A subset of chicken statoacoustic ganglion neurites are repelled by Slit1 and Slit2.

Mechanosensory hair cells in the chicken inner ear are innervated by bipolar afferent neurons of the statoacoustic ganglion (SAG). During development, individual SAG neurons project their peripheral process to only one of eight distinct sensory organs. These neuronal subtypes may respond differently to guidance cues as they explore the periphery in search of their target. Previous gene expression data suggested that Slit repellants might channel SAG neurites into the sensory primordia, based on the presence of robo transcripts in the neurons and the confinement of slit transcripts to the flanks of the prosensory domains. This led to the prediction that excess Slit proteins would impede the outgrowth of SAG neurites. As predicted, axonal projections to the primordium of the anterior crista were reduced 2-3 days after electroporation of either slit1 or slit2 expression plasmids into the anterior pole of the otocyst on embryonic day 3 (E3). The posterior crista afferents, which normally grow through and adjacent to slit expression domains as they are navigating towards the posterior pole of the otocyst, did not show Slit responsiveness when similarly challenged by ectopic delivery of slit to their targets. The sensitivity to ectopic Slits shown by the anterior crista afferents was more the exception than the rule: responsiveness to Slits was not observed when the entire E4 SAG was challenged with Slits for 40 h in vitro. The corona of neurites emanating from SAG explants was unaffected by the presence of purified human Slit1 and Slit2 in the culture medium. Reduced axon outgrowth from E8 olfactory bulbs cultured under similar conditions for 24 h confirmed bioactivity of purified human Slits on chicken neurons. In summary, differential sensitivity to Slit repellents may influence the directional outgrowth of otic axons toward either the anterior or posterior otocyst.

Automated condition-invariable neurite segmentation and synapse classification using textural analysis-based machine-learning algorithms.

High-resolution live-cell imaging studies of neuronal structure and function are characterized by large variability in image acquisition conditions due to background and sample variations as well as low signal-to-noise ratio. The lack of automated image analysis tools that can be generalized for varying image acquisition conditions represents one of the main challenges in the field of biomedical image analysis. Specifically, segmentation of the axonal/dendritic arborizations in brightfield or fluorescence imaging studies is extremely labor-intensive and still performed mostly manually. Here we describe a fully automated machine-learning approach based on textural analysis algorithms for segmenting neuronal arborizations in high-resolution brightfield images of live cultured neurons. We compare performance of our algorithm to manual segmentation and show that it combines 90% accuracy, with similarly high levels of specificity and sensitivity. Moreover, the algorithm maintains high performance levels under a wide range of image acquisition conditions indicating that it is largely condition-invariable. We further describe an application of this algorithm to fully automated synapse localization and classification in fluorescence imaging studies based on synaptic activity. Textural analysis-based machine-learning approach thus offers a high performance condition-invariable tool for automated neurite segmentation.

DSCAM and DSCAML1 function in self-avoidance in multiple cell types in the developing mouse retina.

DSCAM and DSCAM-LIKE1 (DSCAML1) serve diverse neurodevelopmental functions, including axon guidance, synaptic adhesion, and self-avoidance, depending on the species, cell type, and gene family member studied. We examined the function of DSCAM and DSCAML1 in the developing mouse retina. In addition to a subset of amacrine cells, Dscam was expressed in most retinal ganglion cells (RGCs). RGCs had fasciculated dendrites and clumped cell bodies in Dscam(-/-) mice, suggesting a role in self-avoidance. Dscaml1 was expressed in the rod circuit, and mice lacking Dscaml1 had fasciculated rod bipolar cell dendrites and clumped AII amacrine cell bodies, also indicating a role in self-avoidance. Neurons in Dscam or Dscaml1 mutant retinas stratified their processes appropriately in synaptic laminae in the inner plexiform layer, and functional synapses formed in the rod circuit in mice lacking Dscaml1. Therefore, DSCAM and DSCAML1 function similarly in self-avoidance, and are not essential for synaptic specificity in the mouse retina.

Avaliação neurofuncional no pré e pós-operatório de neurólise no dano neural devido à hanseníase / Pre and post operative neurofunctional assessment of neurolysis surgery in nerve damage in leprosy patients

O presente estudo teve como objetivo analisar, por meio do exame neurofuncional, a eficácia da cirurgia de neurólise em pacientes com neurite hansênica. Tratou-se de um estudo prospectivo, do tipo descritivo. Teve como universo de estudo a população portadora de neurite hansênica do Estado do Amazonas, tratada na FUAM-AM. Foram selecionados 27 participantes os quais foram triados para cirurgia eletiva de neurólise no período de outubro de 2004 a janeiro de 2005, correspondendo a 64 cirurgias eletivas. Foi realizada uma avaliação neurofuncional pré-operatória compreendendo o teste de força muscular manual, teste de sensibilidade pelos estesiômetros de Semmes-Weinstein, avaliação da dor por meio de escala analógica visual e classificação do grau de incapacidade.A mesma avaliação foi repetida um mês após a cirurgia. Os principais nervos operados foram, em ordem decrescente, medial e ulnar, fibular comum e tibial posterior. A forma clínica mais comum entre os mesmos foi a virchowiana (67 por cento), seguida da dimorfa-virchoviana (19 por cento), tuberculóide (7 por cento) e dimorfa-tuberculóide (7 por cento). Setenta e quatro por cento dos pacientes operados encontravam-se em alta clínica e 26 por cento ainda realizavam tratamento por poliquimioterapia. A principal queixa apresentada foi dor (55 por cento). Em relação ao grau de incapacidade, 70 por cento apresentaram grau I e 30 por cento apresentaram grau II. No pós-operatório, foi relatada melhora da dor em 64 por cento das cirurgias, 30 por cento não apresentaram alteração e 6 por cento apresentaram piora. Cinqüenta e seis por cento dos nervos com comprometimento motor não apresentaram melhora, 32 por cento melhoraram em até um grau e 12 por cento apresentaram piora em até um grau. Trinta e quatro por cento dos pacientes apresentaram melhora da sensibilidade, e 66 por cento se mostraram com quadro inalterado.

The aim of this study was to analyze, through neurofunctional assessment, the effectiveness of the neurolysis surgery in patients with neuritis due to leprosy. It was a prospective and descriptive study. The subjects of this study were the population with neuritis due to leprosy of Amazon state, treated in Fundação Alfredo da Matta (FUAM-AM). 27 participants were selected for elective surgery of neurolysis from October 2004 to January 2005, corresponding to 64 elective surgeries. A preoperative neurofunctional assessment was carried out including manual muscle strength testing, sensibility test using Semmes-Weinstein nylon filaments, pain assessment using a visual analogical scale and classification of rate disability. The same assessment was repeated one month after surgery. The main nerves that underwent surgery were, in decreasing order, medial and ulnar, fibular and posterior tibial. The clinical form more frequently was the lepromatous form (67 percent), following by the lepromatous-borderline (19 percent), tuberculoid (7 percent) and tuberculoid-borderline (7 percent). Seventy four percent of patients had already finished clinical treatment and 26 percent were still using multidrug therapy. Fifty five percent referred pain complaint. Concerning rate disability, 70 percent had level I and 30 percent level II. During the postoperative, patients referred to get better pain sensation in 64 percent of surgeries, 30 percent did not have alteration and 6 percent were worst. Fifty six percent of motor nerves degeneration did not improve, 32 percent improved one level and 12 percent have become worse one level. Thirty four percent of patients referred improvement of sensibility and 66 percent referred no improvement.

Characterization of glucagon-expressing neurons in the chicken retina.

We recently identified large glucagon-expressing neurons that densely ramify neurites in the peripheral edge of the retina and regulate the proliferation of progenitors in the circumferential marginal zone (CMZ) of the postnatal chicken eye (Fischer et al. [2005] J Neurosci 25:10157-10166). However, nothing is known about the transmitters and proteins that are expressed by the glucagon-expressing neurons in the avian retina. We used antibodies to cell-distinguishing markers to better characterize the different types of glucagon-expressing neurons. We found that the large glucagon-expressing neurons were immunoreactive for substance P, neurofilament, Pax6, AP2alpha, HuD, calretinin, trkB, and trkC. Colocalization of glucagon and substance P in the large glucagon-expressing neurons indicates that these cells are the "bullwhip cells" that have been briefly described by Ehrlich et al. ([1987] J Comp Neurol 266:220-233). Similar to the bullwhip cells, the conventional glucagon-expressing amacrine cells were immunoreactive for calretinin, HuD, Pax6, and AP2alpha. Unlike bullwhip cells, the conventional glucagon-expressing amacrine cells were immunoreactive for GABA. While glucagon-immunoreactive amacrine cells were negative for substance P in central regions of the retina, a subset of this type of amacrine cell was immunoreactive for substance P in far peripheral regions of the retina. An additional type of glucagon/substance P-expressing neuron, resembling the bullwhip cells, was found in far peripheral and dorsal regions of the retina. Based on morphology, distribution within the retina, and histological markers, we conclude that there may be four different types of glucagon-expressing neurons in the avian retina.

Cell adhesion molecule L1 promotes neurite outgrowth of septal neurons.

To establish if the cell adhesion molecule L1 could promote neurite outgrowth of septal neurons, L1-positive substrates were prepared by genetically modifying 3T3 fibroblasts with a retroviral vector encoding human L1 under the control of a negative tetracycline-regulatory system. In several clones of L1-transfected fibroblasts, L1 expression at the cell surface was prominent and efficiently regulated by doxycycline, a tetracycline analogue. In co-culture of septal neurons and fibroblasts, a two-dimensional fractionator probe provided systematic random sampling of the neurites to be measured. Septal neurons, isolated at embryonic Day 17, were found to express L1 in vitro and to extend significantly longer neurites when plated on L1-expressing fibroblasts compared to control fibroblasts. The neurite outgrowth-promoting effect of L1 was inhibited after a doxycycline treatment, which specifically suppressed L1 expression from the modified fibroblasts. The findings that septal neurons at embryonic Day 17 in vitro express L1 and respond to L1-modulation suggest that this molecule is involved in development of the septohippocampal pathway.

Responses of rat trigeminal neurones to dental pulp cells or fibroblasts overexpressing neurotrophic factors in vitro.

The adult dental pulp is innervated by sensory trigeminal axons and efferent sympathetic axons. Rat trigeminal ganglia extend neurites when co-cultivated in vitro with pulpal tissue explants, suggesting that pulpal cells secrete soluble molecules that stimulate the growth of trigeminal ganglion axons. In addition, cultured pulpal cells produce mRNAs for neurotrophins and glial cell line-derived neurotrophic factor-family members. These data suggest that neurotrophic factors are involved in the formation of a pulpal innervation. Here, we examine how pulpal cells and 3T3 fibroblasts overexpressing certain neurotrophic factors (nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, neurotrophin-4, glial cell line-derived neurotrophic factor or neurturin) influence survival and growth of single trigeminal ganglion neurones in vitro in quantitative terms. The results show that most of the neurotrophic factor-overexpressing fibroblasts induce similar neuronal soma diameters, but higher survival rates and neurite lengths compared with pulpal cells. With respect to neurite growth pattern, trigeminal ganglion neurones co-cultured with fibroblasts overexpressing nerve growth factor develop a geometry that is most similar to that seen in co-cultures with pulpal cells. We conclude that none of the fibroblasts overexpressing neurotrophic factors can fully mimic the effects of pulpal cells on trigeminal ganglion neurones, and that nerve growth factor promotes a neurite growth pattern most similar to the picture seen in co-cultures with pulpal cells.

Contribution of bradykinin and nitric oxide to AT2 receptor-mediated differentiation in PC12 W cells.

We investigated the effect of angiotensin II on intracellular cyclic GMP content and neurite outgrowth as an indicator of cell differentiation in PC12 W cells. Neurite outgrowth was examined by phase-contrast microscopy. Outgrown neurites were classified as small, medium and large, and were expressed as neurites per 100 cells. Angiotensin II (10-7 m) increased the outgrowth of medium and large neurites by mean +/- SEM 20.2 +/- 2.3 and 6.6 +/- 1.4 compared with 1.66 +/- 0.5 and 0.1 +/- 0.06 neurites per 100 cells in control. Cellular cyclic GMP content increased by 50-250% with angiotensin II at concentrations of 10-6-10-4 m. Both blockade of AT2 receptors and of nitric oxide synthase markedly reduced angiotensin II-induced neurite outgrowth and cyclic GMP production. In contrast, B2 receptor blockade had no effect or even increased these angiotensin II effects. Sodium nitroprusside and 8-bromo-cyclic GMP both mimicked the effects of angiotensin II on cell differentiation. The protein kinase G inhibitor KT-5823 inhibited the neurite outgrowth induced by both angiotensin II and 8-bromo-cyclic GMP. Our results demonstrate that angiotensin II can stimulate cell differentiation in PC12 W cells by nitric oxide-related and cyclic GMP-dependent mechanisms. The effects of angiotensin II on cell differentiation and cyclic GMP production were mediated via the AT2 receptor and further enhanced by bradykinin B2 receptor blockade.

Disability prevention and management in leprosy: a field experience

Bombay leprosy project has conducted operational research into cost effective ways of using therapeutic management for prevention of disabilities (POD). The goal of achieving this are broadly divided as 1)prevention of impairments and disabilities(POID) and 2)prevention of worsening of disabilities (POWD). About 33-56% of newly registered leprosy patients already have clinically detectable nerve function impairment (NFI), often no longer amenable to MDT. An analysis of 892 leprosy cases treated with WHO-MD stresses the need to focus attention on leprosy patients...