Using C. elegans to decipher the cellular and molecular mechanisms underlying neurodevelopmental disorders

Prova tipográfica (uncorrected proof)Neurodevelopmental disorders such as epilepsy, intellectual disability (ID), and autism spectrum disorders (ASDs) occur in over 2 % of the population, as the result of genetic mutations, environmental factors, or combination of both. In the last years, use of large-scale genomic techniques allowed important advances in the identification of genes/loci associated with these disorders. Nevertheless, following association of novel genes with a given disease, interpretation of findings is often difficult due to lack of information on gene function and effect of a given mutation in the corresponding protein. This brings the need to validate genetic associations from a functional perspective in model systems in a relatively fast but effective manner. In this context, the small nematode, Caenorhabditis elegans, presents a good compromise between the simplicity of cell models and the complexity of rodent nervous systems. In this article, we review the features that make C. elegans a good model for the study of neurodevelopmental diseases. We discuss its nervous system architecture and function as well as the molecular basis of behaviors that seem important in the context of different neurodevelopmental disorders. We review methodologies used to assess memory, learning, and social behavior as well as susceptibility to seizures in this organism. We will also discuss technological progresses applied in C. elegans neurobiology research, such as use of microfluidics and optogenetic tools. Finally, we will present some interesting examples of the functional analysis of genes associated with human neurodevelopmental disorders and how we can move from genes to therapies using this simple model organism.The authors would like to acknowledge Fundação para a Ciência e Tecnologia (FCT) (PTDC/SAU-GMG/112577/2009). AJR and CB are recipients of FCT fellowships: SFRH/BPD/33611/2009 and SFRH/BPD/74452/2010, respectively

The Source of Spontaneous Activity in the Main Olfactory Bulb of the Rat

In vivo, most neurons in the main olfactory bulb exhibit robust spontaneous activity. This paper tests the hypothesis that spontaneous activity in olfactory receptor neurons drives much of the spontaneous activity in mitral and tufted cells via excitatory synapses.Single units were recorded in vivo from the main olfactory bulb of a rat before, during, and after application of lidocaine to the olfactory nerve. The effect of lidocaine on the conduction of action potentials from the olfactory epithelium to the olfactory bulb was assessed by electrically stimulating the olfactory nerve rostral to the application site and monitoring the field potential evoked in the bulb.Lidocaine caused a significant decrease in the amplitude of the olfactory nerve evoked field potential that was recorded in the olfactory bulb. By contrast, the lidocaine block did not significantly alter the spontaneous activity of single units in the bulb, nor did it alter the field potential evoked by electrical stimulation of the lateral olfactory tract. Lidocaine block also did not change the temporal patters of action potential or their synchronization with respiration.Spontaneous activity in neurons of the main olfactory bulb is not driven mainly by activity in olfactory receptor neurons despite the extensive convergence onto mitral and tufted cells. These results suggest that spontaneous activity of mitral and tufted is either an inherent property of these cells or is driven by centrifugal inputs to the bulb

Pharmacological Analysis of Ionotropic Glutamate Receptor Function in Neuronal Circuits of the Zebrafish Olfactory Bulb

Although synaptic functions of ionotropic glutamate receptors in the olfactory bulb have been studied in vitro, their roles in pattern processing in the intact system remain controversial. We therefore examined the functions of ionotropic glutamate receptors during odor processing in the intact olfactory bulb of zebrafish using pharmacological manipulations. Odor responses of mitral cells and interneurons were recorded by electrophysiology and 2-photon Ca2+ imaging. The combined blockade of AMPA/kainate and NMDA receptors abolished odor-evoked excitation of mitral cells. The blockade of AMPA/kainate receptors alone, in contrast, increased the mean response of mitral cells and decreased the mean response of interneurons. The blockade of NMDA receptors caused little or no change in the mean responses of mitral cells and interneurons. However, antagonists of both receptor types had diverse effects on the magnitude and time course of individual mitral cell and interneuron responses and, thus, changed spatio-temporal activity patterns across neuronal populations. Oscillatory synchronization was abolished or reduced by AMPA/kainate and NMDA receptor antagonists, respectively. These results indicate that (1) interneuron responses depend mainly on AMPA/kainate receptor input during an odor response, (2) interactions among mitral cells and interneurons regulate the total olfactory bulb output activity, (3) AMPA/kainate receptors participate in the synchronization of odor-dependent neuronal ensembles, and (4) ionotropic glutamate receptor-containing synaptic circuits shape odor-specific patterns of olfactory bulb output activity. These mechanisms are likely to be important for the processing of odor-encoding activity patterns in the olfactory bulb

Isolation and Culture of Larval Cells from C. elegans

Cell culture is an essential tool to study cell function. In C. elegans the ability to isolate and culture cells has been limited to embryonically derived cells. However, cells or blastomeres isolated from mixed stage embryos terminally differentiate within 24 hours of culture, thus precluding post-embryonic stage cell culture. We have developed an efficient and technically simple method for large-scale isolation and primary culture of larval-stage cells. We have optimized the treatment to maximize cell number and minimize cell death for each of the four larval stages. We obtained up to 7.8×104 cells per microliter of packed larvae, and up to 97% of adherent cells isolated by this method were viable for at least 16 hours. Cultured larval cells showed stage-specific increases in both cell size and multinuclearity and expressed lineage- and cell type-specific reporters. The majority (81%) of larval cells isolated by our method were muscle cells that exhibited stage-specific phenotypes. L1 muscle cells developed 1 to 2 wide cytoplasmic processes, while L4 muscle cells developed 4 to 14 processes of various thicknesses. L4 muscle cells developed bands of myosin heavy chain A thick filaments at the cell center and spontaneously contracted ex vivo. Neurons constituted less than 10% of the isolated cells and the majority of neurons developed one or more long, microtubule-rich protrusions that terminated in actin-rich growth cones. In addition to cells such as muscle and neuron that are high abundance in vivo, we were also able to isolate M-lineage cells that constitute less than 0.2% of cells in vivo. Our novel method of cell isolation extends C. elegans cell culture to larval developmental stages, and allows use of the wealth of cell culture tools, such as cell sorting, electrophysiology, co-culture, and high-resolution imaging of subcellular dynamics, in investigation of post-embryonic development and physiology

Origins of cellular geometry

Cells are highly complex and orderly machines, with defined shapes and a startling variety of internal organizations. Complex geometry is a feature of both free-living unicellular organisms and cells inside multicellular animals. Where does the geometry of a cell come from? Many of the same questions that arise in developmental biology can also be asked of cells, but in most cases we do not know the answers. How much of cellular organization is dictated by global cell polarity cues as opposed to local interactions between cellular components? Does cellular structure persist across cell generations? What is the relationship between cell geometry and tissue organization? What ensures that intracellular structures are scaled to the overall size of the cell? Cell biology is only now beginning to come to grips with these questions

A note on internal and external reference in efficiency wage models

Efficiency wages, Wage rigidity, Internal and external reference wage, E24, E32, J50,

Ca2+-activated Cl− currents are dispensable for olfaction

International audienceCanonical olfactory signal transduction involves the activation of cAMP-activated cation channels that depolarize the cilia of receptor neurons and raise intracellular calcium. Calcium then activates Cl- currents that may be up to 10-fold larger than cation currents and are believed to powerfully amplify the response. We now unambiguously identified Ano2 (Anoctamin2, TMEM16B) as the long-sought ciliary Ca++-activated Cl- channel of olfactory receptor neurons. Ano2 is expressed in the main olfactory epithelium (MOE) and in the vomeronasal organ (VNO) that additionally expresses the related Ano1 channel. Disruption of Ano2 in mice virtually abolished Ca++-activated Cl- currents in the MOE and VNO. Surprisingly, Ano2 disruption reduced fluid phase electroolfactogram responses by only ~40%, did not change air phase electroolfactograms, and did not reduce performance in olfactory behavioral tasks. In contrast to the current view, cyclic nucleotide-gated cation channels do not need a boost by Cl- channels to achieve near-physiological levels of olfaction