Stochastic models and dynamic measures for the characterization of bistable circuits

During the last few years, a great deal of interest has risen concerning the applications of stochastic methods to several biochemical and biological phenomena. Phenomena like gene expression, cellular memory, bet-hedging strategy in bacterial growth and many others, cannot be described by continuous stochastic models due to their intrinsic discreteness and randomness. In this thesis I have used the Chemical Master Equation (CME) technique to modelize some feedback cycles and analyzing their properties, including experimental data. In the first part of this work, the effect of stochastic stability is discussed on a toy model of the genetic switch that triggers the cellular division, which malfunctioning is known to be one of the hallmarks of cancer. The second system I have worked on is the so-called futile cycle, a closed cycle of two enzymatic reactions that adds and removes a chemical compound, called phosphate group, to a specific substrate. I have thus investigated how adding noise to the enzyme (that is usually in the order of few hundred molecules) modifies the probability of observing a specific number of phosphorylated substrate molecules, and confirmed theoretical predictions with numerical simulations. In the third part the results of the study of a chain of multiple phosphorylation-dephosphorylation cycles will be presented. We will discuss an approximation method for the exact solution in the bidimensional case and the relationship that this method has with the thermodynamic properties of the system, which is an open system far from equilibrium.In the last section the agreement between the theoretical prediction of the total protein quantity in a mouse cells population and the observed quantity will be shown, measured via fluorescence microscopy

La dépression synaptique à long terme: un mécanisme pour la mémoire et l’apprentissage au niveau du cervelet

Le cervelet, partie du cerveau qui coordonne la fonction motrice, a un rôle clé dans l’apprentissage moteur, voire dans certains processus cognitifs. Il est aujourd’hui couramment admis que la modification à long terme de l’efficacité de la transmission synaptique est la base cellulaire de la mémoire et de l’apprentissage. La cellule de Purkinje, élément central de l’organisation cyto-architectonique du cervelet et de la fonction cérébelleuse, est le siège d’une plasticité neuronale particulière appelée « dépression à long terme ». En quoi consiste cette « dépression »? Quels en sont les mécanismes cellulaires et moléculaires? Quels éléments permettent de considérer que cette plasticité est le mécanisme de base de l’apprentissage moteur? Telles sont les questions abordées dans cet article.It is commonly thought that a persistent change in the efficacy of the synaptic transmission is the basic mechanism underlying learning and memory. The cerebellum, key structure of the motor function, exhibits a synaptic plasticity named cerebellar long-term depression or LTD. This phenomenon appears in the Purkinje cell when the two main excitatory inputs (one consists of the parallel fibers which relay information on the task to accomplish and the other one includes the climbing fiber which conveys error signals) are activated in combination, resulting in a persistent decrease of the efficacy of the parallel fiber-Purkinje cell synapse. Studies made in the last 20 years show that activation of ionotropic and metabotropic glutamate receptors triggers complex signal transduction processes, leading to the phosphorylation and the internalization of AMPA receptors, a subtype of glutamatergic receptors. The aim of this paper is firstly to present mechanisms involved in LTD induction and maintenance. The second part introduces briefly experimental data that show that LTD is indeed strongly associated with motor learning. Recent studies on the involvement of the cerebellum in cognitive tasks also suggest that LTD may play some role other than that in the sole motor learning

The GABAB1a isoform mediates heterosynaptic depression at hippocampal mossy fiber synapses

GABA(B) receptor subtypes are based on the subunit isoforms GABA(B1a) and GABA(B1b), which associate with GABA(B2) subunits to form pharmacologically indistinguishable GABA(B(1a,2)) and GABA(B(1b,2)) receptors. Studies with mice selectively expressing GABA(B1a) or GABA(B1b) subunits revealed that GABA(B(1a,2)) receptors are more abundant than GABA(B(1b,2)) receptors at glutamatergic terminals. Accordingly, it was found that GABA(B(1a,2)) receptors are more efficient than GABA(B(1b,2)) receptors in inhibiting glutamate release when maximally activated by exogenous application of the agonist baclofen. Here, we used a combination of genetic, ultrastructural and electrophysiological approaches to analyze to what extent GABA(B(1a,2)) and GABA(B(1b,2)) receptors inhibit glutamate release in response to physiological activation. We first show that at hippocampal mossy fiber (MF)-CA3 pyramidal neuron synapses more GABA(B1a) than GABA(B1b) protein is present at presynaptic sites, consistent with the findings at other glutamatergic synapses. In the presence of baclofen at concentrations <or=1 microm, both GABA(B(1a,2)) and GABA(B(1b,2)) receptors contribute to presynaptic inhibition of glutamate release. However, at lower concentrations of baclofen, selectively GABA(B(1a,2)) receptors contribute to presynaptic inhibition. Remarkably, exclusively GABA(B(1a,2)) receptors inhibit glutamate release in response to synaptically released GABA. Specifically, we demonstrate that selectively GABA(B(1a,2)) receptors mediate heterosynaptic depression of MF transmission, a physiological phenomenon involving transsynaptic inhibition of glutamate release via presynaptic GABA(B) receptors. Our data demonstrate that the difference in GABA(B1a) and GABA(B1b) protein levels at MF terminals is sufficient to produce a strictly GABA(B1a)-specific effect under physiological conditions. This consolidates that the differential subcellular localization of the GABA(B1a) and GABA(B1b) proteins is of regulatory relevance

A mouse model for visualization of GABA(B) receptors

GABA(B) receptors are the G-protein-coupled receptors for the neurotransmitter gamma-aminobutyric acid (GABA). Receptor subtypes are based on the subunit isoforms GABA(B1a) and GABA(B1b), which combine with GABA(B2) subunits to form heteromeric receptors. Here, we used a modified bacterial artificial chromosome (BAC) containing the GABA(B1) gene to generate transgenic mice expressing GABA(B1a) and GABA(B1b) subunits fused to the enhanced green fluorescence protein (eGFP). We demonstrate that the GABA(B1)-eGFP fusion proteins reproduce the cellular expression patterns of endogenous GABA(B1) proteins in the brain and in peripheral tissue. Crossing the GABA(B1)-eGFP BAC transgene into the GABA(B1) (-/-) background restores pre and postsynaptic GABA(B) functions, showing that the GABA(B1)-eGFP fusion proteins substitute for the lack of endogenous GABA(B1) proteins. Finally, we demonstrate that the GABA(B1)-eGFP fusion proteins replicate the temporal expression patterns of native GABA(B) receptors in cultured neurons. These transgenic mice therefore provide a validated tool for direct visualization of native GABA(B) receptors

Redistribution of GABAB(1) protein and atypical GABAB responses in GABAB(2)-deficient mice

GABAB receptors mediate slow synaptic inhibition in the nervous system. In transfected cells, functional GABAB receptors are usually only observed after coexpression of GABAB(1) and GABAB(2) subunits, which established the concept of heteromerization for G-protein-coupled receptors. In the heteromeric receptor, GABAB(1) is responsible for binding of GABA, whereas GABAB(2) is necessary for surface trafficking and G-protein coupling. Consistent with these in vitro observations, the GABAB(1) subunit is also essential for all GABAB signaling in vivo. Mice lacking the GABAB(1) subunit do not exhibit detectable electrophysiological, biochemical, or behavioral responses to GABAB agonists. However, GABAB(1) exhibits a broader cellular expression pattern than GABAB(2), suggesting that GABAB(1) could be functional in the absence of GABAB(2). We now generated GABAB(2)-deficient mice to analyze whether GABAB(1) has the potential to signal without GABAB(2) in neurons. We show that GABAB(2)-/- mice suffer from spontaneous seizures, hyperalgesia, hyperlocomotor activity, and severe memory impairment, analogous to GABAB(1)-/- mice. This clearly demonstrates that the lack of heteromeric GABAB(1,2) receptors underlies these phenotypes. To our surprise and in contrast to GABAB(1)-/- mice, we still detect atypical electrophysiological GABAB responses in hippocampal slices of GABAB(2)-/- mice. Furthermore, in the absence of GABAB(2), the GABAB(1) protein relocates from distal neuronal sites to the soma and proximal dendrites. Our data suggest that association of GABAB(2) with GABAB(1) is essential for receptor localization in distal processes but is not absolutely necessary for signaling. It is therefore possible that functional GABAB receptors exist in neurons that naturally lack GABAB(2) subunits

BARP suppresses voltage-gated calcium channel activity and Ca2+-evoked exocytosis

10.1083/jcb.201304101Journal of Cell Biology2052233-249JCLB