Otic placode cell specification and proliferation are regulated by Notch signaling in avian development

Background: The entire inner ear including the cochlear‐vestibular ganglion arises from a simple epithelium, the otic placode. Precursors for the placode originate from a pool of progenitors located in ectoderm next to the future hindbrain, the pre‐otic field, where they are intermingled with future epibranchial and epidermal cells. While the importance of secreted proteins, such as FGFs and Wnts, in imparting otic identity has been well studied, how precursors for these different fates segregate locally is less well understood. Results: (1) The Notch ligand Delta1 and the Notch target Hes5‐2 are expressed in a part of pre‐otic field before otic commitment, indicative of active Notch signaling, and this is confirmed using a Notch reporter. (2) Loss and gain‐of‐function approaches reveal that Notch signaling regulates both proliferation and specification of pre‐otic progenitors. Conclusions: Our results identify a novel function of Notch signaling in cell fate determination in the pre‐otic field of avian embryos. Developmental Dynamics 244:839–851, 2015. © 2015 Wiley Periodicals, Inc.     

Identification of two populations of corticothalamic neurons in cat primary somatosensory cortex

Summary Extracellular and intracellular recordings of corticothalamic (CT) cells were performed in the primary somatosensory cortex of the cat. CT neurons were antidromically activated by electrically stimulating the ventroposterior lateral (VPL) nucleus of the thalamus and were classified into two types according to their physiological properties. Type 1 had no spontaneous activity and no identifiable somatic receptive field. Type 2 fired action potentials spontaneously and responded to mechanical stimulation of the skin or underlying tissues. Axonal conduction velocities were slower for type 1 cells and their cell bodies were located slightly deeper in the cortex than those of type 2 cells. Both types of CT neurons exhibited inhibitory postsynaptic potentials in response to VPL stimulation but an early synaptic excitation and rebound discharge was observed almost exclusively in type 2 cells. These results suggest that only type 2 CT cells can modify the activity of thalamic neurons through a corticothalamic feedback loop.     

Glutamate-like immunoreactive structures in primary sensory neurons in the rat detected by a specific antiserum against glutamate

Summary We found that large cells in the dorsal root and trigeminal ganglia contained glutamate-like immunoreactivity. Immunoreactive neurons were not detected in the superior cervical or pterygopalatine ganglia. These findings indicated that glutamate is a neurotransmitter or neuromodulator of large cells of sensory ganglia.     

Morphological arrangement between astrocytes and trigeminal mesencephalic primary afferent neurons in the rat

Summary The arrangement between astrocytes and the primary afferent neurons of the mesencephalic trigeminal nucleus (Me5) was analyzed in a light and electron microscopy immunocytochemistry study using anti-GFAP antibodies. It appears that each Me5 neuron is almost entirely sheathed with astrocytic processes radiating out from two or more astrocytes. Ultrastructural observations confirmed the embracement of Me5 neurons by astrocytic processes that only allowed some synaptic contacts on the neuronal surface. The possible functional significance of this intimate morphological relationship for the mesencephalic trigeminal neurons is discussed.     

Early morphological changes of primary afferent neurons and their processes in newborn mice after treatment with capsaicin

Degenerating figures of dorsal root ganglion (DRG) neurons and their central and peripheral processes (dorsal root and saphenous nerve) and terminals (central terminals in the superficial dorsal horn and cutaneous nerve of the hind paw dorsal skin) of neonatal mice were examined 30 min, 1, 2 and 5 h, and 2, 3, 5, and 10 days after subcutaneous injection of capsaicin on post-natal day 2. Many small DRG neurons showed degeneration 1 h after treatment. Scarcely any features of degeneration were seen in the DRG and dorsal root 10 days after treatment. The degenerating aspects of terminal axons in the marginal layer of the superficial dorsal horn were characterized by enlarged round axons with closely packed osmiophilic materials, lamellar bodies, and loss of axoplasmic organelles. Two types of central terminals (C-terminals) showed degeneration in the substantia gelatinosa from 30 min after treatment onward. One type consisted of small, round, sinuous or slender dark terminals (CI-terminals), and the other of large, pale, round or angular terminals (CII-terminals). Those that degenerated markedly had homogeneously electron-dense axoplasm with dilated synaptic vesicles and inclusion bodies. Extensive degeneration of terminal axons in the marginal layer occurred 5 h after treatment, whereas conspicuous degeneration of C-terminals occurred from 30 min to 10 days after treatment in the substantia gelatinosa. CI-terminals showed marked degeneration during the first 3 days, whereas marked degeneration of CII-terminals occurred between 5 and 10 days after treatment. This time difference between the peaks of degeneration of CI- and CII-terminals indicates an important difference in the origins of these two types of capsaicin-sensitive, nociceptive fibers in the superficial dorsal horn; CI-terminals are derived from small DRG cells, whereas CII-terminals are derived from larger DRG cells. Unmyelinated axons in the dorsal root, saphenous nerve, and dorsal skin of the hind paw showed similar degeneration patterns 2 h after treatment to those of terminal axons in the marginal layer. Thus, the degenerating profiles in the marginal layer suggest that these axons arose from collaterals of unmyelinated primary axons descending or ascending within the marginal layer. Numerous enlarged degenerating axons showing vacuolation were conspicuous in the dorsal skin 3 days after treatment. The synchronous degeneration of the smaller DRG neurons, their central and peripheral processes, and their CI-terminals in the substantia gelatinosa supports the idea that the smaller DRG neurons are directly influenced by capsaicin, and that their degeneration is followed by centrifugal degeneration.     

4-PBA通过抑制内质网应激减轻原代海马神经元的损伤

目的:探讨内质网应激后原代海马神经元树突棘密度及突触蛋白表达的变化,以及通过内质网应激分子伴侣4-苯基丁酸(4-phenylbutyric acid,4-PBA)抑制内质网应激对这种神经元损伤的抑制作用。方法:原代培养新生大鼠海马神经元,将表达增强型绿色荧光蛋白的质粒转染到原代培养5～7 d(DIV 5～7)的大鼠海马神经元内持续培养,DIV 20时分为对照组、衣霉素(tunicamycin,Tm)处理组和Tm+4-PBA预处理组(Tm处理前1 h给予4-PBA),采用Western blot法检测内质网应激标志蛋白Bi P和突触蛋白的表达水平,激光共聚焦显微镜下观察神经元,分析树突棘密度,采用MTT法分析细胞活力。结果:Tm处理后使Bi P蛋白水平明显升高,而4-PBA预处理使Bi P蛋白水平显著下降(P 0. 05)。Tm引起的原代海马神经元树突棘密度下降及突触蛋白的表达下降能够被4-PBA抑制。Tm引起的细胞活力下降可被4-PBA抑制。结论:Tm能够通过诱导内质网应激而引起原代海马神经元树突棘密度下降及突触蛋白表达下降,而提前给予4-PBA预处理可明显降低内质网应激反应,抑制树突棘密度下降及突触蛋白表达下降,从而减轻原代海马神经元的损伤。     

大鼠皮层神经细胞糖氧剥离体外模型的建立及细胞形态学观察

目的本研究利用培养大鼠皮层神经细胞,观察化学缺氧剂及无糖培养基对神经细胞的急性损伤作用,以建立一简单有效且稳定的神经细胞缺血缺氧模型。方法SD大鼠胎鼠大脑皮层神经元培养,连二亚硫酸钠消除培养基中的氧合并培养基缺糖建立神经细胞缺血缺氧模型,光镜下观察。结果在大剂量耗氧剂以及无糖培养基协同作用下,在较短的时间内就可造成神经细胞的损伤。结论连二亚硫酸钠加无糖培养基制造体外神经细胞损伤是一种行之有效的糖氧剥离模型。     

NMDA对新生大鼠皮层神经元的兴奋毒作用

目的:建立NMDA诱导原代培养皮层神经元兴奋毒损伤模型,探讨NMDA对NMDA受体过度活化诱导兴奋性神经毒的可能途径。方法:原代培养新生大鼠大脑皮层神经元,通过倒置显微镜形态学观察、细胞活力检测(MTT及LDH释放的检测)及胞内Ca2+的动态测定,探索NMDA诱导毒性作用的适当浓度及时间。通过对ROS、NO检测,分析NMDA诱导毒性作用于线粒体的损伤情况。结果:NMDA(100μmol/L/2 h)引起皮层神经元形态学改变,且引起神经元细胞活力时间和浓度依赖性的下降,由同时伴随LDH释放增加(P<0.05),ROS和NO的生成量明显增加(P<0.05),皮层神经元内Ca2+的快速升高,并维持在高水平。结论:NMDA诱发皮层神经元明显的细胞毒性作用,提示NMDA过度活化NMDA受体后通过神经元膜内Ca2+超载造成ROS和NO的生成量增加,导致皮层神经元产生毒性损伤。     

Glycinergic interneurons are functionally integrated into the inspiratory network of mouse medullary slices

Neuronal activity in the respiratory network is functionally dependent on inhibitory synaptic transmission. Using two-photon excitation microscopy, we analyzed the integration of glycinergic neurons in the isolated inspiratory pre-Bötzinger complex-driven network of the rhythmic slice preparation. Inspiratory (96%) and ‘tonic’ expiratory neurons (4%) were identified via an increase or decrease, respectively, of the cytosolic free calcium concentration during the inspiratory-related respiratory burst. Furthermore, in BAC-transgenic mice expressing EGFP under the control of the GlyT2-promoter, 50% of calcium-imaged inspiratory neurons were glycinergic. Inspiratory bursting of glycinergic neurons in the slice was confirmed by whole-cell recording. We also found glycinergic neurons that receive phasic inhibition from other glycinergic neurons. Our calcium imaging data show that glycinergic neurons comprise a large population of inspiratory neurons in the pre-Bötzinger complex-driven network of the rhythmic slice preparation.     

Differential effects of distal and proximal nerve lesions on carbonic anhydrase activity in rat primary sensory neurons,ventral and dorsal root axons

Summary The effect of proximal and distal peripheral nerve injuries on the histochemistry of carbonic anhydrase (CA) in rat dorsal root ganglion (DRG) neurons, and myelinated (MyF) dorsal and ventral root fibers was studied. Sciatic neurectomy induced no change. Contrariwise, 7 days after lumbar spinal nerve section the numbers of CA-stained ventral root MyF and DRG cells at the L₄ and L₅ levels decreased to 73.2% and 51.9% of their original values respectively, although the numbers returned to normal by the 90th postoperative day. Dorsal root MyF followed a similar trend, albeit with some delay. Major morphological changes comprised atrophy of dorsal root sensory neurons and axons, particularly in long term experiments, as well as nuclear eccentricity in DRG neurons. These results suggest that, depending on the site of lesion, the rat peripheral nervous system (PNS) either maintains or quickly restores its capacity to synthesize CA. They stand in contrast to the long-lasting metabolic dysfunctions reported to occur when primary neurons are disconnected from the periphery. It is uncertain whether this difference is due to the critical role of CA in neuronal metabolism.     

Transcallosally mediated inhibition of interneurons within human primary motor cortex

The objective of this study was to investigate interhemispheric transcallosal connections between primary motor cortices noninvasively in awake human subjects. For this purpose, focal transcranial magnetic stimulation was performed on eight healthy, right-handed subjects and one patient with congenital collosal agenesis. Using two magnetic stimulators, we investigated the effect of a conditioning magnetic stimulus applied to the motor cortex of one hemisphere on the duration of the silent period (SP) evoked in the first dorsal interosseus (FDI) muscle by a magnetic test stimulus given over the opposite motor cortex. It is well established that SP reflects activation of inhibitory interneurons within primary motor cortex. In all normal subjects, a conditioning stimulus to one hemisphere produced a significant shortening of SP evoked by the test stimulus when the conditioningtest-interval was 10–20 ms. The effect was also observed when an electrical test stimulus was used. The conditioning coil had to be placed over the hand motor area to obtain the maximal effect. The threshold for eliciting this decrease of SP duration was higher than the threshold for eliciting an early excitatory muscle response in the contralateral FDI. Increasing the intensity of the conditioning stimulus led to linear reduction of SP duration. In the patient with callosal agenesis, no such decreasing effect on SP duration was observed. These results suggest that inhibitory interneurons within primary hand motor cortex receive transcallosal inhibitory input from the opposite motor cortex. We propose that modulation of motor cortical interneurons via transcallosal pathways may provide a gain control for the motor cortical output system and subserve interhemispheric coordination in complex, nonsymmetrical bimanual movements.     

新生大鼠海马神经元原代无血清培养与鉴定

 摘要:目的 建立一种简单、易行的海马神经元无血清体外培养方法以获得高纯度、高活力的海马神经元。方法 取新生24h内SD大鼠,分离海马,经消化后种植于包被有Matrigel基膜的玻片上,24h后换含有N2、B27的neurobasal无血清培养基,于不同时间在倒置相差显微镜下观察细胞的生长状态;采用β-tublinⅢ免疫荧光细胞化学技术鉴定海马神经元纯度。结果 大部分海马神经元于3-24h贴壁且可长出细长突起,3d细胞具有典型神经元的形态特征, 5d神经突起进一步增多并形成稠密的网络连接,7d神经元胞体丰满,趋于成熟。经β-tublinШ免疫荧光技术鉴定纯度为（94.2±3.6）%。结论 此方法简单有效,可获得高纯度的海马神经元。     

番茄红素对原代小鼠皮质神经元的保护作用及机制研究

目的:观察番茄红素(lycopene)对氧化损伤的原代皮质神经元的保护效应并探讨其作用机制。方法:采用原代细胞培养技术体外分离培养小鼠皮质神经元,通过免疫荧光染色法检测微管相关蛋白2(microtubuleassociated protein 2,MAP-2)的表达进行鉴定。将神经元分为4组:正常神经元组、叔丁基过氧化氢(tert-butyl hydroperoxide,t-BHP)处理组、t-BHP+lycopene处理组和lycopene处理组,培养24 h,采用MTT法检测各组神经元的活力;采用流式细胞技术检测各组神经元内ROS的水平;Western blot法检测各组神经元Bax、Bcl-2、caspase-3、cleaved caspase-3及细胞色素C蛋白表达的变化。结果:Lycopene能明显提高t-BHP处理的神经元活性,降低tBHP处理的神经元内ROS含量,同时上调Bcl-2蛋白的表达,降低Bax、cleaved caspase-3和细胞色素C蛋白的表达(P0.05)。结论:Lycopene能够对抗t-BHP诱导的原代皮质神经元的损伤,抑制神经元凋亡,其机制可能与降低神经元内ROS的水平及上调的表达有关。     

Suppression of neurite outgrowth of primary cultured hippocampal neurons is involved in impairment of glutamate metabolism and NMDA receptor function caused by nanoparticulate TiO2

Numerous studies have indicated that nano-titanium dioxide (TiO₂) can induce neurotoxicity in vitro and in vivo, however, it is unclear whether nano-TiO₂ affects neurite outgrowth of hippocampal neurons. In order to investigate the mechanism of neurotoxicity, rat primary cultured hippocampal neurons on the fourth day of culture were exposed to 5, 15, and 30 μg/mL nano-TiO₂ for 24 h, and nano-TiO₂ internalization, dendritic growth, glutamate metabolism, expression of N-methyl-d-aspartate (NMDA) receptor subunits (NR1, NR2A and NR2B), calcium homeostasis, sodium current (I_Na) and potassium current (I_K) were examined. Our findings demonstrated that nano-TiO₂ crossed the membrane into the cytoplasm or nucleus, and significantly suppressed dendritic growth of primary cultured hippocampal neurons in a concentration-dependent manner. Furthermore, nano-TiO₂ induced a marked release of glutamate to the extracellular region, decreased glutamine synthetase activity and increased phosphate-activated glutaminase activity, elevated intracellular calcium ([Ca²⁺]i), down-regulated protein expression of NR1, NR2A and NR2B, and increased the amplitudes of the I_Na and I_K. In addition, nano-TiO₂ increased nitric oxide and nitrice synthase, attenuated the activities of Ca²⁺-ATPase and Na⁺/K⁺-ATPase, and increased the ADP/ATP ratio in the primary neurons. Taken together, these findings indicate that nano-TiO₂ inhibits neurite outgrowth of hippocampal neurons by interfering with glutamate metabolism and impairing NMDA receptor function.     

Functional applications of novel Semliki Forest virus vectors are limited by vector toxicity in cultures of primary neurons in vitro and in the substantia nigra in vivo

The Semliki Forest virus (SFV) system has been shown to be highly efficient in transduction of cell lines and primary cells. We employed a novel noncytotoxic SFV(PD) vector for transduction of primary ventral midbrain floor cultures in vitro and rat substantia nigra in vivo. Rapid protein expression was noted with preferential transduction of neuronal cells including the dopaminergic subpopulation. To examine the suitability of the SFV vector system for functional gene expression, SFV(PD) vectors encoding for antiapoptotic proteins Bcl-X_L and XIAP were designed. Despite effective transgene expression, SFV(PD) vectors were unable to rescue dopaminergic neurons from MPP⁺-induced apoptosis. In vivo, virus injection into substantia nigra resulted in fast onset of transgene expression, but elicited an activation of microglia and an inflammation response. We conclude that the use of novel SFV(PD) vectors is currently limited by persistent neurotoxicity of the vector system. Although SFV(PD) vectors may be useful for protein localization studies in dopaminergic neurons, functional applications will require the development of even less cytopathic vector systems.Funded by Deutsche Forschungsgemeinschaft through the DFG Research Center for Molecular Physiology of the Brain     

原代培养海马神经元Drebrin和Synaptophysin的表达与动态变化

目的:观察原代海马神经元不同时期树突棘的变化以及树突棘蛋白Drebrin和突触囊泡膜蛋白SYN的表达水平。方法:光镜观察原代培养7、14和20 d的海马神经元的形态改变。应用免疫荧光对Drebrin和SYN进行染色,观察树突棘形态和数量的改变。进一步应用Western Blot检测Drebrin和SYN蛋白的表达变化。结果:培养7 d的海马神经元,未见树突棘结构,SYN斑点状阳性产物较少。随着海马神经元培养时间的加长,突起数量逐渐增加,14 d和20 d的树突棘数量和SYN阳性产物均显著增加。Drebrin和SYN蛋白表达水平也随培养时间的加长而显著增加。结论:原代培养的海马神经元在14 d已有树突棘形成,20 d显示出成熟树突棘的形态和数量。Drebrin和SYN的蛋白表达水平反映了树突棘和突触的形成和功能状态。     

肝细胞生长因子延长原代培养大鼠脊髓神经元的生存时间

目的探讨肝细胞生长因子(HGF)对原代培养脊髓神经元的保护作用。方法原代培养脊髓神经元转染不同滴度的绿色荧光蛋白或肝细胞生长因子重组腺病毒(Ad-GFP or Ad-HGF),用流式细胞仪检测转染率,PI-Hoechst双染色观察神经元生长状况。用ELISA、中性红、MTT和NSE-ELISA等方法分别检测Ad-HGF转染脊髓神经元后HGF的表达,以及HGF对神经元的保护和迁移作用。结果在转染复数(MO I)为50时,Ad-GFP可高效转染脊髓神经细胞,且细胞生长状态好。转染Ad-HGF后,HGF能有效表达,同时HGF作用后的神经元活性明显高于对照组(P<0.05)。并观察到HGF对脊髓神经元有明显促迁移作用。结论HGF对体外培养的脊髓神经元有保护和迁移作用。     

损伤性C类初级感觉神经元膜上唾液酸对其兴奋性的影响

 目的:研究坐骨神经损伤后,大鼠背根神经节(dorsal root ganglion,DRG)C类初级感觉神经元膜表面唾液酸含量变化对其电生理特性的影响。方法:制作大鼠慢性压迫性神经损伤(chronic constriction injury,CCI)痛觉模型,以正常大鼠为对照,采用胞内电生理记录法检测损伤及正常C类神经元的电生理特性,随后用Ca²⁺去中和损伤及正常C类神经元膜表面唾液酸所带负电荷或用唾液酸酶(neuraminidase,NA)分解膜表面唾液酸,观察电生理特性的变化。结果:损伤性C类神经元的静息电位(rest potential,RP)较正常C类神经元移向去极化方向,诱发动作电位(action potential,AP)发生率增加,所需阈强度减小,兴奋性增加;使用Ca²⁺和唾液酸酶使损伤性C类神经元膜电位向超极化方向移动,诱发AP所需阈强度增加,兴奋性降低。而Ca²⁺和唾液酸酶对正常C类神经元的电生理特性及兴奋性无影响。结论:损伤C类神经元膜表面唾液酸含量增加,导致其RP 去极化且兴奋性增加。