Primary Culture of Adult Mouse Olfactory Receptor Neurons

Olfactory receptor neurons (ORNs) are unique because they can be replaced by stem cells throughout life. Previous studies have demonstrated that adult mouse olfactory epithelium (OE) injured by exposure to ZnSO₄through nasal irrigation can stimulate stem cell mitotic activityin situ, which continues when placed in culture. We report on an improved ZnSO₄delivery method, mist inhalation, which produces more consistent and greater yields of OE cells. Cultures established following this method contained bipolar, nest, fusiform, and giant cells. The bipolar cells usually underwent asymmetric process development. Some bipolar cells reacted positively to neuron-specific antibodies and were immunonegative for keratin and glia-specific proteins, suggesting that they were ORNs. Those that were negative for the neuron-specific proteins may represent either neuron progenitors or olfactory ensheathing cells. The fusiform cells were relatively small and undifferentiated, exposure to brain-derived neurotrophic factor resulted in their decrease and an increase in bipolar cells. Therefore, they might be the stem cells. The nest cells had morphological characteristics of epithelia and bound keratin antibodies. The giant cells had the morphology of epithelial cells but were negative for keratin; they may represent a unique cell population induced by the ZnSO₄. These results indicate that the major cell types of intact OE are present in our cultures, and each retains characteristics foundin situ. The mist inhalation method provides anin vitropopulation of adult mitotically active neurons for study.     

Dendritic Spine Abnormalities in Hippocampal CA1 Pyramidal Neurons Underlying Memory Deficits in the SAMP8 Mouse Model of Alzheimer's Disease

SAMP8 is a strain of mice with accelerated senescence. These mice have recently been the focus of attention as they show several alterations that have also been described in Alzheimer's disease (AD) patients. The number of dendritic spines, spine plasticity, and morphology are basic to memory formation. In AD, the density of dendritic spines is severely decreased. We studied memory alterations using the object recognition test. We measured levels of synaptophysin as a marker of neurotransmission and used Golgi staining to quantify and characterize the number and morphology of dendritic spines in SAMP8 mice and in SAMR1 as control animals. While there were no memory differences at 3 months of age, the memory of both 6- and 9-month-old SAMP8 mice was impaired in comparison with age-matched SAMR1 mice or young SAMP8 mice. In addition, synaptophysin levels were not altered in young SAMP8 animals, but SAMP8 aged 6 and 9 months had less synaptophysin than SAMR1 controls and also less than 3-month-old SAMP8 mice. Moreover, while spine density remained stable with age in SAMR1 mice, the number of spines started to decrease in SAMP8 animals at 6 months, only to get worse at 9 months. Our results show that from 6 months onwards SAMP8 mice show impaired memory. This age coincides with that at which the levels of synaptophysin and spine density decrease. Thus, we conclude that together with other studies that describe several alterations at similar ages, SAMP8 mice are a very suitable model for studying AD.     

The HIV Protein gp120 Alters Mitochondrial Dynamics in Neurons

Neurotoxicity of human immunodeficiency virus-1 (HIV) includes synaptic simplification and neuronal apoptosis. However, the mechanisms of HIV-associated neurotoxicity remain unclear, thus precluding an effective treatment of the neurological complications. The present study was undertaken to characterize novel mechanisms of HIV neurotoxicity that may explain how HIV subjects develop neuronal degeneration. Several neurodegenerative disorders are characterized by mitochondrial dysfunction; therefore, we hypothesized that HIV promotes mitochondrial damage. We first analyzed brains from HIV encephalitis (HIVE) by electron microscopy. Several sections of HIVE subjects contained enlarged and damaged mitochondria compared to brains from HIV subjects with no neurological complications. Similar pathologies were observed in mice overexpressing the HIV protein gp120, suggesting that this viral protein may be responsible for mitochondrial pathology found in HIVE. To gain more information about the cellular mechanisms of gp120 neurotoxicity, we exposed rat cortical neurons to gp120 and we determined cellular oxygen consumption rate, mitochondrial distribution, and trafficking. Our data show that gp120 evokes impairment in mitochondrial function and distribution. These data suggest that one of the mechanisms of HIV neurotoxicity includes altered mitochondrial dynamics in neurons.     

大鼠胚胎中脑前体细胞培养的研究

目的 :通过胚胎MPC体外培养获取PD患者CRT治疗所需的DA能神经元。方法 :取自E12鼠胚中脑腹侧的MPC悬液在添加bFGF的DMEM/F12 /N2培养液中原代培养 ,鉴定培养细胞的增殖和分化能力。结果 :在添加了bFGF 10ng·mol-1的DMEM/F12 /N2培养液中MPC增殖良好 ,体外培养 5～ 7d后细胞数量扩增到培养前的 9 782± 0 0 47倍 ;培养液中撤去bFGF后细胞可分化成星形胶质细胞和神经元 ,其中多数Tuj1抗原标记阳性的神经元同时呈TH抗原标记阳性 ,培养细胞分化为DA能神经元的比例约为2 4 3 4% (10 2 / 419)。结论 :E12鼠胚MPC原代培养是获取DA能神经元的可靠途径 ,类似的技术应用于临床可能缓解PD患者CRT治疗供体不足的矛盾。     

Chronic Hyperprolactinemia and Changes in Dopamine Neurons

The tuberoinfundibular dopaminergic (TIDA) system is known to inhibit prolactin (PRL) secretion. In young animals this system responds to acute elevations in serum PRL by increasing its activity. However, this responsiveness is lost in aging rats with chronically high serum PRL levels. The purpose of this study was to induce hyperprolactinemia in rats for extended periods of time and examine its effects on dopaminergic systems in the brain. Hyperprolactinemia was induced by treatment with haloperidol, a dopamine receptor antagonist, and Palkovits' microdissection technique in combination with high-performance liquid chromatography was used to measure neurotransmitter concentrations in several areas of the brain. After 6 months of hyperprolactinemia, dopamine (DA) concentrations in the median eminence (ME) increased by 84% over the control group. Nine months of hyperprolactinemia produced a 50% increase in DA concentrations in the ME over the control group. However, DA response was lost if a 9-month long haloperidol-induced hyperprolactinemia was followed by a 11

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month-long extremely high increase in serum PRL levels produced by implantation of MMQ cells under the kidney capsule. There was no change in the levels of DA, norepinephrine (NE), serotonin (5-HT), or their metabolites in the arcuate nucleus (AN), medial preoptic area (MPA), caudate putamen (CP), substantia nigra (SN), and zona incerta (ZI), except for a decrease in 5-hydroxyindoleacetic acid (5-HIAA) in the AN after 6-months of hyperprolactinemia and an increase in DA concentrations in the AN after 9-months of hyperprolactinemia. These results demonstrate that hyperprolactinemia specifically affects TIDA neurons and these effects vary, depending on the duration and intensity of hyperprolactinemia. The age-related decrease in hypothalamic dopamine function may be associated with increases in PRL secretion.     

Activation of Parvalbumin-Positive Neurons in Both Retina and Primary Visual Cortex Improves the Feature-Selectivity of Primary Visual Cortex Neurons

Several recent studies using either viral or transgenic mouse models have shown different results on whether the activation of parvalbumin-positive(PV~+)neurons expressing channelrhodopsin-2(ChR2) in the primary visual cortex(V1) improves the orientation-and direction-selectivity of V1 neurons. Although this discrepancy was thoroughly discussed in a follow-up communication, the issue of using different models to express ChR2 in V1 was not mentioned. We found that ChR2 was expressed in retinal ganglion cells(RGCs) and V1 neurons in ChR2fl/~+; PV-Cre mice. Our results showed that the activation of PV~+RGCs using white drifting gratings alone significantly decreased the firing rates of V1 neurons and improved their direction-and orientation-selectivity. Longduration activation of PV~+interneurons in V1 further enhanced the feature-selectivity of V1 neurons in anesthetized mice, confirming the conclusions from previous findings. These results suggest that the activation of both PV~+RGCs and V1 neurons improves feature-selectivity in mice.     

Neurotrophic Actions of Bone Marrow Stromal Cells on Primary Culture of Dorsal Root Ganglion Tissues and Neurons

Application of adult bone marrow stromal cells (BMSCs) provides therapeutic benefits to the treatment of neurological insults. The aim of this study was to explore the potential of nonhematopoietic BMSCs to produce soluble factors and stimulate signaling pathways in neurons that mediate trophic effects. A combination of enzyme-linked immunosorbent assay and two-dimensional gel electrophoresis coupled with mass spectrometry showed that the BMSCs released into the culture medium an array of soluble factors such as nerve growth factor, brain-derived neurotrophic factor, basic fibroblast growth factor, and ciliary neurotrophic factor, which have been shown to exhibit potent neurotrophic effects on neural cells. Immunochemistry, cell viability assay, and quantitative real-time RT-PCR collectively showed that neurite outgrowth and neurogenesis in cultured rat dorsal root ganglion (DRG) explants and neurons were enhanced after they were cocultured with rat BMSCs. Western blot analysis revealed that BMSC-conditioned medium activated phosphorylation of mitogen-activated protein kinase/extracellular signal-regulated protein kinase and/or phosphoinositide 3-kinase/serine/threonine kinase (PI3K/Akt) in primary culture of rat DRG neurons, which suggested that BMSCs trigger endogenous survival signaling pathways in neurons through their secreted soluble factors. Our data help to elucidate the mechanisms by which BMSCs function as a cell therapy agent in peripheral nerve regeneration.     

Nuclear and Cytoplasmic Triiodothyronine-Binding Sites in Primary Sensory Neurons and Schwann Cells: Radioautographic Study During Development

The effects of the thyroid hormones on target cells are mediated through nuclear T₃ receptors. In the peripheral nervous system, nuclear T₃ receptors were previously detected with the monoclonal antibody 283 rnAb in all the primary sensory neurons throughout neuronal life and in peripheral glia at the perinatal period only (Eur. J. Neurosci. 5, 379, 7993). To determine whether these nuclear T₃ receptors correspond to functional ones able to bind T₃, cryostat sections and in vitro cell cultures of dorsal root ganglion (DRG) or sciatic nerve were incubated with 0.1 nM [1251]-labeled T₃, either alone to visualize the total T₃-binding sites or added with a lo3 fold excess of unlabeled T₃ to estimate the part due to the non-specific T₃-binding. After glutaraldehyde fixation, radioautography showed that the specific T₃-binding sites were largely prevalent. The T₃-binding capacity of peripheral glia in DRG and sciatic nerve was restricted to the perinatal period in vivo and to Schwann cells cultured in vitro. In all the primary sensory neurons, specific T₃-binding sites were disclosed in foetal as well as adult rats. The detection of the T₃-binding sites in the nucleus indicated that the nuclear T₃ receptors are functional. Moreover the concomitant presence of both T₃-binding sites and T₃ receptors CI isoforms in the perikaryon of DRG neurons infers that: 1) [1251]-labeied T₃ can be retained on the T₃-binding ‘E’ domain of nascent CI, isoform molecules newly-synthesized on the perikaryal ribosomes; 2) the CI isoforms translocated to the nucleus are modified by posttranslational changes and finally recognized by 2B3 rnAb as nuclear T₃ receptor. In conclusion, the radioautographic visualization of the T₃-binding sites in peripheral neurons and glia confirms that the nuclear T₃ receptors are functional and contributes to clarify t h e discordant intracellular localization provided by the immunocytochemical detection of nuclear T₃ receptors and T₃ receptor CI isoforms.     

Motor Neurons Pathology After Chronic Exposure to MPTP in Mice

Neurotoxicity Research - The neurotoxin 1-methyl,4-phenyl-1,2,3,6-tetrahydropiridine (MPTP) is widely used to produce experimental parkinsonism in rodents and primates. Among different...     

Managing Alcohol Use Disorder in Primary Health Care

Purpose of Review

The aim of this study is to summarise the current literature on both the impact and the implementation of primary health care-based screening and advice programmes to reduce heavy drinking, as an evidence-based component of managing alcohol use disorder in primary health care.

Recent Findings

Systematic reviews of reviews find conclusive evidence for the impact of primary health care delivered screening and brief advice programmes in reducing heavy drinking. The content, length of advice and which profession delivers the advice seems less important than the actual encounter between provider and patient. Despite the global burden of disease due to heavy drinking and the evidence that this can be reduced by screening and brief advice programmes delivered in primary health care, such programmes remain poorly implemented. Were such programmes widely implemented, there would be substantial health and productivity gains. Systematic reviews and international studies indicate that improved implementation requires tailoring of training and programme content to match the needs of providers, training and ongoing support and embedding of programmes within local community support, championed by local leaders.

Summary

The next stage of implementation and scale-up of evidence-based screening and brief advice programmes should take place embedded within supportive local community action, with appropriate research to demonstrate impact.

     

Lamotrigine Reduces Voltage-Gated Sodium Currents in Rat Central Neurons in Culture

Summary: Purpose : To study the mechanism or mechanisms of action of lamotrigine (LTG) and, in particular, to establish its effects on the function of NA⁺ channels in mammalian central neurons.
Methods : Rat cerebellar granule cells in culture were subjected to the whole-cell mode of voltage clamping under experimental conditions designed to study voltage-gated Na⁺ currents.
Results : Extracellular application of LTG (10–500 μ M , n = 21) decreased in a dose-related manner a tetrodotoxin-sensitive inward current that was elicited by depolarizing commands (from −80 to +20mV). The peak amplitude of this Na⁺-mediated current was diminished by 38.8 ± 12.2% (mean ± SD, n = 6) during application of 100 μ M LTG, and the dose-response curve of this effect indicated an IC₅₀ 145 μM. The reduction in the inward currents produced by LTG was not associate with any signficant change in the current decay, whereas the voltage dependency of the steady-state inactivation shifted toward more negative values (midpoint of the inactivation curve: –47.5 and –59.0 mV under control conditions and during application of 100 μM LTG, respectively, n = 4).
Conclusions : Our findings indicate that LTG reduces the amplitude of voltage-gated Na⁺ inward current in rat cerebellar granule cells and induces a negative shift of the steady-state inactivation curve. Both mechanisms may be instrumental in controlling the repetitive firing of action potentials (AP) that occurs in neuronal networks during seizure activity.     

Neuroprotective Effects of Rosmarinic Acid on Ciguatoxin in Primary Human Neurons

Ciguatoxin (CTX), is a toxic compound produced by microalgae (dinoflagellate) Gambierdiscus spp., and is bio-accumulated and bio-transformed through the marine food chain causing neurological deficits. To determine the mechanism of CTX-mediated cytotoxicity in human neurons, we measured extracellular lactate dehydrogenase (LDH) activity, intracellular levels of nicotinamide adenine dinucleotide (NAD⁺) and H2AX phosphorylation at serine 139 as a measure for DNA damage in primary cultures of human neurons treated with Pacific (P)-CTX-1B and P-CTX-3C. We found these marine toxins can induce a time and dose-dependent increase in extracellular LDH activity, with a concomitant decline in intracellular NAD⁺ levels and increased DNA damage at the concentration range of 5–200 nM. We also showed that pre- and post-treatment with rosmarinic acid (RA), the active constituent of the Heliotropium foertherianum (Boraginaceae) can attenuate CTX-mediated neurotoxicity. These results further highlight the potential of RA in the treatment of CTX-induced neurological deficits.     

Aspirin Protects Dopaminergic Neurons Against Lipopolysaccharide-Induced Neurotoxicity in Primary Midbrain Cultures

Aspirin (ASA) is one of the most widely used nonsteroidal anti-inflammatory drugs. ASA has primarily been used to treat headaches, rheumatic pain, and inflammation, but its therapeutic effects have recently been demonstrated on a range of disorders, including those of the central nervous system. In this study, we investigated whether ASA is neuroprotective in inflammation-mediated neurodegenerative diseases. Pretreatment with ASA reduced the lipopolysaccharide (LPS)-induced degeneration of dopaminergic (DA) neurons in mesencephalic neuron–glia cultures in a dose-dependent manner. The neuroprotective effect of ASA was attributed to the inhibition of microglial activation because of its observed inhibitory effects on LPS-stimulated nitric oxide, tumor necrosis factor-α, and superoxide production by microglial cells. Moreover, ASA increased the production of the anti-inflammatory cytokines transforming growth factor beta-1 and interleukin-10 in neuron–glia cultures after stimulation with LPS. Mechanistic studies revealed that the neuroprotective effects of ASA were mediated through the inhibition of nicotinamide adenine dinucleotide phosphate oxidase (PHOX), a key enzyme for superoxide production in microglia. These results suggest that ASA protects DA neurodegeneration by inhibiting the microglial-mediated oxidative stress/inflammatory response and by regulating the production of anti-inflammatory cytokines.