David Lopes Cardozo

Midbrain dopaminergic neurons from postnatal rat in long-term primary culture

Midbrain dopamine neurons project extensively throughout the vertebrate forebrain and influence a wide variety of brain functions. These neurons, which are believed to form a major brain reward system, are involved in initiation and control of motor programs, addictive behaviors, and determination of mood. Given their critical role in behavioral function, relatively little is known about their fundamental cellular physiological and pharmacological properties. A long-term dissociated culture system for postnatal rat dopamine neurons was developed to permit both acute and chronic studies of these cells. Dopamine neurons were dissociated from slices of ventral midbrain from neonatal rat pups and maintained in cell culture for several months. The dopaminergic phenotype was confirmed by catecholamine fluorescence and by tyrosine hydroxylase immunocytochemistry. After four weeks in culture, dopamine neurons had cell bodies 10-40 microns in diameter, displayed either fusiform or multipolar morphology, and had processes with varicosities of 0.5-2 microns in diameter. Electrophysiological recordings were made from 71 dopamine neurons identified by 5,7-dihydroxytryptamine fluorescence after six to 67 days in culture. The neurons had resting potentials of -51 +/- 5 mV, broad action potentials with durations of 2.9 +/- 1.3 ms, and the majority of the neurons (65%) displayed anomalous rectification. Most dopamine neurons in culture fired spontaneously in a pacemaker-like manner with a frequency of 2.3 +/- 1.3 Hz, or in a bursting pattern, typically having two to seven action potentials per burst. All neurons tested had glutamate and gamma-aminobutyric acid receptors, and 90% of neurons responded to dopamine or quinpirole with inhibition of firing, suggesting the presence of dopamine autoreceptors. Some neurons were inhibited by concentrations of quinpirole as low as 10 nM. The results show that midbrain dopamine neurons can be maintained in dissociated cell culture for periods of several months. These neurons can be identified prior to electrophysiological recording, and they express many of the physiological characteristics that have been reported for midbrain dopamine neurons in vivo.

EGL-36 SHAW CHANNELS REGULATE C. ELEGANS EGG-LAYING MUSCLE ACTIVITY

The C. elegans egl-36 gene encodes a Shaw-type potassium channel that regulates egg-laying behavior. Gain of function [egl-36(gf)] and dominant negative [egl-36(dn)] mutations in egl-36 cause reciprocal defects in egg laying. An egl-36::gfp reporter is expressed in the egg-laying muscles and in a few other tissues. Expression of an egl-36(gf) cDNA in the egg-laying muscles causes behavioral defects similar to those observed in egl-36(gf) mutants. Gain of function EGL-36 subunits form channels that are active at more negative potentials than wild-type channels. The egl-36(gf) alleles correspond to missense mutations in an amino terminal subunit assembly domain (E138K) and in the S6 transmembrane domain (P435S), neither of which were previously implicated in the voltage dependence of channel activation. Altogether, these results suggest that EGL-36 channels regulate the excitability of the egg-laying muscles.

The Isolation, Differentiation, and Survival In Vivo of Multipotent Cells from the Postnatal Rat filum terminale

Neural stem cells (NSCs) are undifferentiated cells in the central nervous system (CNS) that are capable of self-renewal and can be induced to differentiate into neurons and glia. Current sources of mammalian NSCs are confined to regions of the CNS that are critical to normal function and surgically difficult to access, which limits their therapeutic potential in human disease. We have found that the filum terminale (FT), a previously unexplored, expendable, and easily accessible tissue at the caudal end of the spinal cord, is a source of multipotent cells in postnatal rats and humans. In this study, we used a rat model to isolate and characterize the potential of these cells. Neurospheres derived from the rat FT are amenable to in vitro expansion in the presence of a combination of growth factors. These proliferating, FT-derived cells formed neurospheres that could be induced to differentiate into neural progenitor cells, neurons, astrocytes, and oligodendrocytes by exposure to serum and/or adhesive substrates. Through directed differentiation using sonic hedgehog and retinoic acid in combination with various neurotrophic factors, FT-derived neurospheres generated motor neurons that were capable of forming neuromuscular junctions in vitro. In addition, FT-derived progenitors that were injected into chick embryos survived and could differentiate into both neurons and glia in vivo.

The Postnatal Human Filum Terminale Is a Source of Autologous Multipotent Neurospheres Capable of Generating Motor Neurons

BACKGROUND Neural progenitor cells (NPCs) are undifferentiated and mitotic and can be induced to differentiate into neurons and glia, the building blocks of the nervous system. NPCs have great therapeutic potential for nervous system trauma and degenerative disorders. They have been identified in the mammalian central nervous system, but current sources are difficult to access surgically and come from regions that are critical for normal brain function. OBJECTIVE To identify and characterize in detail a novel source of human NPCs in the filum terminale (FT), a vestigial structure at the caudal end of the spinal cord, which is easily accessed and plays no functional role in the postnatal nervous system. METHODS Cells were isolated and cultured in vitro from the FT of terminated fetuses and from children and adolescents who had undergone surgical resections for tethered spinal cords. Cell culture techniques, immunohistochemistry, and immunocytochemistry were applied to examine FT cells. RESULTS : FT cells gave rise to neurospheres that proliferated over extended periods of time in culture. These neurospheres were positive for neural stem/progenitor cell markers by immunocytochemical staining. The neurospheres were able to be induced to differentiate in vitro into neurons and glial cells, which were confirmed by the use of antibodies against the cell type-specific markers. Moreover, they have been induced to form motor neurons capable of innervating striated muscle in vitro. CONCLUSION Multipotent NPC cells from the FT are both accessible and expendable. They may allow autologous cell-based transplantation therapy that circumvents immunological rejection.

A structured PBL tutorial involving small teams for teaching the human nervous system.

Background: The Human Nervous System and Behavior course at Harvard Medical School (HMS) incorporates a hybrid model of problem-based learning. Student preparation for and participation in the tutorial seemed to be insufficient. Aims: We sought to increase student engagement in tutorial by creating a structured approach, which included assigned roles for students, weekly testing, formal cornerstone presentations, and a weekly self-assessment exercise. We wished to determine the students’ and tutors’ satisfaction with this structured approach as compared with the more traditional tutorial experienced in other courses at HMS. Methods: For the first 4 years of the course, students (n = 160) were surveyed concerning their impressions of the quality of the structured approach in comparison with the traditional tutorial. In addition, they were surveyed concerning the cornerstone presentations and the self-assessment exercise. Tutors (n = 10) who had taught in both the traditional and structured tutorial formats were surveyed about their impressions of student performance as well as their own enjoyment in the structured format. Results: Students and tutors found the structured approach superior to the previous method. Both groups noted increased student preparation, participation, and accountability. Tutors preferred teaching in the structured format. Conclusions: The structured approach increased student accountability, preparation, and participation. Students and tutors preferred this tutorial experience over the previous approach.

Diffusion, subdiffusion, and localization of active colloids in random post lattices

Combining experiments and theory, we address the dynamics of self-propelled particles in crowded environments. We first demonstrate that motile colloids cruising at constant speed through random lattices undergo a smooth transition from diffusive to subdiffusive to localized dynamics upon increasing the obstacle density. We then elucidate the nature of these transitions by performing extensive simulations constructed from a detailed analysis of the colloid-obstacle interactions. We evidence that repulsion at a distance and hard-core interactions both contribute to slowing down the long-time diffusion of the colloids. In contrast, the localization transition stems solely from excluded-volume interactions and occurs at the void-percolation threshold. Within this critical scenario, equivalent to that of the random Lorentz gas, genuine asymptotic subdiffusion is found only at the critical density where the motile particles explore a fractal maze.

Direct calculation of the critical Casimir force in a binary fluid.

We show that critical Casimir effects can be accessed through direct simulation of a model binary fluid passing through the demixing transition. We work in the semi-grand-canonical ensemble, in slab geometry, in which the Casimir force appears as the excess of the generalized pressure, P_{⊥}-nμ. The excesses of the perpendicular pressure, P_{⊥}, and of nμ, are individually of much larger amplitude. A critical pressure anisotropy is observed between forces parallel and perpendicular to the confinement direction, which collapses onto a universal scaling function closely related to that of the critical Casimir force.

Use of microsatellite markers in molecular analysis of segregating populations of papaya (Carica papaya L.) derived from backcrossing.

Brazil is the world leader in papaya production. However, only a small number of cultivars are registered for commercial planting, mainly owing to delays in obtaining cultivars and the high costs of the field phase of breeding programs. These costs can be reduced when molecular tools are combined with conventional breeding methods. In the present study, we conducted a molecular analysis of a self-fertilized population of a first backcrossing generation of BC1S1 papaya plants via microsatellite markers both to monitor the level of homozygosity and the gene/allele transfer that confers the Golden trait (fruit color) and to assess the parental genomic proportion in the genotypes studied. Based on the analysis of 20 polymorphic microsatellite loci, 19 genotypes with the Golden trait belonging to BC1S1 were evaluated in addition to the parental genotypes. Genetic distance was estimated through weighted index. The genotypes were then grouped using the hierarchical nearest neighbor method, and the analysis of principal coordinates was used to measure the proportion of parental genomes in the segregating genotypes. The mean value of the inbreeding coefficient was 0.36. The analysis of the principal coordinates revealed that on average, 64% of the recurrent parent genome was present in the population. Together, the analyses allowed the selection of 3 individuals for the next backcross cycle (33BC1S1-18, 34BC1S1-16, and 37BC1S1-10). These individuals had a higher proportion of the recurrent parent and were grouped close to the recurrent parent in the cluster analysis.

Characterization of the Filum terminale as a neural progenitor cell niche in both rats and humans

Neural stem cells (NSCs) reside in a unique microenvironment within the central nervous system (CNS) called the NSC niche. Although they are relatively rare, niches have been previously characterized in both the brain and spinal cord of adult animals. Recently, another potential NSC niche has been identified in the filum terminale (FT), which is a thin band of tissue at the caudal end of the spinal cord. While previous studies have demonstrated that NSCs can be isolated from the FT, the in vivo architecture of this tissue and its relation to other NSC niches in the CNS has not yet been established. In this article we report a histological analysis of the FT NSC niche in postnatal rats and humans. Immunohistochemical characterization reveals that the FT is mitotically active and its cells express similar markers to those in other CNS niches. In addition, the organization of the FT most closely resembles that of the adult spinal cord niche. J. Comp. Neurol. 525:661–675, 2017.

Critical Casimir forces from the equation of state of quantum critical systems

The mapping between a classical length and inverse temperature as imaginary time provides a direct equivalence between the Casimir force of a classical system in