Transduction in Olfactory Receptor Neurons of Xenopus laevis Larvae: Pharmacological Blockage with FM1-43 and Endocannabinoid Modulation

Abstract

The sense of smell is critical for finding food and mediating emotional and social responses. A dysfunction of this sense is often associated with diseases. In the last two decades much progress has been made in this research field and insights in how the sensation of smell is perceived has been gained. This thesis covers two topics of basic research in olfaction and is accordingly subdivided:1. Various olfactory receptor neuron subsets exist in the olfactory epithelium. One classification of these subsets can be made according to the transduction mechanism of odorants. The olfactory receptor neurons belonging to one subset transduce odorants into depolarizations using the cAMP-dependent transduction mechanism. To date, it is not possible to stain this subset or to interfere with their generator channels with potent and specific blockers. In this thesis, the styryl dye FM1-43 was identified as a marker for olfactory receptor neurons endowed with cAMP-dependent transduction machinery. The dye is internalized in neurons by uptake through cyclic nucleotide-gated channels, the generator channel of the cAMP-dependent olfactory receptor neurons. This was proven by interference of FM1-43 uptake with divalent ions and unspecific cyclic nucleotide-gated channel blockers used in high concentrations and further confirmed by evoking responses to cAMP and forskolin in FM1-43-stained cells. Characteristic for FM1-43-stained olfactory receptor neurons is that these cells did not respond to odors or rapidly lost their responsiveness. This suggested that FM1-43 severely interfered with the transduction machinery of stained olfactory receptor neurons. Indeed, 10 µM FM1-43 blocked currents through native cyclic nucleotide-gated channels to approximately 25 % and acts from the extracellular side. This tool thus allows optical differentiation and pharmacological interference with olfactory receptor neurons endowed with cAMP-dependent transduction at the level of the signal transduction.2. The sense of smell is an important input for the search for food and food intake, but the underlying mechanisms for this functional interaction are poorly understood. One key factor for energy homeostasis and nutrition at central stages is the endocannabinoid system. Therefore, it was hypothesized, that the endocannabinoid system may link food intake with olfaction. Recently, cannabinoids were shown to act in the olfactory epithelium. Pharmacological interference at the according cannabinoid receptor modulates odor-evoked responses. In this thesis, it is shown that the endocannabinoid 2-AG is synthesized in the olfactory epithelium and acts on olfactory receptor neurons. Blocking 2-AG synthesis decreased and delayed odorant-induced responses. Analyzing single cells revealed, that there are two sources of 2-AG in the olfactory epithelium: the first are olfactory receptor neurons, where production of 2-AG depends on diacylglycerol lipase β, and the other are sustentacular cells, where production depends on diacylglycerol lipase α. Diacylglycerol lipase α mediated 2-AG-synthesis in sustentacular cells is influenced by the hunger state of the animal. The essential 2-AG effect in olfactory receptor neurons is the control of odorant detection thresholds. An enhanced 2-AG level decreases the detection threshold of an individual olfactory receptor neuron whereas a lowered 2-AG level increases the detection threshold. Thus, hunger renders olfactory receptor neurons more sensitive and endocannabinoid modulation in the nose may therefore substantially influence food seeking behavior. The intracellular effector mediating cannabinoid receptor actions probably is the plasma membrane calcium ATPase. This signaling cascade was not described as yet. However, future experiments have to be performed to identify the complete transduction cascade. Besides the endocannabinoid system, receptors for several other modulatory substances, i.e. orexin, leptin, adiponectin, and dopamine, were found in the olfactory epithel ium. This indicates that already at the most peripheral stage of the olfactory system, the sensation of odors is modulated by many substances.