Networks of Cultured iPSC-Derived Neurons Reveal the Human Synaptic Activity-Regulated Adaptive Gene Program

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Expression of Biologically Active Basic Fibroblast Growth Factor by Genetically Modified Rat Primary Skin Fibroblasts

Abstract: Basic fibroblast growth factor (FGF-2) is normally expressed as a cell-associated protein, and accordingly it is not clear how it exerts its action on target cells in vivo. It has been proposed that cells release, by death or other mechanisms, small amounts of FGF-2 that then acts in an autocrine manner. To address the question of whether it is necessary that FGF-2 remain cell associated or needs to be secreted from cells to have biological activity, we expressed the 18-kDa form of FGF-2 in primary fibroblasts as a cell-associated (FGF-2-B) or as a secreted (FGF-2-S) protein. FGF-2 protein is detected in cell lysates and membrane fractions of both cell types, whereas it is present in significant amounts only in the conditioned medium of FGF-2-S cells. No FGF-2 is detected in control (untransfected) cells. FGF-2-S cells also grow faster than the control or FGF-2-B cells. Yet, when evaluated for their ability to promote the survival of embryonic hippocampal neurons in vitro, both the cell types are active, establishing the activity of the transgene product. We conclude that FGF-2 is active when engineered to be expressed as a cell-associated form or secreted from cells.     

硒对铅暴露致神经损伤的拮抗作用研究

目的:研究铅暴露诱导的神经毒性损伤作用,明确铅暴露引发神经毒性损伤的部分机制以及硒的保护作用。方法:通过哺乳期染铅及补充硒建立铅暴露动物模型;通过TUNEL实验确定铅暴露引发的神经损伤;通过Western blot实验检测Bax、Bcl-2、Caspase-3水平确定铅暴露对凋亡途径的启动;并确证补硒在铅神经毒性作用下对机体的保护作用。结果:1.哺乳期铅暴露能够引起仔鼠海马神经细胞凋亡的发生;2.铅暴露能够诱导Bax/Bcl-2水平改变,激活Caspase-3。3.同时给予硒干预后,机体抗铅神经毒性能力显著增加。结论:1.铅暴露能够诱导海马部位神经毒性损伤,损伤可能通过启动凋亡途径而发生,2.补硒能够通过拮抗凋亡发生从而拮抗铅的神经毒性,产生保护作用。     

Tachykinins Potentiate N-Methyl-D-Aspartate Responses in Acutely Isolated Neurons from the Dorsal Horn

Abstract: Substance P and neurokinin A both potentiated N -methyl- d -aspartate (NMDA)-induced currents recorded in acutely isolated neurons from the dorsal horn of the rat. To elucidate the mechanism underlying this phenomenon, we measured the effects of tachykinins and glutamate receptor agonists on [Ca²⁺]_i in these cells. Substance P, but not neurokinin A, increased [Ca²⁺]_i in a subpopulation of neurons. The increase in [Ca²⁺]_i was found to be due to Ca²⁺ influx through voltage-sensitive Ca²⁺ channels. Substance P and neurokinin A also potentiated the increase in [Ca²⁺]_i produced by NMDA, but not by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, kainate, or 50 m M K⁺. Phorbol esters enhanced the effects of NMDA and staurosporine inhibited the potentiation of NMDA effects by tachykinins. It is concluded that activation of protein kinase C may mediate the enhancement of NMDA effects by tachykinins in these cells. However, the effects of tachykinins on [Ca²⁺]_i can be dissociated from their effects on NMDA receptors.     

大蹄蝠回声定位信号特征与下丘神经元频率调谐

采用超声监测仪录制超声信号和细胞外电生理记录下丘神经元的频率调谐曲线(frequency tuningcurqes,FTCs)的方法,探讨了大蹄蝠(Hipposideros armiger)回声定位信号与下丘(inferior colliculus,IC)神经元频率调谐之间的相关性.结果发现,大蹄蝠回声定位叫声为恒频-调频(consrant frequency-frequenevmodulated,CF-FM)信号,一般含有2-3个谐波,第二谐波为其主频,cF成分频率(Mean±SD,n=18)依次为:(33.3 4±0.2)、(66.5±0.3)、(99.4 4±0.5)kHz;电生理实验共获得72个神经元的频率调谐曲线,Q10-dB值的范围是0.5-95.4(9.2±14.6,rg=72),最佳频率(best frequency,BF)在回声定位主频附近的神经元具有尖锐的频率调谐特性.结果表明,大蹄蝠回声定位信号与下丘神经元频率调谐存在相关性,表现为最佳频率在回声定位信号主频附近的神经元频率调谐曲线的Q10-dB值较大,具有很强的频率分析能力.     

Organization of Synaptic Inputs to Rubro-Spinal Neurons in the Cat: Interaction of the Effects of Stimulation of the Cerebellar Nucl. Interpositus and Thalamic Nucl. Ventrolateralis

We studied the interaction of synaptic effects in efferent (rubro-spinal) neurons (RSN) of the red nucleus (NR) of the cat brain using intracellular recording and different variants of stimulation of the cerebellar nucl. interpositus (NI) and nucl. ventrolateralis (VL) of the thalamus. Combined simultaneous or consequent stimulation of the above two inputs allowed us to use collision of the impulses in pre-synaptic pathways for estimation of the peculiarities of summation of post-synaptic potentials; it was taken into consideration that cerebello-rubral fibers are collaterals of the cerebello-thalamic axons. In the case of relatively low intensities of stimulation of the VL and NI, EPSP evoked by these stimulations in RSN could demonstrate linear summation, which is indicative of the absence of common fibers excited by stimulation of both the thalamic and cerebellar structures and of the absence of interaction of synaptic effects on the membrane of the postsynaptic unit (an RSN). With increased stimulation intensity and definite time relations between the stimuli applied to the above structures, the second EPSP evoked by the two stimuli was significantly suppressed; this resulted from a decrease of the afferent drive coming to the NR due to collision of afferent impulses in presynaptic pathways. When an interstimulus interval was increased, the second EPSP could be facilitated (probably due to generation of repetitive impulses in common presynaptic fibers). Our experiments show that the statement concerning the nature of cerebello-rubral inputs (all these inputs are formed by collaterals of the cerebello-(interposito)-thalamic pathways) should be considered with reservation.     

Predominant Neuritic Pathology Induced by Viral Overexpression of <Emphasis Type="Bold">α</Emphasis>-Synuclein in Cell Culture

1. Alpha-synuclein is known to play an important role in the pathogenesis of Parkinson’s disease (PD). The pathogenicity of α-synuclein is related to its ability to form intraneuronal inclusions. The inclusions, which are found in brains of patients with PD and diffuse Lewy body disease consist partially of C-terminally truncated α-synuclein. This α-synuclein species has an increased ability to form aggregates compared to full length α-synuclein. 2. We have used an adeno-associated virus (AAV) vector system to overexpress either C-terminally truncated or full length α-synuclein containing the A53T mutation, which have both been identified in brains of familial PD patients and transgenic mouse models. Dissociated mesencephalic neurons, cerebellar granule neurons, and organotypic midbrain slice cultures were infected with AAV containing the transgene under the control of the cytomegalovirus promoter. 3. We demonstrate that viral overexpression of α-synuclein(A53T) leads to the formation of distorted neurites, intraneuritic swellings, and granular perikaryal deposits in cultured neurons. Our results indicate that these cell culture models may represent an early phase of PD reflecting pathologic neuritic alterations before significant neuronal cell loss occurs.     

An oxidative stress-mediated positive-feedback iron uptake loop in neuronal cells

Intracellular reactive iron is a source of free radicals and a possible cause of cell damage. In this study, we analyzed the changes in iron homeostasis generated by iron accumulation in neuroblastoma (N2A) cells and hippocampal neurons. Increasing concentrations of iron in the culture medium elicited increasing amounts of intracellular iron and of the reactive iron pool. The cells had both IRP1 and IRP2 activities, being IRP1 activity quantitatively predominant. When iron in the culture medium increased from 1 to 40 microm, IRP2 activity decreased to nil. In contrast, IRP1 activity decreased when iron increased up to 20 microm, and then, unexpectedly, increased. IRP1 activity at iron concentrations above 20 microm was functional as it correlated with increased (55) Fe uptake. The increase in IRP1 activity was mediated by oxidative-stress as it was largely abolished by N-acetyl-L-cysteine. Culturing cells with iron resulted in proteins and DNA modifications. In summary, iron uptake by N2A cells and hippocampus neurons did not shut off at high iron concentrations in the culture media. As a consequence, iron accumulated and generated oxidative damage. This behavior is probably a consequence of the paradoxical activation of IRP1 at high iron concentrations, a condition that may underlie some processes associated with neuronal degeneration and death.     

Pituitary adenylate cyclase-activating polypeptide is up-regulated in cortical pyramidal cells after focal ischemia and protects neurons from mild hypoxic/ischemic damage

The protective effect of pituitary adenylate cyclase-activating polypeptide (PACAP) in stroke models is poorly understood. We studied patterns of PACAP, vasoactive intestinal peptide, and the PACAP-selective receptor PAC1 after middle cerebral artery occlusion and neuroprotection by PACAP in cortical cultures exposed to oxygen/glucose deprivation (OGD). Within hours, focal ischemia caused a massive, NMDA receptor (NMDAR)-dependent up-regulation of PACAP in cortical pyramidal cells. PACAP expression dropped below the control level after 2 days and was normalized after 4 days. Vasoactive intestinal peptide expression was regulated oppositely to that of PACAP. PAC1 mRNA showed ubiquitous expression in neurons and astrocytes with minor changes after ischemia. In cultured cortical neurons PACAP27 strongly activated Erk1/2 at low and p38 MAP kinase at higher nanomolar concentrations via PAC1. In astrocyte cultures, effects of PACAP27 on Erk1/2 and p38 were weak. During OGD, neurons showed severely reduced Erk1/2 activity and dephosphorylation of Erk1/2-regulated Ser112 of pro-apoptotic Bad. PACAP27 stimulation counteracted Erk1/2 inactivation and Bad dephosphorylation during short-term OGD but was ineffective after expanded OGD. Consistently, PACAP27 caused MEK-dependent neuroprotection during mild but not severe hypoxic/ischemic stress. While PACAP27 protected neurons at 1–5 nmol/L, full PAC1 activation by 100 nmol/L PACAP exaggerated hypoxic/ischemic damage. PACAP27 stimulation of astrocytes increased the production of Akt-activating factors and conferred ischemic tolerance to neurons. Thus, ischemia-induced PACAP may act via neuronal and astroglial PAC1. PACAP confers protection to ischemic neurons by maintaining Erk1/2 signaling via neuronal PAC1 and by increasing neuroprotective factor production via astroglial PAC1.