, such as synaptic transmission efficiency and morphology. At the network level, caffeine has the ability to activate cortical neural oscillators that deliver repetitive N-methyl-D-aspartate receptor-dependent signals to surrounding areas, causing strengthening of long-range inter-cortical communications. Caffeine might thus allow reorganization of cortical network functions via synaptic mobilizations.     

Repeated electroconvulsive seizures increase the total number of synapses in adult male rat hippocampus

The underlying mechanism of the therapeutic effect of electroconvulsive therapy (ECT) is still unclear. Here we investigated whether repeated electroconvulsive seizures (ECS), an animal model of ECT, in rats induce neuroplastic changes in the subregions of the hippocampus. ECS or sham treatment was given daily for 10 days to adult male rats. Stereological principles were employed to quantify volumes and the number of neurons and synapses. Volumes of granule cell layer (GCL) and Hilus in Dentate Gyrus of the hippocampus were significantly larger in the ECS treatment group. The neuron numbers in GCL, synapse numbers (including total synapses, spine synapses, and both perforated and nonperforated spine synapse subtypes) and synapse height in CA1 were significantly increased in the ECS treatment group. Our results indicated that repeated ECS induces neurogenesis, synaptogenesis and remodelling of synapses in rat hippocampus. This could provide a potential mechanism to explain the therapeutic effect of ECS.     

Toxic effects of midazolam on differentiating neurons in vitro as a consequence of suppressed neuronal Ca2+-oscillations

Background

In immature neurons anesthetics induce apoptosis and influence neuronal differentiation. Neuronal Ca²⁺-oscillations regulate differentiation and synaptogenesis. We examined the effects of the long-term blockade of hippocampal Ca²⁺-oscillations with midazolam on neuronal synapsin expression.

Material and methods

Hippocampal neurons were incubated at day 15 in culture with the specific GABA_A receptor agonist muscimol (50 μM) or with midazolam (100 and 300 nM), respectively, for 24 h. TUNEL and activated-Caspase-3 staining were used to detect apoptotic neurons. Ca²⁺-oscillations were detected using the Ca²⁺-sensitive dye FURA-2 and dual wavelength excitation fluorescence microscopy. Synapsin was identified with confocal anti-synapsin immunofluorescence microscopy.

Results

Muscimol, when applied for 24 h, decreased the amplitude and frequency Ca²⁺-oscillations significantly. Midazolam concentration-dependently suppressed the amplitude and frequency of the Ca²⁺-oscillations. This was associated by a downregulation of the synapsin expression 24 h after washout.

Conclusion

Neuronal Ca²⁺-oscillations mediate neuronal differentiation and are involved in synaptogenesis. By acting via the GABA_A receptor, midazolam exerts its toxic effect through the suppression of neuronal Ca²⁺-oscillations, a reduction in synapsin expression and consecutively reduced synaptic integrity.     

Basal metallothionein-I/II protects against NMDA-mediated oxidative injury in cortical neuron/astrocyte cultures

N-Methyl-D-aspartate (NMDA) receptor overactivation-mediated oxidative stress has been proposed to contribute to brain injury. Metallothionein-I/II (MT-I/II), a member of cysteine-rich metalloproteins, has been found to express in the central nervous system primarily in cortical tissues and be upregulated following brain injury. To address the role of MT-I/II on NMDA-mediated oxidative injury, we established primary cortical neuron/astrocyte cultures from neonatal MT-I/II deficient (MT?/?) and wild type (MT+/+) mice to test whether basal MT-I/II protects cortical cultures against NMDA-mediated injury. We found that MT-I/II expression was increased by NMDA in MT+/+ cultures but was not detectable in MT?/? cultures. NMDA concentration-dependently induced oxidative injury in both MT+/+ and MT?/? cultures as evidenced by decrease of cell viability, increases of lipid peroxidation and DNA damage. However, these toxic effects were greater in MT?/? than MT+/+ cultures. NMDA significantly increased reactive oxygen species (ROS) generation and disrupted mitochondrial membrane potential in neurons in MT+/+ cultures, and these effects were exaggerated in MT?/? cultures. Our findings clearly show that basal MT-I/II provides protection against NMDA-mediated oxidative injury in cortical neuron/astrocyte cultures, and suggest that the protective effects are possibly associated with inhibition of ROS generation and preservation of mitochondrial membrane potential.     

Efflux of glutathione conjugate of monochlorobimane from striatal and cortical neurons.

Evidence for the presence of a novel transporter in primary cultures of rat striatal neurons and mouse cortical neurons similar in function to the multidrug resistance-associated protein (MRP1) is presented. Functional activity was assessed by efflux studies with the glutathione conjugate of monochlorobimane (B-SG). The glutathione transferase-catalyzed formation of B-SG in rat striatal neurons and mouse cortical neurons was inhibited by ethacrynic acid. The efflux of B-SG from rat striatal neurons and mouse cortical neurons was lower at 20 degrees C than at 37 degrees C and was lower in cells with reduced ATP concentrations compared with cells with constitutive ATP concentrations. In addition, the efflux of B-SG was inhibited by MK-571 in both rat striatal and mouse cortical neurons and by probenecid in rat striatal neurons, but not in mouse cortical neurons. Verapamil did not inhibit B-SG efflux in either rat striatal or mouse cortical neurons. Although functionally similar to MRP1, Western blot analysis with commercially available antibodies directed against human and mouse MRP1 failed to show MRP1-like protein in either whole-cell homogenates of rat striatal neurons or mouse cortical neurons, indicating that the described neuronal transporter differs in structure from human or mouse MRP1 or lacks epitopes in common with MRP1.     

Assessment of PC12 cell differentiation and neurite growth: a comparison of morphological and neurochemical measures

In vitro techniques are used increasingly to screen for and characterize neurotoxicants. In many cases, chemical-induced injury to developing neurons has been examined in vitro by assessing morphological changes in differentiation and neurite growth. This research evaluated the use of proteins associated with axonal growth and synaptogenesis as surrogates for morphological measurement of neuronal differentiation. PC12 cells, which differentiate upon nerve growth factor (NGF) stimulation, were used as the in vitro model. NGF-induced (50 ng/ml) differentiation (cells with at least one neurite with a length equal to the cell body diameter) and neurite growth (length of longest neurite) were determined using light microscopy and computer-based quantitative image analysis. PC12 cell differentiation and neurite growth reached a plateau after 6 days in culture. Expression of the axonal growth associated protein 43 (GAP-43) and the synaptic protein synapsin I were assessed simultaneously by Western blot during cell differentiation. Expression of GAP-43 was low on Culture Day 0 and increased progressively to maximum levels on Culture Day 5. Likewise, synapsin I expression increased slowly on Days 0-4, and then rapidly on Days 5-7 of culture. Pharmacologic inhibitors of NGF-induced signaling were used to test the sensitivity of the proteins to chemical disruption of differentiation. The MAP kinase inhibitor, U0126 (5-30 microM) and the PKC inhibitor, bisindolylmaleimide I (Bis I; 1.25-5 microM) inhibited differentiation and neurite outgrowth in a concentration-dependent manner. U0126 and Bis I significantly decreased GAP-43, but not synapsin I expression. Interestingly, the PI-PLC inhibitor edelfosine (ET-18; 5-30 microM) stimulated differentiation at early times of exposure followed by a significant decrease in neurite length at later time points. However, ET-18 did not alter the expression of GAP-43 or synapsin I. These data suggest that GAP-43 may be a useful indicator of the status of PC12 cell differentiation.     

The involvement of cleavage of neural cell adhesion molecule in neuronal death under oxidative stress conditions in cultured cortical neurons

Neural cell adhesion molecule (NCAM) is a member of the immunoglobulin superfamily with an important function in the central nervous system, particularly in synapse stabilization and neurite outgrowth. Our recent study clearly demonstrated that cleavage of NCAM-180 by matrix metalloproteinase-9 (MMP-9) exacerbated the neuronal damage induced by in vivo ischemic stress. In the present study, we investigated the effect of oxidative stress on the expression levels of full-length NCAM-180 and NCAM-cleavage product (65 kDa) and the relationship between NCAM-180 and MMP-9 in cultured cortical neurons. Primary cultured cortical neurons were exposed to oxidative stress by administration of hydrogen peroxide into the culture medium. After exposure to oxidative stress, cell death of cultured cortical neurons was gradually increased in a time-dependent manner. In parallel to the cell death, levels of full-length NCAM-180 and its cleavage product (65 kDa) were gradually and significantly decreased and increased, respectively, in a time-dependent manner. These changes completely disappeared following addition of an MMP-9 inhibitor, while MMP-9 protein levels were increased only in the early phase of oxidative stress. We conclude that oxidative stress can induce cleavage of NCAM-180 through up-regulation of MMP-9 during the early phase of oxidative stress. These changes might be related to the neuronal death observed under oxidative stress conditions.     

知母皂苷元对体外培养皮层神经元树突发育的影响及信号转导机制

目的探讨知母皂苷元(Sarsasapogenin,SAR)对大脑皮层神经元树突发育的促进作用及其信号转导机制。方法选用出生0～24 h的Sprague-Dawley(SD)大鼠乳鼠,取皮层神经元进行体外细胞培养,4 d后用于实验。倒置相差显微镜测量培养神经元树突分支总长度(TDBL)、一级树突数目(PDN)、最大分支级数(MBO)和神经元胞体面积。West-ern blot法观察神经元p-PDK1、p-Akt473及p-mTOR蛋白表达。结果形态学观察结果显示SAR(10、30、100μmol.L-1)可明显促进树突发育,表现为树突分支总长度增加、一级树突数目增多、最大分支级数增大及胞体面积增大,并呈明显浓度依赖。SAR 30+LY组、SAR 30+TCBN组、SAR 30+Rapa组的神经元树突总长度、一级树突数目、最大分支级数及胞体面积较SAR30μmol.L-1组明显降低。Westernblot结果显示SAR 30也可明显增加p-PDK1、p-Akt473及p-mTOR蛋白表达水平。SAR 30+LY组明显降低神经元p-PDK1、p-Akt473及p-mTOR的蛋白表达水平。SAR 30+TCBN组明显降低神经元p-Akt473及p-mTOR的蛋白表达水平。SAR 30+Rapa组明显降低神经元p-mTOR的蛋白表达水平。结论 SAR对体外培养皮层神经元树突的发育有促进作用,这种作用可能与PI3K/Akt/mTOR信号转导通路有关。     

Neuroprotective effects of a standardized extract of Diospyros kaki leaves on MCAO transient focal cerebral ischemic rats and cultured neurons injured by glutamate or hypoxia

Naoxinqing (NXQ, a standardized extract of Diospyros kaki leaves) is a patented and approved drug of Traditional Chinese Medicine (TCM) used for the treatment of apoplexy syndrome for years in China, but its underlying mechanism remains to be further elucidated. The present study investigates the effects of NXQ against focal ischemia/reperfusion injury induced by middle cerebral artery occlusion (MCAO) in rats and against glutamate-induced cell injury of hippocampal neurons as well as against hypoxia injury of cortical neurons. Oral administrations of NXQ at 20, 40, 80 mg/kg/day for 7 days (3 days before MCAO and 4 days after MCAO) significantly reduced the lesion of the insulted brain hemisphere and improved the neurological behavior of the rats. In primary rat hippocampal neuron cultures, treatment with NXQ at 5 - 20 microg mL concentration protects the neurons against glutamate-induced excitotoxic death in a dose-dependent manner. In primary rat cerebral cortical neuron cultures, pretreatment with 5 - 100 microg/mL NXQ also attenuates hypoxia-reoxygen induced neuron death and apoptosis in a dose-dependent manner. These results suggest that NXQ significantly protects the rats from MCAO ischemic injury in vivo and the hippocampal neurons from glutamate-induced excitotoxic injury as well as cortical neurons from hypoxia injury in vitro by synergistic mechanisms involving its antioxidative effects. NXQ:Naoxinqing CNS:central nervous system MCAO:middle cerebral artery occlusion I/R:ischemia and reperfusion.     

Ethanol protects cultured neurons against amyloid-β and α-synuclein-induced synapse damage

The loss of synapses and a corresponding reduction in synaptic proteins are histopathological features of Alzheimer’s disease that correlate strongly with dementia. Here we report that stable Aβ oligomers secreted by 7PA2 cells reduced the amount of synaptophysin, a protein used as an indicator of synapse density, in cultured cortical and hippocampal neurons. Pre-treatment with physiologically relevant concentrations of ethanol (0.02–0.08%) protected neurons against Aβ-induced synapse damage. Ethanol also protected neurons against synapse damage induced by α-synuclein (αSN), pre-synaptic aggregates of which are characteristic of Parkinson’s disease and dementia with Lewy bodies. Exposure of neurons to ethanol did not affect the accumulation of Aβ at synapses, rather it reduced the Aβ and αSN-induced activation of cytoplasmic phospholipase A₂ (cPLA₂) within synapses. Ethanol did not affect synapse damage caused by platelet-activating factor or prostaglandin E₂, bioactive lipids that are formed following the activation of cPLA₂. These results may help explain epidemiological reports that moderate alcohol consumption protects against the development of dementia in Alzheimer’s and Parkinson’s diseases.     

Neuroprotective effects of curculigoside against NMDA-induced neuronal excitoxicity in vitro

Glutamate is an important excitatory neurotransmitter in the central nervous system. Excessive accumulation of glutamate can cause excitotoxicity, which plays a key role in spinal cord injury, traumatic brain injury, stroke, and neurodegenerative diseases. Curculigoside (CCGS) is a major bioactive compound isolated from the rhizome of Curculigo orchioides Gaertn. CCGS has an extensive biological effect and has been used in Traditional Chinese Medicine. However, little is known about the neuroprotective effects of CCGS on glutamate-induced excitotoxicity. This study aims to evaluate the neuroprotective effects of CCGS in cultured cortical neurons. The results indicated that treatment with 1 and 10 μM CCGS evidently prevented N-methyl-d-aspartate (NMDA)-induced neuronal cell loss and reduced the number of apoptotic and necrotic cells in a time- and concentration-dependent manner. The neuroprotective effects of CCGS are related to down regulating the apoptotic protein levels and reducing the production of intracellular reactive oxygen species in cultured cortical neurons. These findings give a new insight into the development of natural anti-excitotoxicity agents.     

Cannabinoid CB1 receptors in rat medial prefrontal cortex are colocalized with calbindin but not parvalbumin- and calretinin-positive GABA-ergic neurons

In the present study, we investigate putative localization of cannabinoid receptors 1 (CB1) protein on a population of cortical γ-aminobutyric acid (GABA) – positive interneurons characterized by expression of calciumbinding proteins in rat medial prefrontal cortex (MPC). Parvalbumin (PARV)/calretinin (CALR)- and calbindin (CALB)-positive neurons form two distinct populations of GABA-ergic interneurons that comprise the axo-somatic/axo-axonic and axo dendritic inhibitory systems of pyramidal cells. It has been found that CB1 receptor-positive cells are randomly distributed across the rat MPC. All spotted neurons that were positive for CB1 receptors were positive for GABA; however, the number of GABA-positive cells drastically exceeded the number of CB1 receptor-positive neurons. Subsequent experiments with double-labelling of CB1 receptors with PARV and CALR revealed no colocalization. CALB-positive neurons (e.g., double bouquet and bipolar cells) display colocalization: the degree of colocalization among CB1 receptor-positive cells reached 18%. The appearance of CB1 receptors in double bouquet and bipolar neurons indicates that CB1 receptors may control the activity of pyramidal neurons from presynaptic sites in axo dendritic synapses formed on apical and basilar dendrites of pyramidal neurons, as is characteristic for CALB-positive cortical interneurons. The phenotype of GABA-and CB1 receptor-positive but CALB-negative neurons may represent a population of inhibitory neurons that allow axosomatic control of information flow, governed by principal neurons of the MPC.