Tiling among stereotyped dendritic branches in an identified Drosophila motoneuron.

Different types of neurons can be distinguished by the specific targeting locations and branching patterns of their dendrites, which form the blueprint for wiring the brain. Unraveling which specific signals control different aspects of dendritic architecture, such as branching and elongation, pruning and cessation of growth, territory formation, tiling, and self-avoidance requires a quantitative comparison in control and genetically manipulated neurons. The highly conserved shapes of individually identified Drosophila neurons make them well suited for the analysis of dendritic architecture principles. However, to date it remains unclear how tightly dendritic architecture principles of identified central neurons are regulated. This study uses quantitative reconstructions of dendritic architecture of an identified Drosophila flight motoneuron (MN5) with a complex dendritic tree, comprising more than 4,000 dendritic branches and 6 mm total length. MN5 contains a fixed number of 23 dendritic subtrees, which tile into distinct, nonoverlapping volumes of the diffuse motor neuropil. Across-animal comparison and quantitative analysis suggest that tiling of the different dendritic subtrees of the same neuron is caused by competitive and repulsive interactions among subtrees, perhaps allowing different dendritic compartments to be connected to different circuit elements. We also show that dendritic architecture is similar among different wildtype and GAL4 driver fly lines. Metric and topological dendritic architecture features are sufficiently constant to allow for studies of the underlying control mechanisms by genetic manipulations. Dendritic territory and certain topological measures, such as tree compactness, are most constant, suggesting that these reflect the intrinsic molecular identity of the neuron. J. Comp. Neurol. 518:2169-2185, 2010. (c) 2010 Wiley-Liss, Inc.

Differential effects of octopamine and tyramine on the central pattern generator for Manduca flight.

The biogenic amine, octopamine, modulates a variety of aspects of insect motor behavior, including direct action on the flight central pattern generator. A number of recent studies demonstrate that tyramine, the biological precursor of octopamine, also affects invertebrate locomotor behaviors, including insect flight. However, it is not clear whether the central pattern generating networks are directly affected by both amines, octopamine and tyramine. In this study, we tested whether tyramine affected the central pattern generator for flight in the moth, Manduca sexta. Fictive flight was induced in an isolated ventral nerve cord preparation by bath application of the octopamine agonist, chlordimeform, to test potential effects of tyramine on the flight central pattern generator by pharmacological manipulations. The results demonstrate that octopamine but not tyramine is sufficient to induce fictive flight in the isolated ventral nerve cord. During chlordimeform induced fictive flight, bath application of tyramine selectively increases synaptic drive to depressor motoneurons, increases the number of depressor spikes during each cycle and decreases the depressor phase. Conversely, blocking tyramine receptors selectively reduces depressor motoneuron activity, but does not affect cycle by cycle elevator motoneuron spiking. Therefore, octopamine and tyramine exert distinct effects on the flight central pattern generating network.

Analysis of polymorphism at site -174 G/C of interleukin-6 promoter region in multiple myeloma.

It is well established that interleukin-6 (IL-6) is an essential growth factor for multiple myeloma (MM) and patients with increased IL-6 levels have a poor prognosis. In healthy subjects, the presence of the C allele at a polymorphic site (-174 G/C) of the IL-6 gene is related to low IL-6 levels. In view of the potential association of this particular polymorphism with IL-6 concentration, and the relevance of IL-6 in MM pathogenesis, the objective of the present study was to investigate the prevalence of IL-6 (-174 G/C) promoter polymorphism and its association with development of MM in Brazilian individuals. We investigated the prevalence of these alleles in 52 patients and 60 healthy subjects (matched by age, sex, and race) of a Brazilian population. Thirty patients were male (42.4%), 24 (46.2%) were white and the median age at diagnosis was 58.5 years (range: 28 to 84 years). To determine the IL-6 (-174 G/C) polymorphism, molecular analysis was performed by polymerase chain reaction followed by endonuclease restriction digestion. The genotype distributions observed in the group of patients were 4% CC, 42% GC and 54% GG. The C allele frequency was 0.25. These results were similar to the control group, suggesting no impact of this polymorphism on the susceptibility to MM.

Analysis of polymorphism at site -174 G/C of interleukin-6 promoter region in multiple myeloma

It is well established that interleukin-6 (IL-6) is an essential growth factor for multiple myeloma (MM) and patients with increased IL-6 levels have a poor prognosis. In healthy subjects, the presence of the C allele at a polymorphic site (-174 G/C) of the IL-6 gene is related to low IL-6 levels. In view of the potential association of this particular polymorphism with IL-6 concentration, and the relevance of IL-6 in MM pathogenesis, the objective of the present study was to investigate the prevalence of IL-6 (-174 G/C) promoter polymorphism and its association with development of MM in Brazilian individuals. We investigated the prevalence of these alleles in 52 patients and 60 healthy subjects (matched by age, sex, and race) of a Brazilian population. Thirty patients were male (42.4 percent), 24 (46.2 percent) were white and the median age at diagnosis was 58.5 years (range: 28 to 84 years). To determine the IL-6 (-174 G/C) polymorphism, molecular analysis was performed by polymerase chain reaction followed by endonuclease restriction digestion. The genotype distributions observed in the group of patients were 4 percent CC, 42 percent GC and 54 percent GG. The C allele frequency was 0.25. These results were similar to the control group, suggesting no impact of this polymorphism on the susceptibility to MM.

Revisión del tratamiento quimioterápico del cáncer de colon metastásico / A review of chemotherapy for metastatic colon cancer

Objetivo: Mostrar y discutir los resultados de los ensayos clínicospublicados sobre el tratamiento quimioterápico del cáncercolorrectal metastásico (CCRm) con el fin de esclarecer y definirlas líneas de tratamiento.Método: Se realizó una búsqueda en la base de datos biomédicay farmacéutica EMBASE (enero de 1998-enero de 2006) deensayos clínicos en fases II y III o metanálisis que estudiaban el tratamientoquimioterápico del CCRm.Resultados: El papel preponderante que ha mantenido el 5-fluorouracilo en las últimos 40 años se mantiene, y su modulacióncon la adición de ácido folínico o mediante su administración enperfusiones más prolongadas ha conseguido aumentar ligeramentela supervivencia de estos pacientes y mejorar el perfil de toxicidaddel fármaco. El desarrollo del irinotecán primero y posteriormentedel oxaliplatino ha logrado extender en unos meses lasupervivencia cuando se combinan en esquemas con 5-fluorouraciloy folínico. Todos los pacientes debieran tener garantizada ladisponibilidad de ambos fármacos durante el transcurso de suenfermedad ya que ello incrementa su supervivencia. El raltitrexedno consigue mejorar la supervivencia y, además, disminuye la calidadde vida de estos pacientes por lo que su uso queda relegado aensayos clínicos.El bevacizumab, en combinación con los esquemas clásicos dequimioterapia, consigue supervivencias ligeramente superiores.Los resultados de los estudios del cetuximab no son tan contundentesy su uso debería quedar relegado a pacientes en recaídatras el uso de irinotecán y sin otras alternativas de tratamiento.Conclusiones: Los nuevos fármacos contra el cáncer colorrectalmetastásico abren nuevas líneas de tratamiento que permitenconseguir supervivencias cada vez mejores. Sin embargo, losesquemas óptimos, así como el orden de administración no hansido completamente definidos todavía

Objective: To report and discuss the results of clinical trialspublished concerning chemotherapy for metastatic colorectal cancerin order to elucidate and define treatment guidelines.Method: The biomedical and pharmaceutical databaseEMBASE was searched for phase-II and -III clinical trials or metaanalysisthat examined chemotherapy for mCRC (January 1998 -January 2006).Results: 5-fluorouracil still maintains its preponderant roleafter 40 years, and its modulation by adding folinic acid or administeringit in more prolonged infusions has managed to slightlyincrease survival in these patients, as well as to improve the drug’stoxicity profile. The development of irinotecan first, and thenoxaliplatin has lengthened survival by a few months when in combinationwith 5-fluorouracil and folinic acid-based regimens. Allpatients should have access to these two drugs guaranteed duringthe course of the disease, as it will increase survival. Raltitrexedfails to improve survival, and also diminishes quality of life in thesepatients; hence its use has been relegated to clinical trials.Bevacizumab, combined with classic chemotherapy regimens,attains slightly longer survivals. Results from studies with cetuximabare not that conclusive, and its use should be restricted topatients with relapsing disease after irinotecan and no other treatmentoptions left.Conclusions: New drugs for metastatic colorectal canceropen up new therapy lines allowing increasingly improved survival.However, optimal schemes, as well as their order of administration,have yet to be fully devised

Developmental relocation of presynaptic terminals along distinct types of dendritic filopodia.

Dendritic filopodia are long thin protrusions occurring predominantly on developing neurons. Data from different systems suggest a range of crucial functions for filopodia in central circuit formation, including steering of dendritic growth, branch formation, synaptogenesis, and spinogenesis. Are the same filopodia competent to mediate all these processes, do filopodia acquire different functions through development, or do different filopodial types with distinct functions exist? In this study, 3-dimensional reconstructions from confocal image stacks demonstrate the existence of two morphologically and functionally distinct types of filopodia located on the dendritic tips versus the dendritic shafts of the same developing motoneuron. During dendritic growth, both filopodial types undergo a process of stage-specific morphogenesis. Using novel quantification strategies of 3-dimensional co-localization analysis for immunocytochemically labeled presynaptic specializations along postsynaptic filopodia, we find that presynaptic terminals accumulate along filopodia towards the dendrites at both stable dendritic shafts and on growing dendritic tips. On tips, this is likely to reflect synaptotrophic growth of the dendrite. At stable shafts, however, presynaptic sites become relocated along filopodia towards dendritic branches. This indicates the interactive growth of both pre- and postsynaptic partner towards one another during synaptogenesis, using filopodia as guides.

A steroid hormone affects sodium channel expression in Manduca central neurons.

Neuronal differentiation is characterized by stereotypical sequences of membrane channel and receptor acquisition. This is regulated by the coordinated interactions of a variety of developmental mechanisms, one of which is the control by steroid hormones. We have used the metamorphosis of the holometabolous insect, Manduca sexta, as a model to study effects of 20-hydroxyecdysone on the maturation of thoracic neuron membrane channel expression. To test for direct hormone action, neurons were dissociated into primary cell culture on the first day of pupal life. In situ hybridization demonstrated that the amount of expression of the acetylcholine receptor alpha subunit, MARA1, was not affected by 20-hydroxyecdysone. Immunocytochemistry with an antibody directed against the SP19 segment of voltage-gated sodium channels revealed no effect of 20-hydroxyecdysone treatment during the first 6 days in culture. SP19 sodium channel protein was evenly distributed along all neurites. In contrast, after 8 days in culture, 20-hydroxyecdysone increased the amount of SP19 protein expression and strongly affected its distribution in differentiating neurons. In the presence of 20-hydroxyecdysone, patches of high densities of SP19 sodium channel protein were found in growth cones close to the base of filopodia. This is a further step toward unraveling the blend of membrane proteins under the control of steroids during the development of the central nervous system of postembryonic Manduca. Our results, taken together with previous studies, indicate that 20-hydroxyecdysone does not affect the expression of potassium membrane current or of the nicotinic acetylcholine receptor but instead regulates the amplitude of the calcium membrane current and the amount and distribution of SP19 sodium channel protein.

Developmental changes of CaMKII localization, activity and function during postembryonic CNS remodelling in Manduca sexta.

Insect metamorphosis is a compelling example of postembryonic remodelling of neuronal structure and synaptic connectivity as larval and adult behaviours place distinct demands on the CNS. Holometabolous insects such as the moth Manduca sexta have long served as suitable models for the study of steroid effects on CNS remodelling, but activity and calcium-dependent mechanisms have been found to act in concert with hormonal signals. This study examines developmental changes in the localization and the activational state of CaMKII during postembryonic Manduca CNS remodelling. Western blotting, CaMKII purification and autophosphorylation with gamma(32)P-ATP indicate that the lepidopteran CNS may contain only one CaMKII isoform. In situ immunohistochemistry reveals developmental changes in the expression patterns of CaMKII in different types of thoracic neurons and in different neuronal compartments. Early pupal life is characterized by an increase in postsynaptic CaMKII localization, which coincides with a developmental increase in CaMKII activation. Both events correlate temporally with motoneuron dendritic filopodia collapse and rapid synaptogenesis, indicating a possible functional role for CaMKII for the postembryonic development of invertebrate motor circuitry. Substrate phosphorylation assays demonstrate that CaMKII activity in the ventral nerve cord reflects changes in calcium influx through voltage-activated channels as occurring in vivo during normal development.

[A review of chemotherapy for metastatic colon cancer]. / Revisión del tratamiento quimioterápico del cáncer de colon metastásico.

OBJECTIVE: To report and discuss the results of clinical trials published concerning chemotherapy for metastatic colorectal cancer in order to elucidate and define treatment guidelines. METHOD: The biomedical and pharmaceutical database EMBASE was searched for phase-II and -III clinical trials or metaanalysis that examined chemotherapy for mCRC (January 1998-January 2006). RESULTS: 5-fluorouracil still maintains its preponderant role after 40 years, and its modulation by adding folinic acid or administering it in more prolonged infusions has managed to slightly increase survival in these patients, as well as to improve the drug s toxicity profile. The development of irinotecan first, and then oxaliplatin has lengthened survival by a few months when in combination with 5-fluorouracil and folinic acid-based regimens. All patients should have access to these two drugs guaranteed during the course of the disease, as it will increase survival. Raltitrexed fails to improve survival, and also diminishes quality of life in these patients; hence its use has been relegated to clinical trials. Bevacizumab, combined with classic chemotherapy regimens, attains slightly longer survivals. Results from studies with cetuximab are not that conclusive, and its use should be restricted to patients with relapsing disease after irinotecan and no other treatment options left. CONCLUSIONS: New drugs for metastatic colorectal cancer open up new therapy lines allowing increasingly improved survival. However, optimal schemes, as well as their order of administration, have yet to be fully devised.

Progress in functional neuroanatomy: precise automatic geometric reconstruction of neuronal morphology from confocal image stacks.

Dendritic architecture provides the structural substrate for myriads of input and output synapses in the brain and for the integration of presynaptic inputs. Understanding mechanisms of evolution and development of neuronal shape and its respective function is thus a formidable problem in neuroscience. A fundamental prerequisite for finding answers is a precise quantitative analysis of neuronal structure in situ and in vivo. Therefore we have developed a tool set for automatic geometric reconstruction of neuronal architecture from stacks of confocal images. It provides exact midlines, diameters, surfaces, volumes, and branch point locations and allows analysis of labeled molecule distribution along neuronal surfaces as well as direct export into modeling software. We show the high accuracy of geometric reconstruction and the analysis of putative input synapse distribution throughout entire dendritic trees from in situ light microscopy preparations as a possible application. The binary version of the reconstruction module is downloadable at no cost.

Neuromodulatory octopaminergic neurons and their functions during insect motor behaviour. The Ernst Florey memory lecture.

In this article we describe recent advances in functional studies on the role of octopamine released in the periphery by efferent dorsal or ventral unpaired median neurons. In addition to the previously described modulatory effects on the neuromuscular junction, we describe a metabolic regulatory role for these neurons. Due to their activity glycolytic rates in target tissues, such as muscles, are increased. In flight muscles that use carbohydrate catabolism only at take-off but have to switch to lipid oxidation during prolonged flight, these neurons are only active at rest but are inhibited as soon as flight motor patterns are selected.

Characterisation and monitoring of neutralisation-resistant VP2 phenotypes in BTV-1 isolates from northern Australia collected over a twenty-year period.

Phenotypic profiles of the VP2 protein of isolates of bluetongue virus serotype 1 (BTV-1) collected from Queensland and the Northern Territory, Australia, between 1979 and 1986 were analysed using neutralising monoclonal antibodies (MAbs) raised to the prototype isolate of Australian BTV-1 collected in the Northern Territory in 1979. Two distinct profiles were found. Northern Territory isolates exhibited the prototype profile, yet those from Queensland had a significantly different ('resistant') profile. Nucleotide sequencing of gene segment 2 from both groups of isolates was undertaken. When the nucleotide sequences of isolates from a later period in each State were analysed (1997-2001), all exhibited the 'resistant' profile. Thus, a novel VP2 phenotype, already in existence in Queensland, had supplanted a pre-existing VP2 phenotype in the Northern Territory between the two periods. Furthermore, amino acid differences between the resistant and prototype VP2 proteins were analogous to amino acid substitutions known to be associated with neutralisation resistance. The host immune response may therefore have contributed to selection of the 'resistant' phenotype.

Stage-specific activity patterns affect motoneuron axonal retraction and outgrowth during the metamorphosis of Manduca sexta.

During the metamorphosis of holometabolous insects, most larval muscles and sensory neurons are replaced by new adult elements, whereas most motoneurons persist and are remodelled to serve new adult functions. In Manduca sexta, the formation of the anlagen of the adult dorsal longitudinal flight muscle (DLM) is characterized by retraction of axonal terminals and dendrites of persisting larval motoneurons, partial target muscle degeneration and myoblast accumulation during late larval life. Most of these structural changes have been attributed to hormonal control, not only because ecdysteroids govern metamorphosis, but also because motoneurons express ecdysteroid receptors and experimental manipulations of ecdysteroid titres perturb normal development. To test whether activity-dependent mechanisms also came into play, chronic extracellular recordings were conducted in vivo from the five future DLM motoneurons throughout the last 3 days of larval life. Motoneuron activity is regulated developmentally. The types of motoneurons recruited, the number of motor spikes and the duration of bursts change in a stereotypical fashion during different stages, indicating an internal control of motor activity. A characteristic cessation in the activity of the five future DLM motoneurons coincides in time with the retraction of their dendrites and their terminal arborizations, whereas their activation during ecdysis coincides with the onset of new outgrowth. Inducing advanced activity by stimulating the motoneurons selectively with ecdysis-like patterns results in significant outgrowth of their terminal arborizations. Therefore, steroids might act in concert with activity-dependent mechanisms during the postembryonic modifications of neuromuscular systems.

Changes in calcium signaling during postembryonic dendritic growth in Manduca sexta.

Activity-dependent Ca(2+) influx plays crucial roles in adult and developing nervous systems through its influence on signal processing, synaptic plasticity, and neuronal differentiation. The responses to internal Ca(2+) elevations vary depending on the spatial distribution of Ca(2+) accumulation in different cell compartments. In this study, the mechanisms and the distribution of Ca(2+) accumulation are addressed by in situ Ca(2+) imaging of an identified insect motoneuron, MN5, at critical stages of postembryonic life. During metamorphosis of Manduca sexta, MN5 undergoes extensive dendritic regression followed by regrowth. The time course, amplitude, and distribution of Ca(2+) accumulation within MN5 change during development. During the initial stage of rapid dendritic growth and branching, dendritic growth cones are present, and voltage-dependent Ca(2+) currents are small. At this stage, activity-induced elevations of internal Ca(2+) are largest in the distal dendrites, suggesting that the density of voltage-gated Ca(2+) channels is highest in these regions. Later phases of dendritic growth are accompanied by the transient occurrence of prominent Ca(2+) spikes. Single Ca(2+) spikes cause robust Ca(2+) influx of similar amplitudes and time courses in all central compartments of MN5. The resting Ca(2+) levels also increase during development. Ca(2+)-induced Ca(2+) release from intracellular stores did not contribute to the elevations measured at either stage, although Ca(2+) stores are present in the dendrites. These developmental changes of the internal Ca(2+) signaling are consistent with a regulatory role for activity-dependent Ca(2+) influx in postembryonic dendritic growth.

Nerve-muscle interactions regulate motor terminal growth and myoblast distribution during muscle development.

Interactions between motoneurons and muscles influence many aspects of neuromuscular development in all animals. These interactions can be readily investigated during adult muscle development in holometabolous insects. In this study, the development of the dorsolongitudinal flight muscle (DLM) and its innervation is investigated in the moth, Manduca sexta, to address the specificity of neuromuscular interactions. The DLM develops from an anlage containing both regressed larval template fibers and imaginal myoblasts. In the adult, each fiber bundle (DLM1-5) is innervated by a single motoneuron (MN1-MN5), with the dorsal-most fiber bundle (DLM5) innervated by a mesothoracic motoneuron (MN5). The DLM failed to develop following complete denervation because myoblasts failed to accumulate in the DLM anlage. After lesioning MN1-4, MN5 retained its specificity for the DLM5 region of the anlage and failed to rescue DLM1-4. Thus specific innervation of the DLM fiber bundles does not depend on interactions among motoneurons. Myoblast accumulation, but not myonuclear proliferation, increased around the MN5 terminals, producing a hypertrophied adult DLM5. Therefore, motoneurons compete for uncommitted myoblasts. MN5 terminals subsequently grew more rapidly over the hypertrophied DLM5 anlage, indicating that motoneuron terminal expansion is regulated by the size of the target muscle anlage.

The functional role of octopaminergic neurons in insect motor behavior.

The role of efferent, octopaminergic dorsal unpaired median (DUM) neurons in insects is examined by recording from them during motor behaviour. This population of neuromodulatory neurons is divided into sub-populations which are specifically activated or inhibited during ongoing motor behavior. These neurons are always activated in parallel to the respective motor circuits, and in addition to their modulatory effects on synaptic transmission may also cause metabolic changes in their target tissues.

Remodeling of membrane properties and dendritic architecture accompanies the postembryonic conversion of a slow into a fast motoneuron.

The postembryonic acquisition of behavior requires alterations in neuronal circuitry, which ultimately must be understood as specific changes in neuronal structure, membrane properties, and synaptic connectivity. This study addresses this goal by describing the postembryonic remodeling of the excitability and dendritic morphology of an identified motoneuron, MN5, which during the metamorphosis of Manduca sexta (L.) changes from a slow motoneuron that is involved in larval-crawling behavior into a fast adult flight motoneuron. A fivefold lower input resistance, a higher firing threshold, and an increase in voltage-activated K(+) current contribute to a lower excitability of the adult MN5, which is a prerequisite for its newly acquired behavioral role. In addition, the adult MN5 displays larger Ca(2+) currents. The dendrites of MN5 undergo extensive remodeling. Drastic regression of larval dendrites during early pupal stages is followed by rapid growth of new dendrites. Critical changes in excitability take place during the onset of adult dendrite formation. Larval Ca(2+) currents are absent when dendritic remodeling is most dramatic but increase markedly during later development. Changes in Ca(2+) and K(+) currents follow different time courses, allowing the transient occurrence of Ca(2+) spikes during pupal stages when new dendritic branching ceases. The adult MN5 can produce prolonged Ca(2+) spikes after K(+) currents are reduced. We suggest that alterations in Ca(2+) and K(+) currents are necessary for the participation of MN5 in flight behavior and that the transient production of Ca(2+) spikes may influence postembryonic dendritic remodeling.

Postembryonic development of the dorsal longitudinal flight muscle and its innervation in Manduca sexta.

The neuromuscular systems of holometabolous insects must be remodeled during metamorphosis to allow striking behavioral changes, such as the acquisition of flight. The fast contracting dorsal longitudinal flight muscle (DLM) of Manduca arises from an anlage containing both remnants of specific larval dorsal body wall muscles and extrinsic myoblasts. In the mesothorax, the DLM is innervated by five persisting larval motoneurons: one in the mesothoracic and four in the prothoracic ganglion. These motoneurons innervate two slowly contracting body wall muscles in the larva. 2 days before pupation, the DLM template fibers begin to degenerate, whereas other muscles remain intact until pupation. Correspondingly, the motor terminals retract from the template fibers while they remain on other muscle fibers until pupation. Accumulation and proliferation of putative myoblasts also starts 2 days before pupation in close spatial relationship to the retracted motor tufts around the degenerating larval template fibers. Proliferation increases through the early pupal stages, and is detected within the anlage until the ninth day after pupation. 2 days after pupation, the anlage splits into five bundles, each innervated by one motoneuron. Striations occur on the seventh day after pupation when the growing motor axons reach the attachment sites. Subsequently, the muscle grows in volume and higher-order motor branches are formed. Within the central nervous system, there is dramatic regression of larval dendrites followed by growth of new dendrites as the persistent motoneurons assume their new role in flight behavior. Both central and peripheral remodeling follow similar time courses.