An Automated Method for Cell Detection in Zebrafish

Quantification of cells is a critical step towards the assessment of cell fate in neurological disease or developmental models. Here, we present a novel cell detection method for the automatic quantification of zebrafish neuronal cells, including primary motor neurons, Rohon–Beard neurons, and retinal cells. Our method consists of four steps. First, a diffused gradient vector field is produced. Subsequently, the orientations and magnitude information of diffused gradients are accumulated, and a response image is computed. In the third step, we perform non-maximum suppression on the response image and identify the detection candidates. In the fourth and final step the detected objects are grouped into clusters based on their color information. Using five different datasets depicting zebrafish cells, we show that our method consistently displays high sensitivity and specificity of over 95%. Our results demonstrate the general applicability of this method to different data samples, including nuclear staining, immunohistochemistry, and cell death detection.     

腺病毒介导的GDNF基因转移体外表达及生物学活性研究

为利用重组腺病毒介导的胶质细胞源性神经营养因子（GDNF）基因转移治疗帕金森病（PD）提供依据。方法：采用免疫组化、RT－PCR及ELISA定量分析观察人GDNF腺病毒（Ad-GDNF)在大鼠星形胶质 PC12细胞的表达,通过观察病毒直接感染及病毒感染的PC12细胞上清对中脑原代培养细胞中的TH阳性细胞（DA能神经元）生存能力和形态分化的影响来验证其生物学活性。结果Ad-GDNF在星形胶质细胞、PC12细胞及大鼠中脑原代培养细胞均可有效表达,其表达产物对中脑DNA能神经元的生存和形态分化均有显著的促进作用。结论：腺病毒介导的GDNF基因转移可在体外有效表达,且表达产物具有生物学活性,提示该手段在PD治疗方面具有良好的应用前景。     

一种微量CCNU测定方法的建立和评价

利用CCNU在过量酸中的脱亚硝基反应,建立以分光光度法测定体内微量CCNU含量的方法,并对其测试敏感性、可靠性进行分析讨论和评价。结果显示:在血液及脑组织中,CCNU浓度与吸收度间线性相关良好,可测浓度范围分别为0.2～40μg/ml和40～80μg/g。与既往方法相比,本法不失为一种快捷、可靠且敏感的测试方法。     

Effective Normative Samples For the Detection Of Cognitive Impairment in Older Adults

The inclusion of individuals with incipient dementia in normative data contaminates the distinction between normal and pathological aging. Conventional and Robust (excluding persons with incipient dementia) norms were created using data from the Canadian Study of Health and Aging (CSHA). Robust norms were not significantly better at distinguishing between normal and pathological aging. Norms reflecting the relationship between age and the prevalence of dementia revealed a probability of dementia of less than 35%. The results of the norming procedure serve to illustrate the validity of our current measures and methods for identifying cognitive impairment. CSHA Conventional norms are adequate for the identification of cognitive impairment.     

Crossroads in GDNF therapy for Parkinson's disease.

The development of a neuroprotective or neuroregenerative therapy for Parkinson's disease (PD) would be a major therapeutic advance. Unfortunately, results from a recent controlled clinical study delivering the neurotrophic factor, glial-derived neurotrophic factor (GDNF), directly into brain did not demonstrate efficacy and safety of such a treatment. A critical review of available data suggests that there are questions that need to be answered before the future of GDNF as a therapy for PD can be determined.     

Expression of Neurturin, GDNF, and GDNF Family-Receptor mRNA in the Developing and Mature Mouse

The GDNF family of neurotrophic factors currently has four members: neurturin (NRTN), glial cell line-derived neurotrophic factor (GDNF), persephin, and artemin. These proteins are potent survival factors for several populations of central and peripheral neurons. The receptors for these factors are complexes that include the Ret tyrosine kinase receptor and a GPI-linked, ligand-binding component called GDNF family receptor alpha 1-4 (GFRalpha1-4). We have used in situ hybridization to study the mRNA expression of NRTN, GDNF, Ret, GFRalpha1, and GFRalpha2 during embryonic development and in the adult mouse. GDNF receptors were prominently expressed during embryonic development in the nervous system, the urogenital system, the digestive system, the respiratory system, and in developing skin, bone, muscle, and endocrine glands. In some regions, incomplete receptor complexes were expressed suggesting that other, as yet unidentified, receptor components exist or that receptor complexes are formed in trans. NRTN and GDNF were expressed in many trigeminal targets during embryonic development including the nasal epithelium, the teeth, and the whisker follicles. NRTN and GDNF were also expressed in the developing limbs and urogenital system. In the embryo, GDNF factors and receptors were expressed at several sites of mesenchyme/epithelial induction, including the kidney, tooth, and submandibular gland. This expression pattern is consistent with the possibility that the GDNF factors function in inductive processes during embryonic development and with the recently discovered role of NRTN as a necessary trophic factor for the development of some parasympathetic neurons. In the mature animal, receptor expression was more limited than in the embryo. In the adult mouse, NRTN was most prominently expressed in the gut, prostate testicle, and oviduct; GDNF was most prominently expressed in the ovary.     

聚合酶链法检测莱姆病患者尿液中伯氏疏螺旋体DNA(附17例报道)

目的:评估聚合酶链反应(PCR)检测莱姆病患者尿液中伯氏疏螺旋体DNA的诊断价值。方法:2004至2006年来自新疆莱姆病自然疫源地临床疑似莱姆病患者17例(均有蜱暴露史和莱姆病临床证据者)和6例以前确诊并治疗后完全恢复的莱姆病患者,共23例为病例组;另选择25例非疑似莱姆病患者为对照组。应用PCR方法检测尿液中伯氏疏螺旋体DNA,采用间接免疫荧光(IFA)检测血清伯氏疏螺旋体抗体。结果:①17例莱姆病患者临床表现呈多系统损害。12/17例(70.59%)为神经型(周围神经病3例,其中1例合并下肢瘀积性皮炎;游走性红斑并发莱姆脑病2例;脑膜脑炎2例;脑炎2例;脑膜炎、脑干炎和脊髓炎各1例);流感样症状2例,其中伴皮疹1例;余心脏型、精神障碍和肝病型各1例。②PCR伯氏疏螺旋体DNA检测阳性17/17例(总阳性率100%)。4例血清IFA试验伯氏疏螺旋体抗体阳性。以前确诊并治疗者6例及对照组25例尿PCR伯氏疏螺旋体DNA和血清IFA试验伯氏疏螺旋体抗体均阴性。③10例完成抗生素治疗后3个月复查,9例PCR伯氏疏螺旋体DNA检测阴性(包括3例晚期莱姆病患者),1例2年复查时仍阳性;4例血清伯氏疏螺旋体抗体阳性者复查均阴性。④早期莱姆病多采用几周的抗生素治疗方案。结论:PCR检测尿液中伯氏疏螺旋体DNA是莱姆病一个有价值的诊断工具。     

Use of the Wisconsin Card Sorting Test in the Detection of Malingering in Student Simulator and Patient Samples

We analyzed the ability of the Wisconsin Card Sorting Test to distinguish between 41 malingering and 31 normal undergraduates, and 17 probable malingering and 16 brain injured patients. A logistic regression consisting of number of categories (CAT) and failure to maintain set (FMS) distinguished malingering and normal undergraduates with 70.7% sensitivity, 87.1% specificity, and 77.8% overall classification, and distinguished patients suspected of malingering from brain injured controls with 82.4% sensitivity, 93.3% specificity, and 87.5% overall classification.     

A Method for Analyzing Biological Rhythms in Healthy Subjects and Depressed Patients

Abstract: Rectal temperature rhythms of healthy subjects and patients with depression were fitted to a waveform having cosine components of periods 8, 12, and 24 hours using the least squares method. The nadir was then used as an index of phase. The results suggest that this method gives more exact data than the conventional method of analyzing biological rhythms, which uses a least squares fit to a single, 24-hour-period cosine waveform.     

GDNF in Parkinson's disease: the perils of post-hoc power

The practice of performing post-hoc power calculations for studies that do not demonstrate statistically significant results has been widely recognized in the scientific literature as being unhelpful and potentially misleading. However, this practice continues to cause confusion in the interpretation of results from clinical trials and other studies. Here, we examine the re-interpretation of a recent randomized, double-blind, placebo-controlled study of intraputamenally administered GDNF in late-stage Parkinson's disease patients [Hutchinson M, Gurney S, Newson R. GDNF in Parkinson disease: an object lesson in the tyranny of type II. J Neurosci Methods 2007;163:190-2]. Their main criticism is that the study was not large enough to detect clinically worthwhile effects and that the observed non-significant result does not contradict the promising results observed in two previous, small, open-label studies. We have carefully assessed the re-analysis of the data performed by Hutchinson et al. and found their conclusions to be flawed, in part because they are based on post-hoc power calculations. We have reaffirmed that the confidence interval for the treatment effect in the placebo-controlled study of GDNF shows that the trial is capable of excluding effects of GDNF of the magnitudes that were observed in the open-label studies and that the conclusions drawn in the original paper remain scientifically sound.     

GDNF enhances the synaptic efficacy of dopaminergic neurons in culture

Glial cell line-derived neurotrophic factor (GDNF) is known to promote the survival and differentiation of dopaminergic neurons of the midbrain. GDNF also causes an enhancement of dopamine release by a mechanism which is presently unclear. Using isolated dopaminergic neurons of the rat ventral tegmental area in culture, we have tested the hypothesis that GDNF regulates the establishment and functional properties of synaptic terminals. Previous studies have shown that single dopaminergic neurons in culture can co-release glutamate in addition to dopamine, leading to the generation of a fast excitatory autaptic current via glutamate receptors. Using excitatory autaptic currents as an assay for the activity of synapses established by identified dopaminergic neurons, we found that chronically applied GDNF produced a threefold increase in the amplitude of excitatory autaptic currents. This action was specific for dopaminergic neurons because GDNF had no such effect on ventral tegmental area GABAergic neurons. The enhancement of excitatory autaptic current amplitude caused by GDNF was accompanied by an increase in the frequency of spontaneous miniature excitatory autaptic currents. These observations confirmed a presynaptic locus of change. We identified synaptic terminals by using synapsin-1 immunofluorescence. In single tyrosine hydroxylase-positive neurons, the number of synapsin-positive puncta which represent putative synaptic terminals was found to be approximately doubled in GDNF-treated cells at 5, 10 and 15 days in culture. The number of such morphologically identified terminals in isolated GABAergic neurons was unchanged by GDNF. These results suggest that one mechanism through which GDNF may enhance dopamine release is through promoting the establishment of new functional synaptic terminals.     

An Automatic HFO Detection Method Combining Visual Inspection Features with Multi-Domain Features

As an important promising biomarker, high frequency oscillations (HFOs) can be used to track epileptic activity and localize epileptogenic zones. However, visual marking of HFOs from a large amount of intracranial electroencephalogram (iEEG) data requires a great deal of time and effort from researchers, and is also very dependent on visual features and easily influenced by subjective factors. Therefore, we proposed an automatic epileptic HFO detection method based on visual features and non-intuitive multi-domain features. To eliminate the interference of continuous oscillatory activity in detected sporadic short HFO events, the iEEG signals adjacent to the detected events were set as the neighboring environmental range while the number of oscillations and the peak–valley differences were calculated as the environmental reference features. The proposed method was developed as a MatLab-based HFO detector to automatically detect HFOs in multi-channel, long-distance iEEG signals. The performance of our detector was evaluated on iEEG recordings from epileptic mice and patients with intractable epilepsy. More than 90% of the HFO events detected by this method were confirmed by experts, while the average missed-detection rate was < 10%. Compared with recent related research, the proposed method achieved a synchronous improvement of sensitivity and specificity, and a balance between low false-alarm rate and high detection rate. Detection results demonstrated that the proposed method performs well in sensitivity, specificity, and precision. As an auxiliary tool, our detector can greatly improve the efficiency of clinical experts in inspecting HFO events during the diagnosis and treatment of epilepsy.     

GDNF reverses priming for dyskinesia in MPTP-treated, L-DOPA-primed common marmosets

Parkinson's disease (PD) is associated with a progressive loss of dopamine neurons in the substantia nigra and degeneration of dopaminergic terminals in the striatum. Although L-DOPA treatment provides the most effective symptomatic relief for PD it does not prevent the progression of the disease, and its long-term use is associated with the onset of dyskinesia. In rodent and primate studies, glial cell line-derived neurotrophic factor (GDNF) may prevent 6-OHDA- or MPTP-induced nigral degeneration and so may be beneficial in the treatment of PD. In this study, we investigate the effects of GDNF on the expression of dyskinesia in L-DOPA-primed MPTP-treated common marmosets, exhibiting dyskinesia. GDNF or saline was administered by two intraventricular injections, 4 weeks apart, to MPTP-treated, L-DOPA-treated common marmosets primed to exhibit dyskinesia. Prior to GDNF or saline administration, all animals displayed marked dyskinesia when treated with L-DOPA. GDNF administration produced a significant improvement in motor disability and, following the second injection of GDNF, a significant improvement in the locomotor activity was observed. Following the administration of L-DOPA there was a greater reversal of disability and a reduction in the intensity of L-DOPA-induced dyskinesia in GDNF-treated animals compared to saline-treated controls. However, there was no significant difference in L-DOPA's ability to increase locomotor activity between GDNF-treated and saline-treated animals. GDNF treatment caused a significant increase in the number of tyrosine hydroxylase-positive neurons in the substantia nigra, but no change in [(3)H]mazindol binding to dopamine terminals was found in the striatum of GDNF-treated animals compared to saline-treated controls. In GDNF-treated animals a small but significant reduction in enkephalin mRNA was observed in the caudate nucleus but not in the putamen or the nucleus accumbens. Substance P mRNA expression was equally reduced in the caudate nucleus and the putamen of the GDNF-treated animals but not in the nucleus accumbens. Intraventricular administration of GDNF improved MPTP-induced disability and reversed dopamine cell loss in the substantia nigra. GDNF also diminished L-DOPA-induced dyskinesia, which may relate to its ability to partly restore nigral dopaminergic transmission or to modify the activity of striatal output pathways.     

GDNF、TGF-β1联合治疗帕金森病的实验研究

目的采用阳离子脂质体介导的方法,探讨胶质细胞源性神经生长因子(GDNF)、转化生长因子-β1(TGF-β1)基因联合治疗对帕金森病(PD)的疗效和两者的相互影响。方法用6-羟基多巴(6-OHDA)破坏SD大鼠一侧黑质,建立帕金森病模型,并分为对照组、GDNF治疗组、GDNF TGF-β1治疗组。通过立体定向仪将DOTAP脂质体包裹的GDNFcDNA、TGF-β1cDNA注入大鼠的纹状体,观察阿朴吗啡(APO)诱导的旋转行为,应用RT-PCR、Westernblot分别从mRNA和蛋白水平检测GDNF在脑内表达的变化。结果治疗2周后,PD大鼠旋转行为明显减轻(P<0.01)。治疗后4周,GDNF TGF-β1组症状进一步减轻,而GDNF组没有继续改善。RT-PCR、Westernblot显示,与GDNF组相比,联合治疗组GDNF在mRNA、蛋白水平均有较高的表达。结论阳离子脂质体介导GDNF、TGF-β1能改善帕金森病症状,TGF-β1是GDNF重要的联合因子。     

GDNF is a chemoattractant factor for neuronal precursor cells in the rostral migratory stream

Olfactory bulb (OB) interneurons are generated from neuroblast cells derived from the anterior subventricular zone (SVZa) of the forebrain. The mechanisms guiding the rostral migration of these neuronal precursors are not well understood. Here, we show that glial cell line-derived neurotrophic factor (GDNF) is produced in the olfactory bulb but distributed along the rostral migratory stream (RMS) in a pattern concordant with the expression of its GPI-anchored receptor GFRalpha1. We demonstrate that GDNF is a chemoattractant factor for RMS-derived neuronal precursors, but not for SVZa neuroblast cells. In agreement with this, GDNF increased Cyclin-dependent kinase 5 (Cdk5) activity in RMS cells, a kinase critically involved in neuronal migration and guidance. GDNF-mediated cell chemoattraction was abrogated in RMS explants treated with the Cdk5 inhibitor Roscovitine as well as in RMS explants isolated from Ncam mutant mice. Chemical cross-linking assays showed that 125I-GDNF is able to interact directly with NCAM in RMS-derived cells. Taken together, these data demonstrate that GDNF is a direct chemoattractant factor for neuroblast cells migrating along the RMS and support the participation of NCAM during this guidance process.     

Evolution of the GDNF family ligands and receptors

Four different ligand-receptor binding pairs of the GDNF (glial cell line-derived neurotrophic factor) family exist in mammals, and they all signal via the transmembrane RET receptor tyrosine kinase. In addition, GRAL (GDNF Receptor Alpha-Like) protein of unknown function and Gas1 (growth arrest specific 1) have GDNF family receptor (GFR)-like domains. Orthologs of the four GFRalpha receptors, GRAL and Gas1 are present in all vertebrate classes. In contrast, although bony fishes have orthologs of all four GDNF family ligands (GFLs), one of the ligands, neurturin, is absent in clawed frog and another, persephin, is absent in the chicken genome. Frog GFRalpha2 has selectively evolved possibly to accommodate GDNF as a ligand. The key role of GDNF and its receptor GFRalpha1 in enteric nervous system development is conserved from zebrafish to humans. The role of neurturin, signaling via GFRalpha2, for parasympathetic neuron development is conserved between chicken and mice. The role of artemin and persephin that signal via GFRalpha3 and GFRalpha4, respectively, is unknown in non-mammals. The presence of RET- and GFR-like genes in insects suggests that a ProtoGFR and a ProtoRET arose early in the evolution of bilaterian animals, but when the ProtoGFL diverged from existing transforming growth factor (TGFbeta)-like proteins remains unclear. The four GFLs and GFRalphas were presumably generated by genome duplications at the origin of vertebrates. Loss of neurturin in frog and persephin in chicken suggests functional redundancy in early tetrapods. Functions of non-mammalian GFLs and prechordate RET and GFR-like proteins remain to be explored.