Proper response to external stimuli is essential for survival. Upon aversive stimuli animals display a stereotypical sequence of motor responses consisting of flight and/or freeze followed by recovery to baseline activity. While these motor responses cannot be executed simultaneously, they do however, occur sequentially. Aberrant response to aversive stimuli leads to depression, anxiety or addiction in humans. While studies have identified neuronal circuits that mediate aversive behavior in distinct brain regions, the biological correlates for how one circuit is selected over the other, a symmetry-breaking step known as competitive selection, is yet unclear. The evolutionarily conserved habenulo-interpeduncular nucleus (Hb-IPN) pathway has emerged as a crucial circuit that mediates fear and stress-related behaviors. This pathway is composed of two distinct circuits, the cholinergic and the peptidergic non-cholinergic, that innervate adjacent domains in the IPN. We recently found a hardwired mode of negatively correlated activity between the cholinergic and non-cholinergic circuits, whereby the synchronized activation of cholinergic neurons inhibits non-cholinergic neuron activity. This occurs through retrograde GABAB signaling at the IPN via GABAB receptors located on the non-cholinergic terminals. An aversive stimulus, electric shock also induces this mode of negatively correlated activity. We hypothesize that different external stimuli induce competitive selection in the habenulo-interpeduncular nucleus pathway to modulate the stereotypical motor response. By taking advantage of the small size and transparency of the zebrafish larval brain to easily monitor and modify neuronal activity in head-fixed animals during behavior, this project aims to understand how an evolutionarily conserved asymmetric structure in the brain that integrates various external stimuli utilizes an atypical mode of competition to modulate motor responses to aversive stimuli.

One of the central problems in neurobiology, and perhaps in all biological systems, is to understand how nerve cells are organised in a network which is both stable and capable of a great plasticity. In particular, synapses are very stable structural elements which can be modified in a few minutes to strengthen or weaken the connectivity between neurons. Moreover, synapses can be modified individually, independently of other synapses which are connected to different neurons but located only a few micrometres away. The altered capacity for plasticity is probably at the basis of cognitive impairments which characterize cerebral pathologies such as Alzheimer’s disease or autism spectrum disorders. To grant synaptic stability associated with fast plasticity, a number of synaptic constituents, among them postsynaptic receptors, are in a dynamic equilibrium between synaptic and extrasynaptic regions. Work from a number of laboratories in the last fifteen years has shown that this equilibrium is maintained at least at two kinetically and mechanistically different levels. The First level of equilibrium involves the exchange of receptors between the plasma membrane and intracellular compartments through endocytosis, recycling and exocytosis. These phenomena can modify synaptic efficacy within minutes and are essential for the expression of long term synaptic plasticity, in particular NMDA dependent long term potentiation (LTP) and depression (LTD). Secondly, as shown in a large part by the group of Daniel Choquet, director of the laboratory of Partner 1, postsynaptic receptors diffuse rapidly in the plasma membrane between synaptic and extrasynaptic zones. Receptor stabilization critically depends on the interaction between AMPAR accessory subunits and PSD-95, the main protein present in the PSD. This interaction can be modulated, for example by receptor desensitization following glutamate binding. Moreover, these exchanges are sufficiently fast, in the millisecond timescale, to affect the temporal coding of information by synapses. It is currently unknown if modulation of receptor diffusion modulates long term synaptic plasticity. These two levels of regulation are critical for the stability and plasticity of synaptic transmission. Our hypothesis is that they also contribute to synaptic independence by restricting the trafficking of receptors. Moreover, the extent of receptor trafficking could itself be modulated in physiological and pathophysiological situations. To test this hypothesis, we need to be able to visualize and control simultaneously these two levels in the most precise manner. Our objective is thus to optimize optical probes, develop new protocols and validate photo-activatable blockers to obtain an integrated view of receptor diffusion, internalisation and recycling during synaptic transmission and plasticity. We will focus on the modulation of receptor surface trafficking and internalisation during LTD by following overexpressed and endogenous receptors labelled with fluorescent probes (fluorescent proteins, organic fluorophores and new generation nanoparticles, quantum dots and nanoplatelets). These fluorophores will be sensitive to changes in the environment, such as pH or the presence of quenchers, which will be imposed outside of cells and permit to distinguish if the labels are at the cellular surface or inside cells. These new tools will be developed and optimized by our interdisciplinary consortium of cellular neurobiologists, nanocrystal physicists and organic chemists, and used in protocols to measure the activity of individual endocytic zones and the behaviour of single receptors before and after their internalisation. We predict that with this level of detail we will be able to decipher the role of these processes in the establishment of specific and efficient regulation of synaptic strength.