Removal of tissue plasminogen activator does not protect against neuronal degeneration in the cerebellum of the weaver mouse

Tissue plasminogen activator (tPA) is a serine protease that has been shown to be involved in neuronal degeneration. Recently, elevated cerebellar tPA has been reported in a naturally occurring mutant mouse, weaver. Weaver mice suffer extensive degeneration of cerebellar granular neurons during development, leading to severe malformation of the cerebellum as well as abnormal behavior (ataxia). The observations that the developing weaver cerebellum displays a 10-fold increase in tPA activity over wild-type and that a serine protease inhibitor was able to rescue weaver granule cells from premature death in culture suggested that tPA might mediate the death of these mutant neurons. We tested this possibility by introducing the weaver mutation into tPA-deficient mice and comparing the weaver phenotype in the presence or absence of tPA. Analysis at 28 days after birth indicates that tPA-deficient weaver mice are indistinguishable from tPA-containing weaver mice in behavior, cerebellar anatomy, histology, and laminin expression (also reported to be increased in weaver). These results suggest that removal of tPA activity from weaver mice does not protect against neuronal degeneration in the cerebellum and, thus, tPA does not appear to mediate this form of cell death.     

塞来昔布对脂多糖诱导的原代多巴胺能神经元变性的保护作用

目的研究COX-2选择性抑制剂塞来昔布对脂多糖诱导的中脑原代多巴胺能神经元变性的保护作用及其机制。方法将培养7d的孕14d SD大鼠胚胎中脑原代细胞随机分为4组:对照组、脂多糖(LPS)组(20ng/ml)、塞来昔布(20μmol/L) 脂多糖组、单纯塞来昔布组。培养72h后使用免疫荧光染色观察酪氨酸羟化酶(TH)、OX-42阳性细胞数目和形态变化,放免法测定上清液中前列腺素E2(PGE2)和肿瘤坏死因子-α(TNF-α)水平。结果塞来昔布 LPS组和LPS组比较:TH阳性细胞数目明显增多,分别为对照组的69%和48%(P<0.05);OX-42阳性细胞数目明显减少,分别为对照组的2和3.48倍(P<0.05)。形态上分析塞来昔布显著改善LPS对TH阳性细胞的损伤程度和抑制LPS诱导的小胶质细胞体积增大、形态不规则。同时抑制LPS诱导的PGE2和TNF-α含量的增加(P<0.05)。结论塞来昔布通过抑制小胶质细胞激活以及COX-2表达,减少细胞外PGE2、TNF-α水平发挥其神经保护作用。     

Resistance of hypothalamic dopaminergic neurons to neonatal 6-hydroxydopamine toxicity

We studied the effects of neonatal intracisternal administration of the 6-hydroxydopamine (6-OHDA) following desipramine pretreatment on dopaminergic (DA) neurons in the rat hypothalamus and substantia nigra by immunocytochemistry with an antiserum against tyrosine hydroxylase (TH). Neonatal intracisternal 6-OHDA injection induced almost complete loss of the TH-immunoreactivity in the substantia nigra and the caudate-putamen when examined at final (adult) stage. However, in this stage, no difference of TH-immunoreactivity was observed in hypothalamic DA neurons in the arcuate nucleus (A 12), peri ventricular area (A14), zona incerta (A 13), and posterior hypothalamic area (All). In the initial (neonatal) stage after the 6-OHDA injection, nigral DA neurons started to degenerate in 12 h and were almost completely destructed in 96 h, but hypothalamic DA neurons did not show any degenerative change at any time examined. The route of the injection (cistern, third ventricle or lateral ventricle) of the toxin did not influence the distribution of damage. These data show that 6-OHDA is not equally toxic to all brain DA neurons in neonates, and that all hypothalamic DA neuronal groups resist the toxicity of 6-OHDA, despite their anatomical and functional differences.     

Mesencephalic dopaminergic neurons expressing neuromedin K receptor (NK3): a double immunocytochemical study in the rat

By using a double immunocytochemical method we examined the distribution of dopaminergic neurons expressing neuromedin K receptor (NKR; NK₃) in the rat brain. The distribution of NKR-like immunoreactive (-LI) neurons completely overlapped that of tyrosine hydroxylase (TH)-LI neurons in the retrorubral field (A8), substantia nigra (A9), ventral tegmental area and nucleus raphe linealis (A10). Completely or partially overlapping distributions of NKR- and TH-LI neurons were found in certain regions of the hypothalamus (A11–A15) and olfactory bulb (A16). Neurons showing both NKR- and TH-like immunoreactivities, however, were only found in A8–A10: All of the NKR-LI neurons displayed TH-like immunoreactivity, and about 71–86% of the TH-LI neurons expressed NKR-like immunoreactivity. The present results provided morphological evidence for physiological modulation of dopaminergic neurons by tachykinins through NKR in A8–A10.     

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) regulates survival of midbrain dopaminergic neurons

Summary. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the EGF-family of ligands signaling via the EGF-receptor tyrosine kinase. In the present study we show that HB-EGF which is expressed in close proximity of developing mesencephalic dopaminergic neurons promotes the survival of TH-positive neurons in vitro. The survival promoting effect of HB-EGF is mediated via astroglial cells and utilizes the MAPK as well as the Akt-signaling pathway. Most notably endogenous HB-EGF significantly contributes to the survival of TH-+ neurons in control cultures, suggesting a relevant developmental role of HB-EGF for dopaminergic neurons. These findings indicate that HB-EGF may be an important molecule for developing dopaminergic neurons of the ventral midbrain. Received March 12, 2001; accepted April 27, 2001     

多巴胺能神经元保护的研究进展

一提到多巴胺（dopamine,DA）能神经元,我们就会想到帕金森病（Parkinson’SDisease,PD）。人们最初是先认识了PD,而后在对其发病机制的不断探索中,才开始慢慢解开DA能神经元的神秘面纱,并确立了DA能神经元在PD发病机制的中心地位。近年来,人们对PD的治疗逐渐倾向于神经保护治疗,除了保护DA受体、抑制DA降解及干细胞替代治疗等,保护DA能神经元是关键措施。本文先简单介绍DA能神经元的解剖生理基础,并就目前DA能神经元保护的热点研究做一综述。     

Chronic administration of (+)-amphetamine alters the reactivity of midbrain dopaminergic neurons to prefrontal cortex stimulation in the rat

Repeated intermittent administration of (+)-amphetamine produces sensitisation to many of the behavioural effects of the drug. Evidence suggests that excitatory amino acidergic projections from the prefrontal cortex (PFC) to dopaminergic (DA) neurons in the ventral midbrain may be partly involved in the maintenance of sensitisation once induced. The present study was designed to investigate whether chronic amphetamine administration produces any alteration to this input, by assessing the impact of single pulse electrical stimulation of the PFC (0.25 and 0.5 mA) on the extracellular activity of individual midbrain DA neurons in drug and vehicle treated rats. Animals were administered amphetamine according to a schedule known to produce sensitisation (2.5 mg/kg free base, once daily for 6 days; s.c.), and the effect of PFC stimulation was assessed on withdrawal days 2 and 10. In addition to single spike firing patterns, the ability of the stimulation to elicit stimulus bound (time-locked) burst events was also noted. In the majority of cases, the elicited responses could be broadly categorised into two types — ones characterised by an initial excitation (E responses) and ones characterised by excitation following an initial inhibition (IE responses). On withdrawal day 2, IE responses were affected such that, in those responses which contained time-locked bursts in their excitatory phases, the stimulus produced a time-locked burst on a greater percentage of trials. On withdrawal day 10, the principal change was that E responses were more likely to occur in amphetamine-treated animals than controls (0.25 mA; 57.1% vs. 41.2% of responses, respectively; 0.5 mA; 36.7% vs. 23.5% of responses, respectively). It is argued that an increase in the proportion of excitatory responses in drug animals indicates a potentiation of the excitatory drive to the DA neurons. Insofar as sensitisation in the longer term relies upon an enhancement of amphetamine-induced dopamine release in the forebrain, this may be one mechanism by which it is achieved.     

神经营养因子对体外培养中脑多巴胺能神经元存活和分化的影响

目的观察不同神经营养因子对体外培养中脑多巴胺能神经元(DN)存活和分化的作用。方法选取14d孕鼠,无菌条件下取出胎鼠,采用酶消化法培养中脑DN神经元,在培养过程中,分别加入不同浓度的胶质细胞源性神经营养因子(GDNF)、神经营养因子3(NT3)、脑源性神经营养因子(BDNF)和神经生长因子(NGF),通过细胞形态学和免疫荧光方法进行细胞纯度鉴定,观察不同作用条件下TH阳性细胞率确定细胞存活。结果以10~60ng/L的GDNF或BDNF持续培养10d,中脑多巴胺能神经元的存活率明显高于NGF和NT3作用组,浓度为20ng/m l的GDNF作用最强,能够维持60%的DN神经元存活。此外BDNF和GDNF能够增加DN神经元的数目,但未发现明显的剂量依赖效应,当GDNF与BDNF联合应用时,未见DN神经元的保护作用增强。结论GDNF和BDNF对原代培养的多巴胺能神经元存活具有较强的促进作用,并能诱导神经前体细胞分化为DN神经元。     

Opioid receptors in midbrain dopaminergic regions of the rat II. Kappa and delta receptor autoradiography

Summary Opiates and opioid peptides are known to influence the dopaminergic (DA) neurons in the midbrain. The purpose of this study was to map and quantify the density of kappa and delta opioid receptor subtypes in the retrorubral field, substantia nigra, and ventral tegmental area and related nuclei, which contain DA nuclei A8, A9, and A10, respectively. Sections through the rostral-caudal extent of the rat midbrain were stained with an antibody against tyrosine hydroxylase, as a DA cell marker, and comparable sections were processed for in vitro receptor autoradiography using the kappa-selective ligand, U-69593, and the delta-selective ligand, D-Pen², D-Pen⁵-enkephalin. In general, both kappa and delta ligands exhibited low levels of specific binding in regions occupied by the midbrain DA neurons.Kappa binding (4–8 fmol/mg tissue) was high throughout the rostral-caudal extent of the substantia nigra, in rostral portions of the ventral tegmental area, and in the nucleus paranigralis; low binding occurred in the retrorubral field and central linear nucleus raphe.Delta binding (6–18 fmol/mg tissue) was high in the caudal portion of the substantia nigra pars reticulata, and in the medial terminal nucleus of the accessory optic system (a region previously shown to contain DA dendrites). The kappa and delta receptor binding is heterogeneously distributed in regions occupied by midbrain dopaminergic neurons, and several fold lower than the binding of mu opioid receptors in the same brain regions.     

Opioid receptors in midbrain dopaminergic regions of the rat I. MU receptor autoradiography

Summary Several lines of evidence indicate that an interaction exists between opioid peptides and midbrain dopaminergic neurons. The purpose of this study was to map and quantify the density of the mu opioid receptor subtype relative to the location of the dopaminergic (DA) neurons in the retrorubral field (nucleus A8), substantia nigra (nucleus A9), and ventral tegmental area and related nuclei (nucleus A10) in the rat. Sections through the rostral-caudal extent of the midbrain were stained with an antibody against tyrosine hydroxylase, as a DA cell marker, and comparable sections were processed for in vitro receptor autoradiography using the mu-selective ligand,³H-Tyr-D-Ala-N-MePhe-Gyl-ol enkephalin. In the nucleus A8 region, there were low levels of mu binding. In the rostral portion of nucleus A9, there was prominent mu binding both in the ventral pars compacta, which contains numerous DA neurons, and in regions that correspond to the location of the DA dendrites which project ventrally into the underlying substantia nigra pars reticulata. In the caudal portion of nucleus A9, mu binding was greatest in the substantia nigra pars reticulata, but also in the same region that contains DA neurons. In nucleus A10, mu receptor densities differed depending upon the nucleus A10 subdivision, and the rostral-caudal position in the nucleus. Low receptor densities were observed in rostral portions of the ventral tegmental area and interfascicular nucleus, and there was negligible binding in the parabrachial pigmented nucleus and paranigral nucleus at the level of the interpeduncular nucleus; all regions where there are high densities of DA somata. Mu binding was relatively high in the central linear nucleus, and in the dorsal and medial divisions of the medial terminal nucleus of the accessory optic system, which has been shown to contain DA dendrites. These data indicate that mu opioid receptors are located in certain regions occupied by all three midbrain DA nuclei, but in a highly heterogeneous fashion.     

Serotonin axon terminals in the ventral tegmental area of the rat: fine structure and synaptic input to dopaminergic neurons

The serotoninergic (5-hydroxytryptamine, 5-HT) innervation of the rat ventral tegmental area (VTA) was examined by light and electron microscopic radioautography following intraventricular infusion of [³H]5-HT. The [³H]5-HT labeled processes were characterized with respect to their regional distribution, ultrastructure and relationships with all neurons, including dopaminergic neurons, identified in the same sections using immunocytochemistry for the localization of the catecholamine-synthesizing enzyme, tyrosine hydroxylase (TH). By light microscopy, [³H]5-HT labeled axons and axonal varicosities were detected throughout the interfascicular nucleus and ventral portion of the VTA. By electron microscopy, [³H]5-HT-labeled axons were found to be mainly small and unmyelinated, although a few showed several lamellae of myelin. The labeled varicosities measured 0.6 μm in mean diameter and contained many small, round or flattened agranular vesicles and a few large granular vesicles. More than 18% showed synaptic specializations in single thin sections. Most of these synapses were asymmetric and established on dendritic shafts. Based on the probability of seeing such synaptic specializations in single thin sections, it was estimated that as many as 50% of the labeled 5-HT terminals formed synaptic contacts in the VTA. In dually labeled light microscopic sections, [³H]5-HT-accumulating processes often appeared adjacent to TH-immunoreactive perikarya and proximal dendrites. Electron microscopy demonstrated that terminals with radioautographic labeling for 5-HT formed conventional synapses both with TH-labeled and unlabeled dendrites in the VTA. Many additional 5-HT terminals lacking recognizable synaptic densities were directly apposed to TH-labeled dendrites and were isolated from the rest of the neuropil by thin glial leaflets. These results suggest that 5-HT neurons innervate both dopaminergic and non-dopaminergic neurons in the VTA and may influence mesocortical and mesolimbic efferent systems through synaptic as well as non-synaptic mechanisms.     

Generation of embryonic stem cells and transgenic mice expressing green fluorescence protein in midbrain dopaminergic neurons

We have generated embryonic stem (ES) cells and transgenic mice with green fluorescent protein (GFP) inserted into the Pitx3 locus via homologous recombination. In the central nervous system, Pitx3-directed GFP was visualized in dopaminergic (DA) neurons in the substantia nigra and ventral tegmental area. Live primary DA neurons can be isolated by fluorescence-activated cell sorting from these transgenic mouse embryos. In culture, Pitx3-GFP is coexpressed in a proportion of ES-derived DA neurons. Furthermore, ES cell-derived Pitx3-GFP expressing DA neurons responded to neurotrophic factors and were sensitive to DA-specific neurotoxin N-4-methyl-1, 2, 3, 6-tetrahydropyridine. We anticipate that the Pitx3-GFP ES cells could be used as a powerful model system for functional identification of molecules governing mDA neuron differentiation and for preclinical research including pharmaceutical drug screening and transplantation. The Pitx3 knock-in mice, on the other hand, could be used for purifying primary neurons for molecular studies associated with the midbrain-specific DA phenotype at a level not previously feasible. These mice would also provide a useful tool to study DA fate determination from embryo- or adult-derived neural stem cells.     

Intranasal administration of rotenone in mice attenuated olfactory functions through the lesion of dopaminergic neurons in the olfactory bulb

Many environmental chemicals are thought to affect brain function. It was reported that chemicals in the nasal cavity directly reach the brain through the connection between olfactory neurons and the olfactory bulb (OB). In this ‘olfactory transport,’ xenobiotics absorbed at the nasal mucosa reach the brain by bypassing some physical barriers and defenses, and thus olfactory transport is suspected to be a vulnerable mechanism of the brain against invasion threats of environmental chemicals. In this study, we focused on the neuronal toxicity of rotenone administered intranasally to mice. The results showed that the mice that were administered rotenone had attenuated olfactory functions. We also found that intranasally administered rotenone induced acute mitochondrial stress at the OB. The repeated administration of rotenone resulted in a decrease in the number of dopaminergic neurons, which are inhibitory interneurons in the OB. Taken together, our findings suggest that the inhalation of environmental toxins induces the neurodegeneration of cranial neurons through olfactory transport, and that olfactory dysfunction may be induced as an earliest symptom of neurodegeneration caused by inhaled neurotoxins.     

Trifluoperazine and calcium antagonists accelerate cholinergic desensitization inAplysia neurons

The onset of desensitization of excitatory acetylcholine responses in isolated, voltage clampedAplysia RB neurons to microperfused agonist was accelerated by trifluoperazine and the calcium antagonists SKF-525A and D-600. In the absence of these drugs the time course of desensitization is described as the sum of two exponentials plus a constant. All 3 compounds accelerated the fast exponential component of desensitization, but had opposite effects on the slow component: trifluoperazine and SKF-525A accelerated the slow component, whereas this phase was greatly slowed by D-600. These results provide further evidence that the two components of cholinergic desensitization may be independent and influenced by agents which may affect calcium binding to the acetylcholine receptor.     

Idiopathic Parkinson's disease patient‐derived induced pluripotent stem cells function as midbrain dopaminergic neurons in rodent brains

Patient‐specific induced pluripotent stem cells (iPSCs) are a promising source for cell transplantation therapy. In Parkinson's disease (PD) patients, however, their vulnerability and the transmission of pathological α‐Synuclein are possible drawbacks that may prevent PD‐specific iPSCs (PDiPSCs) from being used in clinical settings. In this study, we generated iPSCs from idiopathic PD patients and found that there was no significant vulnerability between dopaminergic (DA) neurons generated from healthy individuals and idiopathic PD patients. PDiPSC‐derived DA neurons survived and functioned in the brains of PD model rats. In addition, in the brains of α‐Synuclein transgenic mice, PDiPSC‐derived DA neurons did not cause pathological α‐Synuclein accumulation in the host brain or in the grafts. These results suggested that iPSCs derived from idiopathic PD patients are feasible as donor cells for autologous transplantation to treat PD. © 2017 Wiley Periodicals, Inc.     

Changes in ionic conductances induced by cAMP in Helix neurons

Adenosine 3',5'-cyclic monophosphate (cAMP) was injected by a fast and quantitative pressure injection method into voltage-clamped identified Helix neurons. The intracellular elevation of cAMP caused an inward current which was not accompanied by a significant change in membrane conductance in a negative potential range with little activation of voltage-dependent membrane conductances. Near resting potential Na+ ions were the main carrier of the cAMP-induced inward current as measured with ion-selective microelectrodes. TTX did not affect the Na+ influx. K+ and less effective Ca2+ could substitute for Na+ in carrying the inward current. In the presence of Na+, divalent cations such as Ca2+ and Mg2+, and also La3+ exerted an inhibitory influence on the cAMP-induced inward current, and Ca2+ as measured with ion-selective microelectrodes did not contribute significantly to the current. Thus, the inward current was of a non-specific nature. Simultaneously to this cAMP action, the membrane permeability for K+ ions was decreased by cAMP. This effect became particularly obvious when K+ currents were activated by long-lasting, depolarizing voltage steps. In this situation a reduced K+ efflux following cAMP injection was observed by means of K+-selective microelectrodes located near the external membrane surface. Outward K+ currents were less reduced by cAMP if external Ca2+ was replaced by Ni2+. The nearly compensatory increase and decrease of two membrane conductances in the same neuron explained the lack of change in the cell input resistance despite the considerable depolarizing action of intracellularly elevated cAMP.     

DNA聚合酶β抑制剂齐墩果酸对帕金森病小鼠模型的保护作用

目的 观察DNA聚合酶β抑制剂齐墩果酸对帕金森病小鼠模型的保护作用.方法 利用MPTP腹腔注射法制作小鼠帕金森病模型,在MPTP注射前后分别给予DNA聚合酶β抑制剂齐墩果酸干预,用免疫组织化学染色法检测小鼠中脑酪氨酸羟化酶（TH）阳性神经元,免疫印迹法检测中脑腹侧TH蛋白和活化的Caspase-3表达水平,HPLC法检测纹状体中多巴胺及其代谢产物的水平.结果 小鼠腹腔注射MPTP后出现行为学异常,中脑黑质多巴胺能神经元损伤,中脑腹侧TH表达降低,活化的caspase-3水平增高,同时纹状体多巴胺及其代谢产物3,4二羟基苯乙酸（DOPAC）水平降低.齐墩果酸干预能够显著改善小鼠的行为学评分,增加黑质多巴胺能神经元数量,抑制caspase-3活化,并增加纹状体多巴胺和DOPAC水平.结论 齐墩果酸可能对帕金森病小鼠多巴胺能神经元具有保护作用.     

Octopamine-containing neurons in the alimentary tract of the earthworm (Eisenia fetida)

Octopamine-containing nerve cells have been demonstrated in the enteric plexus of the earthworm (Eisenia fetida), applying immunocytochemistry and HPLC assay. A few octopamine-immunoreactive neurons occurred in the fore- and hindgut, whereas their number in the midgut was considerably greater. Octopamine levels detected by HPLC correlated with the distribution of octopamine-containing nerve cells. A regulatory role for these intrinsic octopaminergic neurons is suggested in the enteric plexus in the earthworm alimentary tract. This is the first report on the occurrence of octopamine-containing nerve cells in the peripheral nervous system of an invertebrate.