Didier Chatenay

Constrained synaptic connectivity in functional mammalian neuronal networks grown on patterned surfaces

The use of ordered neuronal networks in vitro is a promising approach to study the development and the activity of small neuronal assemblies. However, in previous attempts, sufficient growth control and physiological maturation of neurons could not be achieved. Here we describe an original protocol in which polylysine patterns confine the adhesion of cellular bodies to prescribed spots and the neuritic growth to thin lines. Hippocampal neurons in these networks are maintained healthy in serum free medium up to 5 weeks in vitro. Electrophysiology and immunochemistry show that neurons exhibit mature excitatory and inhibitory synapses and calcium imaging reveals spontaneous activity of neurons in isolated networks. We demonstrate that neurons in these geometrical networks form functional synapses preferentially to their first neighbors. We have, therefore, established a simple and robust protocol to constrain both the location of neuronal cell bodies and their pattern of connectivity. Moreover, the long term maintenance of the geometry and the physiology of the networks raises the possibility of new applications for systematic screening of pharmacological agents and for electronic to neuron devices.

Automated imaging of neuronal activity in freely behaving Caenorhabditis elegans.

In order to understand how neuronal circuits control locomotory patterns it is necessary to record neuronal activity of freely behaving animals. Here, using a new automated system for simultaneous recording of behavior and neuronal activity in freely moving Caenorhabditis elegans on standard agar plates, we show that spontaneous reversals from forward to backward locomotion reflect precisely the activity of the AVA command interneurons. We also witness spontaneous activity transients in the PLM sensory neurons during free behavior of the worm in standard conditions. We show that these activity transients are coupled to short spontaneous forward accelerations of the worm.

Probing complex RNA structures by mechanical force

Abstract.RNA secondary structures of increasing complexity are probed combining single molecule stretching experiments and stochastic unfolding/refolding simulations. We find that force-induced unfolding pathways cannot usually be interpreted by solely invoking successive openings of native helices. Indeed, typical force-extension responses of complex RNA molecules are largely shaped by stretching-induced, long-lived intermediates including non-native helices. This is first shown for a set of generic structural motifs found in larger RNA structures, and then for Escherichia coli’s 1540-base long 16S ribosomal RNA, which exhibits a surprisingly well-structured and reproducible unfolding pathway under mechanical stretching. Using out-of-equilibrium stochastic simulations, we demonstrate that these experimental results reflect the slow relaxation of RNA structural rearrangements. Hence, micromanipulations of single RNA molecules probe both their native structures and long-lived intermediates, so-called “kinetic traps”, thereby capturing -at the single molecular level- the hallmark of RNA folding/unfolding dynamics.

Molecular and Sensory Basis of a Food Related Two-State Behavior in C. elegans

Most animals display multiple behavioral states and control the time allocation to each of their activity phases depending on their environment. Here we develop a new quantitative method to analyze Caenorhabditis elegans behavioral states. We show that the dwelling and roaming two-state behavior of C. elegans is tightly controlled by the concentration of food in the environment of the animal. Sensory perception through the amphid neurons is necessary to extend roaming phases while internal metabolic perception of food nutritional value is needed to induce dwelling. Our analysis also shows that the proportion of time spent in each state is modulated by past nutritional experiences of the animal. This two-state behavior is regulated through serotonin as well as insulin and TGF-beta signaling pathways. We propose a model where food nutritional value is assessed through internal metabolic signaling. Biogenic amines signaling could allow the worm to adapt to fast changes in the environment when peptide transcriptional pathways may mediate slower adaptive changes.

Subnanometric measurements of evanescent wave penetration depth using total internal reflection microscopy combined with fluorescent correlation spectroscopy

In total internal reflection microscopy (TIRM), quantitative interpretation of results often requires a precise knowledge of the penetration depth of the evanescent wave. Standard TIRM practice is to calculate this depth from the microscope’s geometry, but this can introduce significant errors. We show how to calibrate the penetration depth of an evanescent wave in TIRM. An evanescent wave is obtained by illuminating a surface at an incident angle greater than the critical angle. Its penetration depth generally depends on the wavelength and the incident angle of the illumination, and on the indices of refraction on either side of the reflecting suface, but cannot be larger than the field of view. By introducing a fluorescent species (such as fluorescein) and measuring its diffusion time, it is possible to measure very precisely the penetration depth of the evanescent wave.

Reversible and irreversible unfolding of eukaryote chromosomes by force

We have carried out studies of metaphase chromosomes in primary explant cultures of newt lung epithelia, using in situ microsurgery and force measurement. The chromosomes are elastic over a five-fold range of extension, with a force constant of about one nanonewton (nN). For larger extensions, chromosomes are permanently elongated; if stretched beyond 30 times, their cross-sectional diameter is increased after relaxation. Fluorescence studies indicate that histone proteins are not being removed. We conclude that we are first unfolding and then breaking chromosome-folding proteins by extension.