Mechanism of activation and function of the odorant receptor expressed at the axon terminus-growth cone of olfactory sensory neurons

A unique feature in the topographic organization of the olfactory bulb is the “dual role” of the odorant receptor. It detects odorants and it has been suggested to play a critical role in the axonal convergence of olfactory sensory neurons to form glomeruli in specific loci of the olfactory bulb. This spatial segregation of sensory afferents results in the sensory map. A role of the odorant receptors in axon guidance was suggested by genetic experiments demonstrating that manipulations of odorant receptor sequences perturb the sensory map (Wang et al., 1998). This hypothesis was confirmed by subsequent works (Barnea et al., 2004, Strotmann et al., 2004) that revealed the presence of the olfactory receptor in the most distal portion of the axon and at the growth cone. The open question to be addressed was whether the odorant receptor expressed at the axon terminal was functional and if yes, what was the signalling pathway coupled to its activation. In a previous study, Maritan et al., 2009, demonstrated, for the first time, that the odorant receptor expressed at the axon terminus - growth cone is functional and coupled to local increases of Ca2+ and cAMP. Although cAMP and Ca2+ are the primary second messengers produced upon activation of the odorant receptor, cGMP is also synthesized and takes part in several key processes such as adaptation, neuronal development and long term cellular responses to odorant stimulation. Many aspects of the regulation of cGMP in olfactory sensory neurons (OSNs) were still unknown, as the mechanism of coupling to odorant receptors (ORs) and downstream targets. To address these points, we investigated the dynamics and the intracellular distribution of cGMP in living rat OSNs in culture transfected with a genetically encoded sensor for cGMP. We demonstrated that OSNs treated with pharmacological stimuli able to activate particulate or soluble guanylyl cyclases (pGC and sGC) presented an increase in cGMP in the whole neuron, from cilia - dendrite to the axon terminus - growth cone. Upon odorant stimulation, a rise in cGMP was again found in the entire neuron, including the axon terminal, where it is locally synthesized. The odorant - dependent rise in cGMP is due to sGC activation by NO and requires an increase of cAMP. The link between cAMP and NO synthase appears to be the rise in [Ca2+]c elicited by either plasma membrane Ca2+ channel activation and Ca2+ mobilization from stores via the guanine nucleotide exchange factor Epac. Finally we show that a cGMP rise can elicit the phosphorylation of nuclear CREB both in vitro and in vivo. The local synthesis of cGMP, coupled to the OR expressed at the axon terminal, suggested that not only cAMP, but also cGMP can contribute to OSN axonal convergence. The question then arose on the mechanism of activation, i.e. the possible natural ligands, of the olfactory receptor at the axon terminal. We hypothesized that a few molecules expressed in gradient in the olfactory bulb could bind and activate the odorant receptor expressed at the axon terminal, regulating in this way the axon pathfinding to its final target. To test our hypothesis we studied the spatio - temporal dynamics of Ca2+ and cAMP in response to molecules from the bulb. We found that a pool of these molecules is capable of eliciting a rise in Ca2+ and cAMP in the axon terminus - growth cone of OSNs loaded with fura-2 or transfected with the sensor for cAMP. To assess whether the Ca2+ and cAMP rises were due to the activation of the olfactory receptor at the axon terminal, we expressed specific odorant receptors in HEK cells. The Ca2+ rise was observed only in HEK cells transfected with specific receptors, but not in HEK cells transfected with the empty vector (controls). All together, our data demonstrate the presence of a pool of active molecules in the bulb able to activate the OR expressed at the axon terminus - growth cone. To assess the physiological meaning of the variation in Ca2+ and cAMP levels on the turning behaviour of the olfactory sensory neuron axons, real - time imaging experiments on isolated olfactory sensory neurons were performed. We analyzed the behaviour of olfactory sensory neuron growth cone in response to gradients of molecules capable of modulating Ca2+ and cAMP levels at the axon terminus - growth cone, such as forskolin, a generic activator of adenylyl cyclase, odors and the active pool of molecules from the olfactory bulb. We found that all these molecules, including the ones from the bulb, were able to regulate the turning behaviour of the olfactory sensory neuron axons. All together our data suggest that molecules from the olfactory bulb, via activation of the odorant receptor expressed at the axon terminus – growth cone, contribute in providing the olfactory sensory neuron axons with instruction to reach the proper target in the olfactory bulb