正常大鼠脑动脉平滑肌细胞电压依赖性钙通道电流特征

目的 研究正常大鼠脑动脉平滑肌细胞电压依赖性钙通道(VDCCs)电流的电生理和药理学特征,为相关的生理或疾病研究提供理论依据.方法 酶促消化法急性分离大鼠脑动脉平滑肌细胞,运用膜片钳电流钳技术研究其静息电位,膜片钳电压钳技术研究其电压依赖性钙通道电流以及二价阳离子载荷离子浓度和Nifedipine对其的影响.结果 静息电位为(50.42±0.26) mV,阶跃电压10 mV和斜坡电压(9.80±0.92) mV时最大VDCCs电流密度分别为(-5.22 ±0.51)pA/pF和(-4.89 ±0.65)pA/pF(10 mmol/L BaCl2).VDCCs峰电流完全由高电压激活(HVA-VDCCs)电流构成(P=0.17),无低电压激活(LVA)-VDCCs电流.VDCCs电流密度与细胞外液Ba2+浓度正相关(P＜0.05),Nifedipine对VDCCs电流的最大抑制作用为(86.13±0.76)％,IC50为6.02 nmol/L.结论 本研究证实脑动脉平滑肌细胞的VDCCs电流来源于HVA-VDCCs,以及脑动脉平滑肌细胞存在Nifedipine不敏感电流(NICCs),NICCs所依赖的离子通道以及在脑血管张力和脑血流自身调节中的作用需要进一步的研究.     

Treatment of aged rat sensory neurons in short-term, serum-free culture with nerve growth factor reverses the effect of aging on neurite outgrowth, calcium currents, and neuronal survival

Impaired NGF production and release has been documented in aged animals, suggesting that decreased NGF receptor stimulation may be one factor contributing to neuronal dysfunction with aging. Other studies have suggested that aging may be associated with impaired intracellular responses to NGF. Because aging-associated neuronal dysfunction contributes to morbidity and mortality in the geriatric population, it is important to determine whether the effects of aging on sensory neuron function and survival are reversible. In the present study, we observed significantly decreased neurite outgrowth and neuronal survival in short-term cultures (0-96 h) of dorsal root ganglion (DRG) neurons from aged (>22 months) Fisher 344 x Brown Norway F1 hybrid rats, compared to young (4-6 month) and middle-aged (14 month) animals. From 24 to 96 h in culture, diminished survival of aged neurons appeared to be due to an increased rate of apoptotic cell death. DRG neurons from aged animals also exhibited significantly decreased whole cell, high-threshold voltage-dependent calcium currents, with a larger proportion of L-type current, compared to youthful and middle-aged animals. Treatment of aged DRG neurons with NGF restored neurite outgrowth, neuronal survival and calcium current amplitude and subtype distribution to those observed in youthful DRG neurons.     

Diffusible factor(s) from adult rat sciatic nerve increases cell number and neurite outgrowth of cultured embryonic ventral mesencephalic tyrosine hydroxylase-positive neurons

Dissociated embryonic rat ventral mesencephalon containing the developing A8-A10 dopamine (DA) neurons was cultured alone or in the presence of a 10 mm segment of adult rat sciatic nerve that had been explanted and maintained in separate culture for 72 hours prior to introduction to mesencephalic cultures. Nerve segments were contained in a co-culture basket, so that midbrain cells and nerve shared medium but were not in physical contact. The number and morphology of cultured DA neurons was assessed via immunocytochemistry for tyrosine hydroxylase (TH). Co-cultures of ventral midbrain tissue and nerve exhibited an increased number of TH-positive neurons, with larger neuronal perikarya, and increased length and complexity of neurites, than cultures of midbrain tissue alone. Increased number and growth of TH-positive neurons was obtained with as little as 2 days of exposure to nerve. This evidence suggests that a diffusible, soluble factor(s) from sciatic nerve can enhance the number and development of TH-positive neurons detected in cultures of embryonic ventral mesencephalon.     

Differential down-regulation of voltage-gated calcium channel currents by glutamate and BDNF in embryonic cortical neurons

In the embryonic brain, post-mitotic cortical neurons migrate from their place of origin to their final location. Various external factors such as hormones, neurotransmitters or peptides regulate their migration. To date, however, only a few studies have investigated the effects of these external factors on the electrical properties of the newly formed embryonic cortical neurons. The aim of the present study was to determine whether glutamate and brain-derived neurotrophic factor (BDNF), known to regulate neuronal cell migration, could modulate currents through voltage-gated calcium channels (ICa) in cortical neurons isolated from embryonic day 13 (E13) mouse foetuses. Whole cell recordings of ICa showed that E13 cortical cells kept 1 day in vitro expressed functional low- and high-voltage activated (LVA and HVA) Ca2+ channels of T-, L- and N-types. A 1-day glutamate treatment non-specifically inhibited LVA and HVA ICa whereas BDNF down-regulated HVA with N-type ICa being more depressed than L-type ICa. The glutamate-induced ICa inhibition was mimicked by NMDA. BDNF exerted its action by recruiting trkB receptors and SKF-96365-sensitive channels. BAPTA prevented the glutamate- and the BDNF-dependent inhibition of Ica, indicating a Ca2+-dependent mechanism of action. It is proposed that an influx of Ca2+ through NMDA receptors depresses the expression of LVA and HVA Ca2+ channels whereas a Ca2+ influx through SKF-96365-sensitive TRPC (transient receptor potential protein of C subtype) channels preferentially inhibits the expression of HVA Ca2+ channels. Glutamate and BDNF appear as potent modulators of the electrical properties of early post-mitotic neurons. By down-regulating ICa they could exert a neuroprotective action on embryonic cortical neurons.     

Effects of calcium ion on neurite outgrowth of rat spinal cord neurons in vitro: the role of non-neuronal cells in regulating neurite sprouting

The interactions of nerve cells with their environment and other cells are specific to different stages of cellular differentiation. Neurite outgrowth was measured from cultured spinal cord neurons under the influence of different Ca²⁺ concentrations. We used fluorodeoxyuridine (FuDr), an antimitotic agent which reduces significantly the proportion of non-neuronal cells in spinal cord cell cultures, to examine the effects of non-neuronal cells on neurite outgrowth. Spinal cord neurons responded to changes in their environment by means of two types of neurite outgrowth: sprouting and elongation. The concurrent presence of non-neuronal cells led to increased sprouting of neurites in certain ionic environments, thus lending support to the idea that non-neuronal cells release diffusible factors which influence sprouting and guide neurite outgrowth.     

Modulation of voltage-dependent calcium currents by serotonin in acutely isolated rat amygdala neurons

The modulation of voltage-dependent calcium currents (I(Ca)) by serotonin (5-HT) was studied in rat acutely dissociated amygdala neurons using whole-cell patch-clamp recording techniques. 5-HT inhibited I(Ca) in a concentration-dependent manner with a ED50 of approximately 1 microM and a maximal inhibition of approximately 50%. The inhibition was mimicked by the selective 5-HT1A agonist 8-hydroxy-dipropylaminotetralin (8-OH-DPAT) and was reduced by the 5-HT1A antagonist NAN-190, indicating its mediation by 5-HT1A receptors. Pretreatment of neurons with the alkylating agent N-ethylmaleimide (NEM) or pertussis toxin (PTX) markedly reduced the action of 5-HT. The modulation was partially reversed by strong depolarization and was not seen in cell-attached patches when the agonist was applied outside the recorded patch, suggesting a membrane-delimited, G-protein-mediated signaling pathway. Nimodipine (1 microM) reduced the I(Ca) by approximately 30% without reducing inhibition of current by 5-HT significantly, ruling out L-type channels as the target of modulation. 5-HT-mediated inhibition after exposure to omega-conotoxin-GVIA (omega-CgTX, 1 microM) or omega-agatoxin-IV (omega-AgTX, 200 nM), which blocked 26% and 21% of the total I(Ca), respectively, was significantly decreased, suggesting involvement of the N- and P/Q-type channels. In the combined presence of omega-CgTX and omega-AgTX, 5-HT still caused a small but significant reduction of I(Ca), suggesting a possible involvement of R-type channels. Stimulation of beta-adrenergic receptor with isoproterenol (Iso) or activation of adenylyl cyclase with forskolin resulted in an enhancement of I(Ca). 5-HT caused the same degree of inhibition with or without Iso or forskolin pretreatment. On the other hand, application of 8-OH-DPAT inhibited I(Ca) and blocked Iso- and Sp-cAMPS-induced enhancement. These results provide the first evidence showing a dominant effect of 5-HT-mediated inhibition over Iso-mediated enhancement of I(Ca).     

Modulation of calcium currents in mouse ventral horn neurons by extracellular pH

Neuronal activity has been shown to modulate the pH of the extracellular environment. Since neuronal circuits in the ventral horn of the spinal cord are highly active during patterned movements, and voltage-gated calcium channels play an important role in the production of spinal motoneuron output, the effects of changes in extracellular pH (pH(e)) on calcium currents in ventral horn neurons of the mouse spinal cord were examined. It is demonstrated that these channels are sensitive to modulation by pH(e). The amplitude of the current mediated by these channels increased as the pH(e) was elevated. The elevated pH(e) also led to a hyperpolarizing shift in the voltage dependence of both activation and inactivation. The opposite effects were seen for a decrease in pH(e). It was also noted that a decrease in pH(e) was associated with a faster inactivation of the current. It is concluded that voltage-gated calcium currents in ventral horn neurons are modulated by changes in pH(e), and that this modulation may play a physiologically important role in determining motoneuronal excitability during behaviors such as locomotion.     

Effects of calcium ion on neurite outgrowth of rat spinal cord neurons in vitro: The role of non-neuronal cells in regulating neurite sprouting

The interactions of nerve cells with their environment and other cells are specific to different stages of cellular differentiation. Neurite outgrowth was measured from cultured spinal cord neurons under the influence of different Ca²⁺ concentrations. We used fluorodeoxyuridine (FuDr), an antimitotic agent which reduces significantly the proportion of non-neuronal cells in spinal cord cell cultures, to examine the effects of non-neuronal cells on neurite outgrowth. Spinal cord neurons responded to changes in their environment by means of two types of neurite outgrowth: sprouting and elongation. The concurrent presence of non-neuronal cells led to increased sprouting of neurites in certain ionic environments, thus lending support to the idea that non-neuronal cells release diffusible factors which influence sprouting and guide neurite outgrowth.     

Aged median eminence glial cell cultures promote survival and neurite outgrowth of cocultured neurons

We have recently shown that tanycytes, a particular type of glial cell that has morphological and biochemical similarities with radial glial cells, constitute a preferential support for the regeneration of lesioned neurohypophysial axons. The present study was designed to explore the possible neurotrophic role of tanycytes in vitro. Glial cells derived from the median eminence or from the cerebral cortex of 10-day-old rats were cultured for 4–7 weeks. At these times the majority of the cells identified in the median eminence cultures exhibited immunostaining patterns of tanycytes, as detected in the mediobasal hypothalamus of 10-day-old and adult rats, i.e., they were immunoreactive to vimentin (VIM), to DARPP-32 (a dopamine- and adenosine 3′:5′-monophosphate-regulated phosphoprotein), and to a lesser extent to glial fibrillary acidic protein (GFAP) antibodies. On the other hand, the majority of cells in cortex cultures showed immunostaining patterns of astrocytes, i.e., they were intensely immunoreactive to GFAP and VIM antibodies but negative to DARPP-32. Cells obtained from the dissociation of 3-day-old rat mesencephalon, cortex, and hypothalamus were cocultured on these glial monolayers, and the number of surviving neurons and their neurite length were quantified after 8 days. Our data showed that, when compared with astrocytes, tanycytes greatly improved both survival (six- to ten-fold higher) and neurite outgrowth (two- to five-fold longer) of cocultured neurons whatever their origin. Experiments performed by coculturing neurons on millicell inserts placed above the glial monolayers showed that diffusible factors from median eminence glial cells slightly increased survival (1.7-fold higher) of cocultured neurons but had no significant effect on neurite outgrowth. These observations indicate: 1) that aged tanycytes have a capacity to support survival and neurite outgrowth for a variety of postnatal neurons; and 2) that this neurotrophic effect is exerted mainly by means of specific molecules bound to the tanycytic plasmalemma limiting membrane and/or to the extracellular matrix. © 1996 Wiley-Liss, Inc.     

High- and low-voltage activated calcium currents are expressed by neurons cultured from embryonic rat neostriatum

Current-clamp studies have shown that voltage-dependent Ca currents are present in rat neostriatal neurons. Although these studies have provided evidence for the presence of high-voltage activated Ca channels, it has been unclear whether low-voltage activated channels are also present. Using the whole-cell variant of the patch-clamp technique, we have studied isolated Ca currents in an attempt to answer this question. We have found that both high- and low-voltage activated calcium currents are expressed by neostriatal neurons cultured from embryonic rat brain. These currents are similar in voltage-dependence and pharmacology to those found in other brain neurons.     

Development of voltage-dependent calcium, sodium, and potassium currents in Xenopus spinal neurons

Action potentials of embryonic nerve and muscle cells often have a different ionic dependence and longer duration than those of mature cells. The action potential of spinal cord neurons from Xenopus laevis exhibits a prominent calcium component at early stages of development that diminishes with age as the impulse becomes principally sodium dependent. Whole-cell voltage-clamp analysis has been undertaken to characterize the changes in membrane currents during development of these neurons in culture. Four voltage-dependent currents of cells were identified and examined during the first day in vitro, when most of the change in the action potential occurs. There are no changes in the peak density of the calcium current (ICa), its voltage dependence, or time to half-maximal activation; a small increase in inactivation is apparent. The major change in sodium current (INa) is a 2-fold increase in its density. In addition, more subtle changes in the kinetics of the macroscopic sodium current were noted. The peak density of voltage-dependent potassium current (IKv) increases 3-fold, and this current becomes activated almost twice as fast. No changes were noted in the extent of its inactivation. The calcium-dependent potassium current (IKc) consists of an inactivating and a sustained component. The former increases 2-fold in peak current density, and the latter increases similarly at less depolarized voltages. The changes in these currents contribute to the decrease in duration and the change in ionic dependence of the impulse.     

Stretch-activated cation channels of leech neurons: characterization and role in neurite outgrowth.

The goal of this study was to characterize the stretch-activated ion channels (SACs) of adult identified neurons of the leech Hirudo medicinalis and to test the role of SACs in neurite outgrowth of isolated cells. Using cell-attached patch recording, we established that SACs are densely distributed in the growth cone membrane of cultured neurons. In excised patches, we found that these channels are permeable to Ca2+, as well as to monovalent cations. The channels are blocked by the extracellular application of gadolinium (Gd3+), amiloride and gentamicin. Amiloride and gentamicin, respectively, induce a partial and complete voltage-dependent block. Time-lapse video recordings of neurite outgrowth from single cultured neurons were used to study the effects of blocking SACs with gentamicin. Within 20 h of plating in the presence of the aminoglycoside, the total length of neuronal arborization was significantly greater than that measured in its absence. The amount of assembled axon per unitary surface area remained constant over 40 h and did not differ significantly with or without gentamicin. Our findings show that SACs of leech neurons admit Ca2+, are densely distributed in the growth cone membrane and exhibit typical pharmacological features of mechanotransducer ion channels. In addition, our data suggest that these cation channels participate in the early interaction between growing neurites and culture substrate.     

Effects of low calcium and calcium antagonists on skeletal muscle staircase and fatigue

The purpose of this investigation was to determine the effects of low extracellular calcium and calcium antagonists on skeletal muscle staircase and fatigue. Initial experiments revealed that, brief exposure (10 minutes) of single frog sartorius muscle to diltiazem, D-600 (5 and 30 microM) and low calcium Ringer's solution (LCR, calcium replaced by magnesium and EGTA) had little effect on isometric twitches evoked every 30 seconds. However, when stimulated at 1 per second for 15 minutes, the calcium antagonists significantly decreased the magnitude and time course of the staircase, whereas LCR decreased only the time course. Each experimental condition significantly increased the rate of fatigue while diltiazem and D-600 both increased the magnitude of fatigue. Following the stimulation period, caffeine (10 mM) elicited contractures from all muscles whereas high potassium (180 mM) elicited contractures from control muscles only. These results indicate that calcium channel antagonists depress the skeletal muscle staircase response. They also indicate that these compounds as well as LCR enhance the fatigue process. Extracellular calcium influx may therefore have some influence on skeletal muscle twitches during prolonged repetitive activity.     

Sirt1 overexpression in neurons promotes neurite outgrowth and cell survival through inhibition of the mTOR signaling

The mammalian nicotinamide-adenine dinucleotide (NAD)-dependent deacetylase Sirt1 impacts different processes involved in the maintenance of brain integrity and in the pathogenic pathways associated with several neurodegenerative disorders, including Alzheimer's disease. Here we used human Sirt1 transgenic mice to demonstrate that neuron-specific Sirt1 overexpression promoted neurite outgrowth and improved cell viability under normal and nutrient-limiting conditions in primary culture systems and that Sirt1-overexpressing neurons exhibited higher tolerance to cell death or degeneration induced by amyloid-β1-42 oligomers. Coincidentally, we found that enhanced Sirt1 expression in neurons downregulated the mammalian target of rapamycin (mTOR) protein levels and its phosphorylation without changes in its mRNA levels, which was accompanied by concomitant inhibition of the mTOR downstream signaling activity as revealed by decreased p70S6 kinase (p70S6K) phosphorylation at Thr389. Consistently with this, using a Sirt1 siRNA transfection approach, we observed that reduction of endogenous mouse Sirt1 led to increased levels of mTOR and phosphorylation of itself and p70S6K as well as impaired cell survival and neurite outgrowth in wild-type mouse primary neurons, corroborating a suppressing effect of mTOR by Sirt1. Correspondingly, the mTOR inhibitor rapamycin markedly improved neuronal cell survival in response to nutrient deprivation and significantly enhanced neurite outgrowth in wild-type mouse neurons. The protective effect of rapamycin was extended to neurons even with Sirt1 siRNA knockdown that displayed developmental abnormalities compared with siRNA control-treated cells. Collectively, our findings suggest that Sirt1 may act to promote growth and survival of neurons in the central nervous system via its negative modulation of mTOR signaling.     

BDNF,NT-3对体外培养的胚鼠脊髓AChE和NADPH-d阳性神经元生长发育的影响

利用AChE和NADPH-d酶组织化学染色法研究了脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)和神经营养因子-3(neurotrophin-3,NT-3)对离体培养的胚胎大鼠脊髓胆碱能神经元和一氧化氮能神经元生长发育的影响.结果显示:BDNF处理组和NT-3处理组AChE阳性神经元数和NADPH-d阳性神经元数均显著高于对照组(P＜0.05).BDNF组AChE阳性神经元和NADPH-d阳性神经元胞体平均直径、每细胞突起数和最长突起长度均显著高于对照组(P＜0.05).NT-3组NADPH-d阳性神经元的生长发育与对照组无明显差异,仅AChE阳性神经元的每细胞突起数和最长突起长度显著高于对照组(P＜0.05),对胞体发育无影响.结果提示:BDNF,NT-3促进脊髓神经元的存活和生长发育,二者的作用具有选择性和特异性.     

一氧化氮供体SNP和DEA对培养海马神经元L-型钙电流的影响

目的观察NO供体DEA和SNP对大鼠海马培养神经元L-型钙电流的影响并对其机理进行初步探讨。方法L-型钙电流用膜片钳的全细胞模式进行记录。结果DEA和SNP这二种NO供体的主要效应为抑制作用。3 mmol/L DEA可抑制L-型钙电流,当电压去极化至10mV时,电流被抑制了29%。SNP对通道也主要起抑制作用,并且呈现剂量依赖性。当用NEM预处理以阻断S-亚硝化通路后,SNP仍对L-型钙电流产生相似的抑制作用,表明S-亚硝化机制不参与该调控作用。用10μmol/L ODQ预处理以阻断cGMP途径后,SNP仍对通道有抑制作用,表明存在另一种非cGMP通路的抑制性机制。结论上述结果表明,NO供体DEA和SNP对海马神经元L-型钙电流具有抑制作用,其作用机理主要是通过与cGMP途径和S-亚硝化修饰无关的途径。     

Effects of cool storage on survival and function of intrastriatal ventral mesencephalic grafts

Ongoing clinical trials with fetal tissue transplants in Parkinson's disease would be facilitated by an effective tissue storage technique that would allow for temporal separation of the procurement of the fetal donor tissue and implantation surgery. In order to develop such a method, we grafted rat or human fetal ventral mesencephalic tissue to the dopamine-depleted striatum of rats either directly, or following pregraft refrigeration in a 'hibernation' medium at 4 °C. Rat tissue transplants were found to normalize amphetamine-induced circling behavior at 6 weeks post-transplantation after having been hibernated for either 2 or 5 days. The number of tyrosine hydroxylase immunoreactive neurons in these hibernated grafts did not differ significantly from that found in matched grafts of fresh tissue. Hibernation for 10 days resulted both in an absence of functional effects and in decreases of graft survival down to 10-20% of control values. Volume assessment of fresh and hibernated grafts prepared from human fetal tissue revealed no adverse effects of a 3 day hibernation interval at 3 weeks after transplantation into immunosuppressed rats. The results indicate that hibernation of neural tissue may be a convenient and simple tool, which can help to guarantee tissue availability at the planned time of implantation in patients and facilitate transport and bacteriological examination. Furthermore, the method offers a simple means which permits prolonged exposure of the neural tissue to trophic factors and specific markers prior to grafting in experimental animals.