Dual spatio-temporal regulation of axon growth and microtubule dynamics by RhoA signaling pathways.

RhoA plays a crucial role in neuronal polarization, where its action restraining axon outgrowth has been thoroughly studied. We now report that RhoA has not only an inhibitory but also a stimulatory effect on axon development depending on when and where exerts its action and the downstream effectors involved. In cultured hippocampal neurons, FRET imaging revealed that RhoA activity selectively localized in growth cones of undifferentiated neurites, whereas in developing axons it displayed a biphasic pattern, being low in nascent axons and high in elongating ones. RhoA-Rho kinase (ROCK) signaling prevented axon initiation but had no effect on elongation, whereas formin inhibition reduced axon extension without significantly altering initial outgrowth. In addition, RhoA-mDia signaling promoted axon elongation by stimulating growth cone microtubule stability and assembly, as opposed to RhoA-ROCK signaling, which restrained growth cone microtubule assembly and protrusion.

Extracellular matrix stiffness negatively affects axon elongation, growth cone area and F-actin levels in a collagen type I 3D culture.

Three dimensional (3D) in vitro neuronal cultures can better reproduce physiologically relevant phenotypes compared to 2D-cultures, because in vivo neurons reside in a 3D microenvironment. Interest in neuronal 3D cultures is emerging, with special attention to the mechanical forces that regulate axon elongation and sprouting in three dimensions. Type I collagen (Col-I) is a native substrate since it is present in the extracellular matrix and hence emulates an in vivo environment to study axon growth. The impact of its mechanical properties needs to be further investigated. Here, we generated Col-I 3D matrices of different mechanical stiffness and evaluated axon growth in three dimensions. Superior cervical ganglion (SCG) explants from neonatal rats were cultured in soft and stiff Col-I 3D matrices and neurite outgrowth was assessed by measuring: maximum neuritic extent; neuritic halo area and fasciculation. Axonal cytoskeletal proteins were examined. Axon elongation in stiff Col-I 3D matrices was reduced (31%) following 24 h in culture compared to soft matrices. In stiff matrices, neurites fasciculated and formed less dense halos. Consistently, almost no F-actin rich growth cones were recognized, and F-actin staining was strongly reduced in the axonal compartment. This study shows that stiffness negatively affects 3D neurite outgrowth and adds insights on the cytoskeletal responses upon mechanic interactions of axons with a 3D environment. Our data will serve to facilitate the development of model systems that are mechanically well-behaved but still mimic key physiologic properties observed in vivo.

Microcephaly gene Cenpj regulates axonal growth in cortical neurons through microtubule destabilization.

Neocortex development comprises of a complex series of time- and space-specific processes to generate the typical interconnected six-layered architecture of adult mammals. Axon growth is required for the proper establishment of cortical circuits. Malformations in axonal growth and pathfinding might lead to severe neuropathologies, such as corpus callosum dysgenesis. Cenpj, a microcephaly gene, encodes a scaffold protein that regulates centrosome biogenesis and microtubule stabilization. During corticogenesis, Cenpj regulates progenitor division and neuronal migration. Since microtubule stabilization is crucial for axon extension, we investigated the role of Cenpj in axon growth during cortical development in a mouse model. Through loss- and gain-of-function assays ex vivo and in utero, we quantified callosal axonal length, branching, and growth cone size compared to controls. We observed that silencing Cenpj results in an increased axonal length. Ex vivo, we assessed the number of branches, the area of growth cones and the stability of microtubules. In silenced Cenpj axons, there were more branches, larger growth cone area, and more stable microtubules. Rescue experiments confirmed that neurons present axonal length comparable to controls. Here we propose that Cenpj regulates axon growth by destabilizing microtubules during cortical development. Finally, our findings suggest that Cenpj might be a novel target for axonal regeneration.

Unraveling Axon Guidance during Axotomy and Regeneration.

During neuronal development and regeneration axons extend a cytoskeletal-rich structure known as the growth cone, which detects and integrates signals to reach its final destination. The guidance cues "signals" bind their receptors, activating signaling cascades that result in the regulation of the growth cone cytoskeleton, defining growth cone advance, pausing, turning, or collapse. Even though much is known about guidance cues and their isolated mechanisms during nervous system development, there is still a gap in the understanding of the crosstalk between them, and about what happens after nervous system injuries. After neuronal injuries in mammals, only axons in the peripheral nervous system are able to regenerate, while the ones from the central nervous system fail to do so. Therefore, untangling the guidance cues mechanisms, as well as their behavior and characterization after axotomy and regeneration, are of special interest for understanding and treating neuronal injuries. In this review, we present findings on growth cone guidance and canonical guidance cues mechanisms, followed by a description and comparison of growth cone pathfinding mechanisms after axotomy, in regenerative and non-regenerative animal models.

Branched actin networks are assembled on microtubules by adenomatous polyposis coli for targeted membrane protrusion.

Cell migration is driven by pushing and pulling activities of the actin cytoskeleton, but migration directionality is largely controlled by microtubules. This function of microtubules is especially critical for neuron navigation. However, the underlying mechanisms are poorly understood. Here we show that branched actin filament networks, the main pushing machinery in cells, grow directly from microtubule tips toward the leading edge in growth cones of hippocampal neurons. Adenomatous polyposis coli (APC), a protein with both tumor suppressor and cytoskeletal functions, concentrates at the microtubule-branched network interface, whereas APC knockdown nearly eliminates branched actin in growth cones and prevents growth cone recovery after repellent-induced collapse. Conversely, encounters of dynamic APC-positive microtubule tips with the cell edge induce local actin-rich protrusions. Together, we reveal a novel mechanism of cell navigation involving APC-dependent assembly of branched actin networks on microtubule tips.

Remodeling of the Actin/Spectrin Membrane-associated Periodic Skeleton, Growth Cone Collapse and F-Actin Decrease during Axonal Degeneration.

Axonal degeneration occurs in the developing nervous system for the appropriate establishment of mature circuits, and is also a hallmark of diverse neurodegenerative diseases. Despite recent interest in the field, little is known about the changes (and possible role) of the cytoskeleton during axonal degeneration. We studied the actin cytoskeleton in an in vitro model of developmental pruning induced by trophic factor withdrawal (TFW). We found that F-actin decrease and growth cone collapse (GCC) occur early after TFW; however, treatments that prevent axonal fragmentation failed to prevent GCC, suggesting independent pathways. Using super-resolution (STED) microscopy we found that the axonal actin/spectrin membrane-associated periodic skeleton (MPS) abundance and organization drop shortly after deprivation, remaining low until fragmentation. Fragmented axons lack MPS (while maintaining microtubules) and acute pharmacological treatments that stabilize actin filaments prevent MPS loss and protect from axonal fragmentation, suggesting that MPS destruction is required for axon fragmentation to proceed.

Identification of Dp71 Isoforms Expressed in PC12 Cells: Subcellular Localization and Colocalization with ß-Dystroglycan and α1-Syntrophin.

Several dystrophin Dp71 messenger RNA (mRNA) alternative splice variants have been described. According to the splicing of exon 78 or intron 77, Dp71 proteins are grouped as Dp71d, Dp71f, and Dp71e, and each group has a specific C-terminal end. In this study, we explored the expression of Dp71 isoforms at the complementary DNA (cDNA) level and the subcellular localization of recombinant Myc-Dp71 proteins in PC12 cells. We determined that PC12 cells express Dp71a, Dp71c, Dp71ab, Dp71e, and Dp71ec mRNA splice variants. In undifferentiated and nerve growth factor-differentiated PC12 Tet-ON cells, Dp71a, Dp71ab, and Dp71e were found to localize and colocalize with ß-dystroglycan and α1-syntrophin in the periphery/cytoplasm, while Dp71c and Dp71ec were mainly localized in the cell periphery and showed less colocalization with ß-dystroglycan and α1-syntrophin. The levels of Dp71a, Dp71e, and Dp71ec were increased in the nucleus of differentiated PC12 Tet-ON cells compared to undifferentiated cells. Dp71 isoforms were also localized in neurite extensions and growth cones.

Neuronal development and axon growth are altered by glyphosate through a WNT non-canonical signaling pathway.

The growth and morphological differentiation of neurons are critical events in the establishment of proper neuronal connectivity and functioning. The developing nervous system is highly susceptible to damage caused by exposure to environmental contaminants. Glyphosate-containing herbicides are the most used agrochemicals in the world, particularly on genetically modified plants. Previous studies have demonstrated that glyphosate induces neurotoxicity in mammals. Therefore, its action mechanism on the nervous system needs to be determined. In this study, we report about impaired neuronal development caused by glyphosate exposure. Particularly, we observed that the initial axonal differentiation and growth of cultured neurons is affected by glyphosate since most treated cells remained undifferentiated after 1 day in culture. Although they polarized at 2 days in vitro, they elicited shorter and unbranched axons and they also developed less complex dendritic arbors compared to controls. To go further, we attempted to identify the cellular mechanism by which glyphosate affected neuronal morphology. Biochemical approaches revealed that glyphosate led to a decrease in Wnt5a level, a key factor for the initial neurite development and maturation, as well as inducing a down-regulation of CaMKII activity. This data suggests that the morphological defects would likely be a consequence of the decrease in both Wnt5a expression and CaMKII activity induced by glyphosate. Additionally, these changes might be reflected in a subsequent neuronal dysfunction. Therefore, our findings highlight the importance of establishing rigorous control on the use of glyphosate-based herbicides in order to protect mammals' health.

Fatores psicossociais e socioeconômicos relacionados à insônia e menopausa: Estudo Pró-Saúde / Psychosocial and socioeconomic factors related to insomnia and menopause: Pró-Saúde Study / Factores psicosociales y socioeconómicos relacionados con el insomnio y la menopausia: Estudio Pro-Salud

Foi avaliada a associação entre menopausa e insônia e a influência de variáveis socioeconômicas e psicossociais, em estudo transversal com 2.190 funcionárias de uma universidade (Estudo Pró-Saúde), a partir de um questionário autopreenchível com variáveis sobre menopausa, insônia, transtorno mental comum, eventos de vida estressantes, apoio social e variáveis socioeconômicas. Odds ratios foram calculados por meio de regressão logística multivariada, com desfecho politômico. Após ajuste para potenciais confundidoras sociodemográficas, mulheres na menopausa há mais de 60 meses apresentaram maior chance de reportar queixas de sono frequentes (OR entre 1,53 e 1,86) do que as que estavam na menopausa há menos de 60 meses. Após os ajustes, no primeiro grupo, para as variáveis psicossociais, a magnitude dos ORs reduziu para 1,53 (IC95%: 0,92-2,52) para dificuldade em iniciar o sono, 1,81 (IC95%: 1,09-2,98) para dificuldade em manter o sono e 1,71 (IC95%: 1,08-2,73) para queixa geral de insônia. Fatores psicossociais podem mediar a manifestação da insônia em mulheres na menopausa.

This study evaluated the association between insomnia and menopausal status and the influence of socioeconomic and psychosocial variables on this association in a cross-sectional analysis of 2,190 university employees (the Pró-Saúde Study). A self-administered questionnaire was used, covering menopausal status, complaints of insomnia, common mental disorders, stressful life events, social support, and socioeconomic variables. Odds ratios were calculated using logistic regression with a polytomous outcome. After adjusting for potential socio-demographic confounders, women who had entered menopause more than 60 months previously were more likely to report complaints with sleep (OR 1.53-1.86) as compared to women in menopause for less than 60 months. After adjusting for psychosocial variables, in the first group the ORs decreased to 1.53 (95%CI: 0.92-2.52) for difficulty initiating sleep, 1.81 (95%CI: 1.09-2.98) for difficulty maintaining sleep, and 1.71 (95%CI: 1.08-2.73) for general complaints of insomnia. Psychosocial factors can mediate the manifestation of insomnia among menopausal women.

En este estudio se evaluó la asociación entre insomnio y menopausia y la influencia de las variables socioeconómicas y psicosociales, en un estudio transversal con 2.190 mujeres de una universidad (Estudio Pro-Salud), a partir de un cuestionario autoadministrado con variables de la menopausia, insomnio, trastornos mentales, situaciones de estrés vital, apoyo social y variables socioeconómicas. Se calcularon los odds ratio mediante regresión logística multivariante con desenlace politómico. Después de ajustar por factores de confusión sociodemográficos potenciales, las mujeres menopáusicas desde hace más de 60 meses fueron más propensas a reportar quejas frecuentes de sueño (OR entre 1,53 y 1,86) que las menopáusicas hace menos de 60 meses. Después de los ajustes, en el primer grupo, para las variables psicosociales la magnitud de los OR se redujo a 1,53 (IC95%: 0,92-2,52) para la dificultad para iniciar el sueño, un 1,81 (IC95%: 1,09-2,98) para mantener el sueño y un 1,71 (IC95%: 1,08-2,73) para las quejas de insomnio en general. Los factores psicosociales pueden mediar en la manifestación del insomnio en las mujeres menopáusicas.

Controlled lateral packing of insulin monolayers influences neuron polarization in solid-supported cultures.

Neurons are highly polarized cells, composed of one axon and several branching dendrites. One important issue in neurobiology is to understand the molecular factors that determine the neuron to develop polarized structures. A particularly early event, in neurons still lacking a discernible axon, is the segregation of IGF-1 (Insulin like Growth Factor-1) receptors in one neurite. This receptor can be activated by insulin in bulk, but, it is not known if changes of insulin organization as a monomolecular film may affect neuron polarization. To this end, in this work we developed solid-supported Langmuir-Blodgett films of insulin with different surface packing density. Hyppocampal pyramidal neurons, in early stage of differentiation, were cultured onto those substrates and polarization was studied after 24 h by confocal microscopy. Also we used surface reflection interference contrast microscopy and confocal microscopy to study attachment patterns and morphology of growth cones. We observed that insulin films packed at 14 mN/m induced polarization in a similar manner to high insulin concentration in bulk, but insulin packed at 44 mN/m did not induce polarization. Our results provide novel evidence that the neuron polarization through IGF-1 receptor activation can be selectively modulated by the lateral packing of insulin organized as a monomolecular surface for cell growth.

Frizzled receptors in neurons: from growth cones to the synapse.

The Wnt signaling pathway has been implicated in several different aspects of neural development and function, including dendrite morphogenesis, axonal growth and guidance, synaptogenesis and synaptic plasticity. Here, we studied several Frizzled Wnt receptors and determined their differential expression during hippocampal development. In cultured hippocampal neurons, the cellular distributions of Frizzleds vary greatly, some of them being localized at neurites, growth cones or synaptic sites. These findings suggest that the Wnt signaling pathway might be temporally and spatially fine tuned during the development of neuronal circuits through specific Frizzled receptors.

Secreted human amyloid precursor protein binds semaphorin 3a and prevents semaphorin-induced growth cone collapse.

The amyloid precursor protein (APP) is well known for giving rise to the amyloid-ß peptide and for its role in Alzheimer's disease. Much less is known, however, on the physiological roles of APP in the development and plasticity of the central nervous system. We have used phage display of a peptide library to identify high-affinity ligands of purified recombinant human sAPPα(695) (the soluble, secreted ectodomain from the main neuronal APP isoform). Two peptides thus selected exhibited significant homologies with the conserved extracellular domain of several members of the semaphorin (Sema) family of axon guidance proteins. We show that sAPPα(695) binds both purified recombinant Sema3A and Sema3A secreted by transfected HEK293 cells. Interestingly, sAPPα(695) inhibited the collapse of embryonic chicken (Gallus gallus domesticus) dorsal root ganglia growth cones promoted by Sema3A (K(d)≤8·10(-9) M). Two Sema3A-derived peptides homologous to the peptides isolated by phage display blocked sAPPα binding and its inhibitory action on Sema3A function. These two peptides are comprised within a domain previously shown to be involved in binding of Sema3A to its cellular receptor, suggesting a competitive mechanism by which sAPPα modulates the biological action of semaphorins.

Análisis morfométrico del proceso de diferenciación in vitro de neuronas del hipocampo / Morphometric analysis of the differentiation process of hippocampal neurons in vitro

Los cultivos neuronales del sistema nervioso central se han venido usando ampliamente para el estudio de los mecanismos que conducen el proceso de diferenciación neuronal, así como también se han empleado como modelos in vitro para evaluar drogas y desarrollar nuevas terapias, de allí la importancia profundizar en la caracterización de dicho proceso. En este estudio, se prepararon cultivos primarios de células del hipocampo para estudiar los tipos celulares desarrollados, el desarrollo de dendritas y axones, la densidad de vesículas sinápticas y el desarrollo de los conos de crecimiento. Mediante inmunofluorescencia usando anticuerpos y marcadores no inmunológicos, se observaron los cambios experimentados por las estructuras de interés durante diferentes estadios temporales (1-21 días). Observamos una mayor proporción de neuronas sobre glias, desarrollo normal de las redes neuronales (conformadas por dendritas y axones), incremento en la longitud de dendritas y el establecimiento de sinapsis. Las vesículas sinápticas también experimentaron un incremento en su densidad a medida que aumentaba el tiempo de cultivo. Finalmente, se estudiaron los cambios morfológicos de los conos de crecimiento observándose que al inicio del cultivo en su mayoría se encontraban cerrados, pero a medida que maduraban las neuronas la proporción de conos de crecimiento abiertos aumentó. Este trabajo representa un avance en la caracterización morfométrica de los cultivos neuronales puesto que recoge de manera simultánea y cuantitativa los principales aspectos que marcan el proceso de diferenciación neuronal. En este estudio, la medición de estas características morfológicas hizo posible establecer parámetros cuantitativos que ayudarán a distinguir las principales etapas de la diferenciación neuronal.

Neuronal cultures of the central nervous system are widely used to study the molecular mechanisms that rule the differentiation process. These cultures have also been used to evaluate drugs and to develop new therapies. From this we can infer the relevance of performing an extended characterization that involves the main aspects driving such process. To carry out such characterization in the present study we prepared primary cultures from hippocampal cells to study cell identity, development of neuronal processes (dendrites and axons), density of synaptic vesicles and development of growth cones. Using immunofluorescence techniques, specific antibodies and non-immunological probes, we studied the changes experienced by the structures under study during different temporal stages (1-21 days). We observed a major proportion of neurons over glia, normal development of neuronal networks (formed by dendrites and axons), increase in the length of dendrites and axons and establishment of synaptic connections. Synaptic vesicles also showed an increase in their densities as long as the time of the culture progressed. Finally, we studied the morphological changes of the growth cones and observed that those were mostly closed at the beginning of the culture period. As neurons matured we observed an increase in the proportion of open growth cones. This work represents an advance in the morphometric characterization of neuronal cultures, since it gathers the main aspects that outline the neuronal differentiation process. In this study, measurement of these morphological features made possible to establish quantitative markers that will allow establishing more precisely the different stages of neuronal differentiation.

Evidence for the involvement of Lfc and Tctex-1 in axon formation.

RhoA and Rac play key and opposite roles during neuronal polarization. We now show that Lfc, a guanosine nucleotide exchange factor (GEF), localizes to the Golgi apparatus and growth cones of developing neurons and negatively regulates neurite sprouting and axon formation through a Rho signaling pathway. Tctex-1, a dynein light chain implicated in axon outgrowth by modulating actin dynamics and Rac activity, colocalizes and physically interacts with Lfc, thus inhibiting its GEF activity, decreasing Rho-GTP levels, and functionally antagonizing Lfc during neurite formation.

Timed changes of synaptic zinc, synaptophysin and MAP2 in medial extended amygdala of epileptic animals are suggestive of reactive neuroplasticity.

Repeated seizures induce permanent alterations of the brain in experimental models and patients with intractable temporal lobe epilepsy (TLE), which is a common form of epilepsy in humans. Together with cell loss and gliosis in many brain regions, synaptic reorganization is observed principally in the hippocampus. However, in the amygdala this synaptic reorganization has been not studied. The changes in Zn density, synaptophysin and MAP(2) as markers of reactive synaptogenesis in medial extended amygdala induced by kainic acid (KA) as a model of TLE was studied. Adult male rats (n=6) were perfused at 10 days, 1, 2, 3 and 4 months after KA i.p. injection (9 mg/kg). Controls were injected with saline. The brains were processed by the Timm's method to reveal synaptic Zn and analyzed by densitometry. Immunohistochemistry was used to reveal synaptophysin and MAP(2) expression. A two-way ANOVA was used for statistics, with a P<0.05 as a significance limit. Normal dark staining was seen in all medial extended amygdala subdivisions of control animals. At 10 days post KA injection a dramatic loss of staining was observed. A slow but steady recovery of Zn density can be followed in the 4 month period studied. Parallel, from 10 days to 2 months stronger synaptophysin expression could be observed, whereas MAP(2) expression increased from 1 month with peak levels at 3-4 months. The results suggest that a process of sprouting exists in surviving neurons of medial extended amygdala after status epilepticus and that these neurons might be an evidence of a reactive synaptogenesis process.

[Morphometric analysis of the differentiation process of hippocampal neurons in vitro]. / Análisis morfométrico del proceso de diferenciación in vitro de neuronas del hipocampo.

Neuronal cultures of the central nervous system are widely used to study the molecular mechanisms that rule the differentiation process. These cultures have also been used to evaluate drugs and to develop new therapies. From this we can infer the relevance of performing an extended characterization that involves the main aspects driving such process. To carry out such characterization in the present study we prepared primary cultures from hippocampal cells to study cell identity, development of neuronal processes (dendrites and axons), density of synaptic vesicles and development of growth cones. Using immunofluorescence techniques, specific antibodies and non-immunological probes, we studied the changes experienced by the structures under study during different temporal stages (1-21 days). We observed a major proportion of neurons over glia, normal development of neuronal networks (formed by dendrites and axons), increase in the length of dendrites and axons and establishment of synaptic connections. Synaptic vesicles also showed an increase in their densities as long as the time of the culture progressed. Finally, we studied the morphological changes of the growth cones and observed that those were mostly closed at the beginning of the culture period. As neurons matured we observed an increase in the proportion of open growth cones. This work represents an advance in the morphometric characterization of neuronal cultures, since it gathers the main aspects that outline the neuronal differentiation process. In this study, measurement of these morphological features made possible to establish quantitative markers that will allow establishing more precisely the different stages of neuronal differentiation.

Schwann cell chondroitin sulfate proteoglycan inhibits dorsal root ganglion neuron neurite outgrowth and substrate specificity via a soma and not a growth cone mechanism.

Sensory axons do not regenerate into or within the spinal cord because of the presence of the axon regeneration inhibitor chondroitin sulfate proteoglycan (CSPG) on activated astrocytes. In the peripheral nervous system, CSPG associated with denervated Schwann cells retards axon regeneration, but regeneration occurs because the balance of regenerating, inhibiting, and promoting factors favors regeneration. The present experiments were aimed at determining the mechanism by which Schwann cells inhibit adult human dorsal root ganglia (H-DRG) neuron growth cone elongation and substrate specificity, restricting the growth cones to Schwann cell membranes and inhibiting their growth onto a poly-l-lysine/laminin substrate. Neurites of H-DRG neurons free of soma contact with Schwann cells, or after the Schwann cell membranes' CSPG had been digested, were 11.1-fold longer than those of neurons in soma contact with untreated Schwann cells. Growth cones of DRG neuron somas without Schwann cell CSPG showed no outgrowth inhibition or substrate specificity. These results indicate that the Schwann cell CSPG influences act via contact with neuron somas but not growth cones. These results suggest that eliminating CSPG associated with Schwann cells within DRG in vivo will make the neurons' growth cones insensitive to the regeneration inhibitory influences of CSPG, allowing them to regenerate through the dorsal root entry zone and into and within the spinal cord, where they can establish appropriate and functional synaptic connections.

Microtubule and cell contact dependency of ER-bound PTP1B localization in growth cones.

PTP1B is an ER-bound protein tyrosine phosphatase implied in the regulation of cell adhesion. Here we investigated mechanisms involved in the positioning and dynamics of PTP1B in axonal growth cones and evaluated the role of this enzyme in axons. In growth cones, PTP1B consistently localizes in the central domain, and occasionally at the peripheral region and filopodia. Live imaging of GFP-PTP1B reveals dynamic excursions of fingerlike processes within the peripheral region and filopodia. PTP1B and GFP-PTP1B colocalize with ER markers and coalign with microtubules at the peripheral region and redistribute to the base of the growth cone after treatment with nocodazole, a condition that is reversible. Growth cone contact with cellular targets is accompanied by invasion of PTP1B and stable microtubules in the peripheral region aligned with the contact axis. Functional impairment of PTP1B causes retardation of axon elongation, as well as reduction of growth cone filopodia lifetime and Src activity. Our results highlight the role of microtubules and cell contacts in the positioning of ER-bound PTP1B to the peripheral region of growth cones, which may be required for the positive role of PTP1B in axon elongation, filopodia stabilization, and Src activity.

Retinal pigment epithelial cells promote spatial reorganization and differentiation of retina photoreceptors.

Retina differentiation involves the acquisition of a precise layered arrangement, with RPE cells in the first layer in intimate contact with photoreceptors in the second layer. Here, we developed an in vitro coculture model, to test the hypothesis that RPE cells play a pivotal role in organizing the spatial structure of the retina. We cocultured rat retinal neurons with ARPE-19 epithelial cells under various experimental conditions. Strikingly, when seeded over RPE cells, photoreceptors attached to their apical surfaces and proceeded with their development, including the increased synthesis of rhodopsin. Conversely, when we seeded RPE cells over neurons, the RPE cells rapidly detached photoreceptors from their substrata and positioned themselves underneath, thus restoring the normal in vivo arrangement. Treatment with the metalloproteinase inhibitor TIMP-1 blocked this reorganization, suggesting the involvement of metalloproteinases in this process. Reorganization was highly selective for photoreceptors because 98% of photoreceptors but very few amacrine neurons were found to redistribute on top of RPE cells. Interestingly, RPE cells were much more efficient than other epithelial or nonepithelial cells in promoting this reorganization. RPE cells also promoted the growth of photoreceptor axons away from them. An additional factor that contributed to the distal arrangement of photoreceptor axons was the migration of photoreceptor cell bodies along their own neurites toward the RPE cells. Our results demonstrate that RPE and photoreceptor cells interact in vitro in very specific ways. They also show that in vitro studies may provide important insights into the process of pattern formation in the retina.

Endurance and resistance exercise training programs elicit specific effects on sciatic nerve regeneration after experimental traumatic lesion in rats.

OBJECTIVE: To evaluate the effects of endurance, resistance, and a combination of both types of exercise training on hindlimb motor function recovery and nerve regeneration after experimental sciatic nerve lesion in rats. METHODS: Sciatic nerve crush was performed on adult male rats, and after 2 weeks of the nerve lesion, the animals were submitted to endurance, resistance, and a combination of endurance-resistance training programs for 5 weeks. Over the training period, functional recovery was monitored weekly using the Sciatic Functional Index (SFI) and histological and morphometric nerve analyses were used to assess the nerve regeneration at the end of the trainings. RESULTS: The SFI values of the endurance-trained group reached the control values from the first posttraining week and were significantly better than both the resistance-trained group at the first, second, and third posttraining weeks and the concurrent training group at the first posttraining week. At the distal portion of the regenerating sciatic nerve, the endurance-trained group showed a greater degree of the myelinated fiber maturation than the sedentary, resistance-trained, and concurrent training groups. Furthermore, the endurance-trained group showed a smaller percentage area of endoneurial connective tissue and a greater percentage area of myelinated fibers than the sedentary group. CONCLUSION: These data provide evidence that endurance training improves sciatic nerve regeneration after an experimental traumatic injury and that resistance training or the combination of 2 strategies may delay functional recovery and do not alter sciatic nerve fiber regeneration.