神经元再生:抑郁症治疗的新策略

成年哺乳动物一生中,海马等脑区神经元是可以再生的,而海马脑区神经元再生的减少和增多分别是抑郁症发生和恢复的重要因素。如果神经元再生过程被抑制,在抑郁症的动物模型上抗抑郁剂将会失去其行为学效应。长期给予不同种类的抗抑郁剂可以显著地促进动物海马神经元再生。随着对神经元再生调节机制研究的不断深入,为进一步探讨抑郁症的发生机制,以及发展新型抗抑郁治疗药物提供了新的思路与视角。     

Robust circadian rhythmicity of Drosophila melanogaster requires the presence of lateral neurons: a brain-behavioral study of disconnected mutants

Mutations at the disconnected (disco) locus of Drosophila melanogaster disrupt neural cell patterning in the visual system, leading to the loss of many optic lobe neurons. Drosophila's presumptive circadian pacemaker neurons – the dorsal and ventral lateral neurons – are usually among the missing cells, and most disco flies are behaviorally arrhythmic. In this study, I show that ventral lateral neurons (LN_vs) are occasionally present and provoke robust circadian rhythmicity in disco mutants. Of 357 individual disco flies four animals with robust circadian rhythmicity were found. All four retained LN_vs together with terminals in the superior protocerebrum. Residual or bi-circadian rhythmicity was found in about 20% of all flies; the remaining flies were completely arrhythmic. One of the flies with residual rhythmicity and two of the arrhythmic flies also had some LN_vs stained. However, these flies lacked the LN_v fibers in the superior protocerebrum. The results suggest that the presence of single LN_vs is sufficient to provoke robust circadian rhythmicity in locomotor activity if the LN_v terminals reach the superior protocerebrum. The presence of residual or bi-circadian rhythmicity in 20% of the flies without LN_vs indicates that also other cells contribute to the rhythmic control of locomotor activity. Accepted: 17 September 1997     

A major bristle QTL from a selected population of Drosophila uncovers the zinc-finger transcription factor poils-au-dos, a repressor of achaete-scute

Traditional screens aiming at identifying genes regulating development have relied on mutagenesis. Here, we describe a new gene involved in bristle development, identified through the use of natural variation and selection. Drosophila melanogaster bears a pattern of 11 macrochaetes per heminotum. From a population initially sampled in Marrakech, a strain was selected for an increased number of thoracic macrochaetes. Using recombination and single nucleotide polymorphisms, the factor responsible was mapped to a single locus on the third chromosome, poils au dos, that encodes a zinc-finger-ZAD protein. The original, as well as new, presumed null, alleles of poils au dos, is associated with ectopic achaete-scute expression that results in the additional bristles. This suggests a possible role for Poils au dos as a repressor of achaete and scute. Ectopic expression appears to be independent of the activity of known cis-regulatory enhancer sequences at the achaete-scute complex that mediate activation at specific sites on the notum. The target sequences for Poils au dos activity were mapped to a 14 kb region around scute. In addition, we show that pad interacts synergistically with the repressor hairy and with Dpp signaling in posterior and anterior regions of the notum, respectively.     

Developing a strategy for the rapid identification of genetically altered mice: an olfactory system perspective

The mouse genome contains a wide array of uncharacterized genes that are involved in olfactory system development and cellular function. The author describes how random mutagenesis of these genes combined with rapid screening techniques afford the identification of the particular olfactory-related genes that have been altered.     

A developmental study of serotonin-immunoreactive neurons in the embryonic brain of the Marbled Crayfish and the Migratory Locust: Evidence for a homologous protocerebral group of neurons

It is well established that the brains of adult malacostracan crustaceans and winged insects display distinct homologies down to the level of single neuropils such as the central complex and the optic neuropils. We wanted to know if developing insect and crustacean brains also share similarities and therefore have explored how neurotransmitter systems arise during arthropod embryogenesis. Previously, Sintoni et al. (2007) had already reported a homology of an individually identified cluster of neurons in the embryonic crayfish and insect brain, the secondary head spot cells that express the Engrailed protein. In the present study, we have documented the ontogeny of the serotonergic system in embryonic brains of the Marbled Crayfish in comparison to Migratory Locust embryos using immunohistochemical methods combined with confocal laser-scan microscopy. In both species, we found a cluster of early emerging serotonin-immunoreactive neurons in the protocerebrum with neurites that cross to the contralateral brain hemisphere in a characteristic commissure suggesting a homology of this cell cluster. Our study is a first step towards a phylogenetic analysis of neurotransmitter system development and shows that, as for the ventral nerve cord, traits related to neurogenesis in the brain can provide valuable hints for resolving the much debated question of arthropod phylogeny.     

Drosophila growth cones: A genetically tractable platform for the analysis of axonal growth dynamics

The formation of neuronal networks, during development and regeneration, requires outgrowth of axons along reproducible paths toward their appropriate postsynaptic target cells. Axonal extension occurs at growth cones (GCs) at the tips of axons. GC advance and navigation requires the activity of their cytoskeletal networks, comprising filamentous actin (F‐actin) in lamellipodia and filopodia as well as dynamic microtubules (MTs) emanating from bundles of the axonal core. The molecular mechanisms governing these two cytoskeletal networks, their cross‐talk, and their response to extracellular signaling cues are only partially understood, hindering our conceptual understanding of how regulated changes in GC behavior are controlled. Here, we introduce Drosophila GCs as a suitable model to address these mechanisms. Morphological and cytoskeletal readouts of Drosophila GCs are similar to those of other models, including mammals, as demonstrated here for MT and F‐actin dynamics, axonal growth rates, filopodial structure and motility, organizational principles of MT networks, and subcellular marker localization. Therefore, we expect fundamental insights gained in Drosophila to be translatable into vertebrate biology. The advantage of the Drosophila model over others is its enormous amenability to combinatorial genetics as a powerful strategy to address the complexity of regulatory networks governing axonal growth. Thus, using pharmacological and genetic manipulations, we demonstrate a role of the actin cytoskeleton in a specific form of MT organization (loop formation), known to regulate GC pausing behavior. We demonstrate these events to be mediated by the actin‐MT linking factor Short stop, thus identifying an essential molecular player in this context. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 2010     

Non-linear selection response in Drosophila: a strategy for testing the rare-alleles model of quantitative genetic variability

Quantitative genetic theory predicts that variation due to rare alleles at many loci will generate a transient acceleration in the response to directional selection. We have tested this prediction by constructing experimental lines ofDrosophila melanogaster that carry positively selected ethanol resistance alleles at low frequencies, and then subjecting the lines to directional selection for ethanol resistance. Approximately 468,000 files were subjected to artificial selection over 30 generations. The predicted non-linear selection responses were observed in all experimental lines and replicates, on three genetic backgrounds. In contrast, un-selected controls and lines carrying random alleles at low frequencies on the same genetic backgrounds exhibited linear selection responses. These results demonstrate that non-linearities due to rare alleles are detectable and repeatable, provided that experiments are done on a sufficiently large scale. The results suggest that it may be possible to test for rare-alleles as a component of naturally occurring genetic variation by careful examination of selection response curves.     

Spatiotemporal switching signals for cancer stem cell activation in pediatric origins of adulthood cancer:Towards a watch-and-wait lifetime strategy for cancer treatment

Pediatric origin of cancer stem cell hypothesis holds great promise and potential in adult cancer treatment, however; the road to innovation is full of obstacles as there are plenty of questions left unanswered. First, the key question is to characterize the nature of such stem cells (concept). Second, the quantitative imaging of pediatric stem cells should be implemented(technology). Conceptually, pediatric stem cell origins of adult cancer are based on the notion that plasticity in early life developmental programming evolves local environments to cancer. Technologically, such imaging in children is lacking as all imaging is designed for adult patients. We postulate that the need for quantitative imaging to measure space-time changes of plasticity in early life developmental programming in children may trigger research and development of the imaging technology. Such quantitative imaging of pediatric origin of adulthood cancer will help develop a spatiotemporal monitoring system to determine cancer initiation and progression. Clinical validation of such speculative hypothesis-that cancer originates in a pediatric environment-will help implement a wait-andwatch strategy for cancer treatment.     

Direct introduction of gene constructs into the pronucleus-like structure of cloned embryos: a new strategy for the generation of genetically modified pigs

Due to a rising demand of porcine models with complex genetic modifications for biomedical research, the approaches for their generation need to be adapted. In this study we describe the direct introduction of a gene construct into the pronucleus (PN)-like structure of cloned embryos as a novel strategy for the generation of genetically modified pigs, termed “nuclear injection”. To evaluate the reliability of this new strategy, the developmental ability of embryos in vitro and in vivo as well as the integration and expression efficiency of a transgene carrying green fluorescence protein (GFP) were examined. Eighty percent of the cloned pig embryos (633/787) exhibited a PN-like structure, which met the prerequisite to technically perform the new method. GFP fluorescence was observed in about half of the total blastocysts (21/40, 52.5%), which was comparable to classical zygote PN injection (28/41, 68.3%). In total, 478 cloned embryos injected with the GFP construct were transferred into 4 recipients and from one recipient 4 fetuses (day 68) were collected. In one of the fetuses which showed normal development, the integration of the transgene was confirmed by PCR in different tissues and organs from all three primary germ layers and placenta. The integration pattern of the transgene was mosaic (48 out of 84 single-cell colonies established from a kidney were positive for GFP DNA by PCR). Direct GFP fluorescence was observed macro- and microscopically in the fetus. Our novel strategy could be useful particularly for the generation of pigs with complex genetic modifications.     

Correlated responses to selection for faster development and early reproduction in Drosophila: the evolution of larval traits

Studies on selection for faster development in Drosophila have typically focused on the trade-offs among development time, adult weight, and adult life span. Relatively less attention has been paid to the evolution of preadult life stages and behaviors in response to such selection. We have earlier reported that four laboratory populations of D. melanogaster selected for faster development and early reproduction, relative to control populations, showed considerably reduced preadult development time and survivorship, dry weight at eclosion, and larval growth rates. Here we study the larval phase of these populations in greater detail. We show here that the reduction in development time after about 50 generations of selection is due to reduced duration of the first and third larval instars and the pupal stage, whereas the duration of the second larval instar has not changed. About 90% of the preadult mortality in the selected populations is due to larval mortality. The third instar larvae, pupae, and freshly eclosed adults of the selected populations weigh significantly less than controls, and this difference appears during the third larval instar. Thereafter, percentage weight loss during the pupal stage does not differ between selected and control populations. The minimum amount of time a larva must feed to subsequently complete development is lower in the selected populations, which also exhibit a syndrome of reduced energy expenditure through reduction in larval feeding rate, larval digging and foraging activity, and pupation height. Comparison of these results with those observed earlier in populations selected for adaptation to larval crowding and faster development under a different protocol from ours reveal differences in the evolved traits that suggest that the responses to selection for faster development are greatly affected by the larval density at which selection acts and on details of the selection pressures acting on the timing of reproduction.     

Homoeosis in Drosophila: Evidence for a maternal effect of the polycomb locus

When heterozygous, dominant mutant alleles of the Polycomb locus are associated with a variety of adult homoeotic effects. Zygotes homozygous for these alleles die as late embryos showing homoeotic transformation of head, thoracic, and abdominal segments. This study shows that embryos homozygous for Pc³ are more extreme than those homozygous for Pc¹ or Pc². Moreover, Pc¹/Pc³ heterozygotes are more extensively transformed if their mothers were Pc³/ + than if they were Pc¹/ +; this effect does not depend on zygotic genetic background and must be maternal in nature. Embryos homozygous for Pc³ are less extreme if they arise from Pc³/ + / + than from Pc³/ + mothers. These results strongly suggest that the Polycomb locus acts maternally as well as zygotically to affect early determinative decisions.     

Crossing the frontier: a hypothesis for the origins of meristic constraint in mammalian axial patterning

Serially homologous systems with high internal differentiation frequently exhibit meristic constraints, although the developmental basis for constraint is unknown. Constraints in the counts of the cervical and lumbosacral vertebral series are unique to mammals, and appeared in the Triassic, early in their history. Concurrent adaptive modifications of the mammalian respiratory and locomotor systems involved a novel source of cells for muscularization of the diaphragm from cervical somites, and the loss of ribs from lumbar vertebrae. Each of these innovations increased the modularity of the somitic mesoderm, and altered somitic and lateral plate mesodermal interactions across the lateral somitic frontier. These developmental innovations are hypothesized here to constrain the anteroposterior transposition of the limbs along the column, and thus also cervical and thoracolumbar count. Meristic constraints are therefore regarded here as the nonadaptive, secondary consequences of adaptive respiratory and locomotor traits.     

Perfusion of Immobilized Isolated Nerve Terminals as a Model for the Regulation of Transmitter Release: Release of Different, Endogenous Transmitters, Repeated Stimulation, and High Time Resolution

To study the release of neurotransmitters, i.e., the recruitment of transmitters for release and the regulation of the release process, isolated nerve terminals (synaptosomes) of the rat forebrain were immobilized in Sephadex gel inside a perfusion chamber. In this way, the following were achieved: (a) A very limited pressure stress was exerted on the synaptosomes, so that these remained viable for long periods (greater than 30 min) inside the chamber and did not elute from the chamber, which allowed long-term experiments with repeated stimulations; (b) estimation of the release of various endogenous transmitters, both in a Ca(2+)-dependent (exocytotic) and Ca(2+)-independent manner; (c) a step-like stimulation with depolarizing agents (rise time, 3-4 s) and a high time resolution (600-ms sampling); and (d) negligible reuptake of transmitter into the terminals or extracellular breakdown. It is concluded that this perfusion setup helps to provide new insights in the presynaptic stimulus-secretion coupling, co-transmission, and the exo-endocytosis cycle.     

A rapid method for constructing precaution maps based on a simple virtual ecology model: a case study on the range expansion of the invasive aquatic species Limnoperna fortunei

Before invasion, or in its early stages, information on the invader in target areas is generally extremely limited. In such situations, managers must select focal areas in which to concentrate control and mitigation efforts. Here, we discuss a rapid method for selecting areas in which to control invasive aquatic species based on limited information. We used a simple cellular automata model that does not require species-specific information, but simulates the process of invasive species expansion and includes observed expansion progress to detect keystone areas. As a case study, we simulated the expansion of an invasive aquatic mussel, Limnoperna fortunei, in Ibaraki Prefecture, Japan, and detected the areas in which control efforts should be concentrated. To some extent, our model was able to predict the expansion of L. fortunei from the initial detected invasion to the current distribution. We predicted areas with a high potential of spreading and areas that would suffer from high propagule pressure. Results revealed a mismatch between areas with high spread potential and those with high propagule pressure. Managers should concentrate their invasion prevention efforts in the former because these are likely to have a greater long-term influence. Additionally, we predicted future expansion from the current distribution and showed that current scattered populations could merge naturally. Our approach is useful for establishing a management plan before or in the early stages of invasion.     

Conformational perturbation of interleukin-2: a strategy for the design of cytokine analogs

Interleukin-2 (IL-2) is a representative of a growing family of small proteins termed lymphokines which are responsible for mediating cell differentiation, growth and function in the immune system. Many of these proteins are being evaluated for their clinical potential. From the perspective of drug development, structure-function analysis of these molecules and their receptors require the use methodologies different than those traditionally employed for small peptides and other natural products. However, similar pharmacologic principles apply and an understanding of ligand-receptor interactions and the associated responses is required in order to efficiently pursue agonist and antagonist design. Although IL-2 is a protein of only 133 amino acid residues for which a low resolution X-ray structure does exist, the complexity of its receptor system has provided an added challenge to structure-function studies. Consequently, little is known concerning the receptor contact residues for this protein. We have attempted to utilize established principles of protein and peptide structure to manipulate the conformation of IL-2 in a manner which has provided analogs helpful for receptor interaction studies. These proteins have not only providing useful information on the nature of the IL-2 receptor but have also revealed potential strategies for the design of IL-2 agonists and antagonists.     

Glutamatergic synapses of Drosophila neuromuscular junctions: a high-resolution model for the analysis of experience-dependent potentiation

The glutamatergic synapses of developing neuromuscular junctions (NMJ) of Drosophila larvae are readily accessible, morphologically simple, and physiologically well-characterized. They therefore have a long and highly successful tradition as a model system for the discovery of genetic and molecular mechanisms of target recognition, synaptogenesis, NMJ development, and synaptic plasticity. However, since the development and the activity-dependent refinement of NMJs are concurrent processes, they cannot easily be separated by the widely applied genetic manipulations that mostly have chronic effects. Recent studies have therefore begun systematically to incorporate larval foraging behavior into the physiological and genetic analysis of NMJ function in order to analyze potential experience-dependent changes of glutamatergic transmission. These studies have revealed that recent crawling experience is a potent modulator of glutamatergic transmission at NMJs, because high crawling activities result after an initial lag-phase in several subsequent phases of experience-dependent synaptic potentiation. Depending on the time window of occurrence, four distinct phases of experience-dependent potentiation have been defined. These phases of potentiation can be followed from their initial induction (phase-I) up to the morphological consolidation (phase-III/IV) of previously established functional changes (phase-II). This therefore establishes, for the first time, a temporal hierarchy of mechanisms involved in the use-dependent modification of glutamatergic synapses.     

A role for glutamate decarboxylase during tomato ripening: the characterisation of a cDNA encoding a putative glutamate decarboxylase with a calmodulin-binding site

A tomato fruit cDNA library was differentially screened to identify mRNAs present at higher levels in fruit of the tomato ripening mutant rin (ripening inhibitor). Complete sequencing of a unique clone ERT D1 revealed an open reading frame with homology to several glutamate decarboxylases. The deduced polypeptide sequence has 80% overall amino acid sequence similarity to a Petunia hybrida glutamate decarboxylase (petGAD) which carries a calmodulin-binding site at its carboxyl terminus and ERT D1 appears to have a similar domain. ERT D1 mRNA levels peaked at the first visible sign of fruit colour change during normal tomato ripening and then declined, whereas in fruit of the ripening impaired mutant, rin, accumulation of this mRNA continued until at least 14 days after the onset of ripening. This mRNA was present at much lower levels in other tissues, such as leaves, roots and stem, and was not increased by wounding. Possible roles for GAD, and its product -aminobutyric acid (GABA) in fruit, are discussed.