Computational Model of the Insect Pheromone Transduction Cascade

A biophysical model of receptor potential generation in the male moth olfactory receptor neuron is presented. It takes into account all pre-effector processes—the translocation of pheromone molecules from air to sensillum lymph, their deactivation and interaction with the receptors, and the G-protein and effector enzyme activation—and focuses on the main post-effector processes. These processes involve the production and degradation of second messengers (IP3 and DAG), the opening and closing of a series of ionic channels (IP3-gated Ca2+ channel, DAG-gated cationic channel, Ca2+-gated Cl− channel, and Ca2+- and voltage-gated K+ channel), and Ca2+ extrusion mechanisms. The whole network is regulated by modulators (protein kinase C and Ca2+-calmodulin) that exert feedback inhibition on the effector and channels. The evolution in time of these linked chemical species and currents and the resulting membrane potentials in response to single pulse stimulation of various intensities were simulated. The unknown parameter values were fitted by comparison to the amplitude and temporal characteristics (rising and falling times) of the experimentally measured receptor potential at various pheromone doses. The model obtained captures the main features of the dose–response curves: the wide dynamic range of six decades with the same amplitudes as the experimental data, the short rising time, and the long falling time. It also reproduces the second messenger kinetics. It suggests that the two main types of depolarizing ionic channels play different roles at low and high pheromone concentrations; the DAG-gated cationic channel plays the major role for depolarization at low concentrations, and the Ca2+-gated Cl− channel plays the major role for depolarization at middle and high concentrations. Several testable predictions are proposed, and future developments are discussed

Event Timing in Associative Learning

Associative learning relies on event timing. Fruit flies for example, once trained with an odour that precedes electric shock, subsequently avoid this odour (punishment learning); if, on the other hand the odour follows the shock during training, it is approached later on (relief learning). During training, an odour-induced Ca++ signal and a shock-induced dopaminergic signal converge in the Kenyon cells, synergistically activating a Ca++-calmodulin-sensitive adenylate cyclase, which likely leads to the synaptic plasticity underlying the conditioned avoidance of the odour. In Aplysia, the effect of serotonin on the corresponding adenylate cyclase is bi-directionally modulated by Ca++, depending on the relative timing of the two inputs. Using a computational approach, we quantitatively explore this biochemical property of the adenylate cyclase and show that it can generate the effect of event timing on associative learning. We overcome the shortage of behavioural data in Aplysia and biochemical data in Drosophila by combining findings from both systems

Signal transduction in olfactory sensory neurons of vertebrates and tools for the computer simulation

Cílem této práce je shrnout poznatky o čichové transdukci obratlovců. Tato rešerše je rozdělena do čtyř částí, přičemž každá část je zaměřena na jiný aspekt čichové transdukce. Nejprve je proveden přehled základních elektrofyziologických metod používaných pro výzkum transdukce. Následuje podrobný popis celé transdukce na molekulární úrovni. Další část práce shrnuje, jaké jsou typy modelů a jak se dají využít při modelování čichové transdukce. V závěru této části je proveden podrobný popis dvou modelů. Jeden z nich popisuje počátek čichové transdukce od vazby odorantu na receptor po produkci cAMP a druhý se zabývá zpětnou negativní vazbou Ca2+. Rešerše je zakončena přehledem softwarových nástrojů pro vytváření a analýzy modelů z předešlé části.The purpose of this thesis is summing up the information about olfactory transduction of vertebrates. This review is divided into four parts, each part focuses on a different aspect of olfactory transduction. First there is an overview of basic electrophysiological methods used for transduction research, followed by a description of a complete transduction on a molecular level. Next is a summary of model types and their use in olfactory transduction simulation, including a detailed description of two models: One of them describes the beginning of olfactory transduction, from the odorant binding on the receptor to the cAMP production, the other deals with the negative feedback of Ca2+. Finally there is an overview of software products designed to create and analyze the models from the preceding section.Katedra fyziologieDepartment of PhysiologyPřírodovědecká fakultaFaculty of Scienc

Dynamical Modeling of the Moth Pheromone-Sensitive Olfactory Receptor Neuron within Its Sensillar Environment

In insects, olfactory receptor neurons (ORNs), surrounded with auxiliary cells and protected by a cuticular wall, form small discrete sensory organs – the sensilla. The moth pheromone-sensitive sensillum is a well studied example of hair-like sensillum that is favorable to both experimental and modeling investigations. The model presented takes into account both the molecular processes of ORNs, i.e. the biochemical reactions and ionic currents giving rise to the receptor potential, and the cellular organization and compartmentalization of the organ represented by an electrical circuit. The number of isopotential compartments needed to describe the long dendrite bearing pheromone receptors was determined. The transduction parameters that must be modified when the number of compartments is increased were identified. This model reproduces the amplitude and time course of the experimentally recorded receptor potential. A first complete version of the model was analyzed in response to pheromone pulses of various strengths. It provided a quantitative description of the spatial and temporal evolution of the pheromone-dependent conductances, currents and potentials along the outer dendrite and served to determine the contribution of the various steps in the cascade to its global sensitivity. A second simplified version of the model, utilizing a single depolarizing conductance and leak conductances for repolarizing the ORN, was derived from the first version. It served to analyze the effects on the sensory properties of varying the electrical parameters and the size of the main sensillum parts. The consequences of the results obtained on the still uncertain mechanisms of olfactory transduction in moth ORNs – involvement or not of G-proteins, role of chloride and potassium currents – are discussed as well as the optimality of the sensillum organization, the dependence of biochemical parameters on the neuron spatial extension and the respective contributions of the biochemical and electrical parameters to the overall neuron response

Event Timing in Associative Learning: From Biochemical Reaction Dynamics to Behavioural Observations

Associative learning relies on event timing. Fruit flies for example, once trained with an odour that precedes electric shock, subsequently avoid this odour (punishment learning); if, on the other hand the odour follows the shock during training, it is approached later on (relief learning). During training, an odour-induced Ca++ signal and a shock-induced dopaminergic signal converge in the Kenyon cells, synergistically activating a Ca++-calmodulin-sensitive adenylate cyclase, which likely leads to the synaptic plasticity underlying the conditioned avoidance of the odour. In Aplysia, the effect of serotonin on the corresponding adenylate cyclase is bi-directionally modulated by Ca++, depending on the relative timing of the two inputs. Using a computational approach, we quantitatively explore this biochemical property of the adenylate cyclase and show that it can generate the effect of event timing on associative learning. We overcome the shortage of behavioural data in Aplysia and biochemical data in Drosophila by combining findings from both systems

Neurofly 2008 abstracts : the 12th European Drosophila neurobiology conference 6-10 September 2008 Wuerzburg, Germany

This volume consists of a collection of conference abstracts

Olfactory coding in vertebrates: a novel tuning mechanism for receptor affinity and evolution of the olfactory receptor repertoire

Information about our environment is to a large extent carried by the chemical senses, and in particular the olfactory sense. Vertebrates perceive thousands of diverse odor molecules with a supply of a wide range of essential information ranging from localising prey or food, avoiding predators, mating behaviour, to social communication. Because olfactory receptor proteins play such an essential role in the specific recognition of diverse stimuli, understanding how they interact with and transduce their cognate ligands is a high priority. This constitutes one of the most complex ligand/receptor binding problems in biology due to the sheer quantity of potential odor molecules facing a limited albeit huge number of different olfactory receptors. Most olfactory receptors are G-protein coupled receptors and form large gene families. One type of olfactory receptors is the trace amine-associated receptor family (TAAR). TAARs generally recognize amines and one particular member of the zebrafish TAAR family, TAAR13c, is a high affinity receptor for the death-associated odor cadaverine, which induces aversive behavior. Here we have modeled the cadaverine/TAAR13c interaction by multistep docking. By exchanging predicted binding residues via site-directed mutagenesis, and measuring the activity of the mutant receptors, we confirmed a binding site for cadaverine at the external surface of the receptor, in addition to an internal binding site, whose mutation resulted in complete loss of activity. Elimination of the external binding site generated supersensitive receptors which suggests this site to act as a gate, limiting access of the ligand to the internal binding site and thereby downregulating the affinity of the native receptor. Potentially related mechanisms have been described for non-olfactory G-protein coupled receptors. The topology of TAAR-expressing neurons in the teleost olfactory epithelium has not been described yet. We have investigated representative taar genes from three classes to test the principle of partial spatial segregation known from other olfactory receptor families for the TAAR family. We report that expression of taar genes is intermingled with expression zones of odorant receptor genes, which in fish share a single sensory surface with TAARs. Individual taar genes show distinct, albeit broadly overlapping expression zones. In the third part of my thesis I investigated the genome of a cartilaginous fish, Scyliorhinus canicula, commonly known as small spotted catshark in order to delineate its chemosensory receptor repertoire: OR, V1R/V2R, TAAR, and T1R/T2R. This is the first repertoire described for a true shark, an important intermediate in the evolution of vertebrates. In contrast to bony vertebrates, but very similar to a chimera (elephant shark), the olfactory receptor repertoire of catshark is dominated by the V2R family

Differentiering av GnRH neuroner från humana pluripotenta stamceller

The onset of puberty and sexual development as well as normal reproductive function are dependent on pulsatile secretion of gonadotropin-releasing hormone (GnRH). GnRH is secreted from the GnRH neuron nerve terminals in the hypothalamic median eminence into the portal vessels that lead to the anterior pituitary. The pulsatile secretion of GnRH stimulates the release of gonadotropins, luteinizing hormone (LH), and follicle-stimulating hormone (FSH), which, in turn, regulate various gonadal functions. In rare occasions, the onset of puberty is delayed or completely absent. This can be caused by disrupted development, migration, or function of GnRH neurons, resulting in defects in sexual development and infertility. Congenital GnRH deficiency is termed congenital hypogonadotropic hypogonadism (CHH), and CHH combined with hyposmia or anosmia (reduced or absent sense of smell) is known as Kallmann syndrome (KS). CHH and KS are genetically heterogeneous diseases, with over 30 genes reported in association with KS and CHH to date. How mutations in these genes cause GnRH deficiency is not yet comprehensively understood, but several are postulated to affect GnRH neuron development. Human pluripotent stem cells (hPSCs) are the equivalent of undifferentiated cells in the early embryo, and able to give rise to all tissues and cell types in the human body. Thus, hPSCs have become a widely used tool for studying the differentiation of specialized cell types and the causes for human diseases in vitro. Developing methods for directed differentiation of hPSCs into GnRH neurons requires insight into the events which lead to the specification of GnRH neurons during embryonic development. GnRH neurons are born in the olfactory placodes in the nasal area of the developing embryo. After their delamination from the olfactory neuroepithelium, the differentiated postmitotic GnRH neurons take an upward migratory route along the axon fibers of the terminal nerve around the olfactory bulb, cross the cribriform plate to the forebrain, and finally make a ventral turn into to the preoptic area of the hypothalamus. The exact cell type within the olfactory placodes that gives rise to GnRH neurons is not entirely known. Its precursors have been proposed to be of both preplacodal ectoderm and neural crest origin. The aim of this work was to create a model in which to study the molecular mechanism of GnRH neuron differentiation and the mechanisms of CHH-associated genetic mutations on GnRH neurogenesis in humans. The literature review of this thesis addresses the relevant background in the field of GnRH neuron development; from neural crest and preplacodal development at gastrulation stages, to ontogeny, migration, and maturation of GnRH neurons at puberty. This thesis presents experimental validation of the methods for in vitro generation of GnRH neurons from human pluripotent stem cells, and the findings include the discovery of several genes and proteins expressed during GnRH neuron differentiation.Människans pubertetsutveckling samt fertilitet styrs av ett noggrannt reglerat system av hormonella signaler, dvs. hypotalamus-hypofys-gonad-axeln (HPG-axeln). Gonadotropinfrisättande hormon (eng. Gonadotropin-releasing hormone, GnRH), utsöndras av en grupp specialiserade nervceller i hypotalamus, GnRH neuronerna. Hormonet GnRH fungerar som en startsignal för utsöndring av follikelstimulerande och luteiniserande hormon (FSH och LH) från hypofysen, som i sin tur stimulerar tillverkningen av könshormon. Eftersom GnRH neuroner har en central roll i HPG-axeln, kan avbrott i deras utveckling eller funktion leda till pubertetsstörningar och infertilitet. Den sällsynta genetiska sjukdomen hypogonadotropisk hypogonadism (CHH) beror på nedsatt utsöndring av GnRH i hypotalamus och leder till försenad eller utebliven pubertet, begränsad könskaraktärsutveckling och infertilitet. Kallmann Syndrom är en form av CHH som förekommer i kombination med nedsatt luktsinne. Det finns ett påvisat samband mellan utvecklingen av luktsinnets celler och GnRH neuroner. Både olfaktoriska receptorceller och GnRH neuroner uppkommer i nasala plakoderna, i neuroepitelet som senare kommer att bilda i embryots luktepitel. Under veckorna 6-8 av fosterutvecklingen migrerar de nybildade GnRH neuronerna från näsan till hypotalamus i hjärnan tillsammans med olfaktoriska receptorcellers axonfibrer. Mekanismer som stör utvecklingen i nasala plakoderna, neuroners migration, eller cellernas mognad, kan därmed vara orsaker till Kallmann Syndrom. Mutationer i över 30 gener har påvisats vara förknippade med CHH och Kallmann Syndrom, men de molekylära mekanismer som orsakar avvikelser i GnRH neuroners fysiologi är till stor del okända. Humana pluripotenta stamceller (hPSC) är ospecialiserade celler som förekommer under den tidiga embryonalutvecklingen, och som genom differentiering ger upphov till alla specialiserade celltyper i kroppens vävnader och organ. Genom att odla hPSC celler i laboratoriemiljö är det möjligt att återskapa samt studera utvecklingen av specialiserade celler och utforska biologiska mekanismer som gynnar eller förhindrar cellernas uppkomst och funktion. Denna avhandling beskriver utvecklingen av metoder för differentiering av GnRH neuroner från hPSC in vitro, samt presenterar användningen av differentieringsmetoden som forskningsmodell i studier om GnRH neuroners utveckling. För övrigt diskuteras flera tidigare obeskrivna gener och protein som uttrycks under differentieringsprocessen, och deras potentiella roller i regleringen av humana GnRH neuroners uppkomst och funktion, och därmed även i regleringen av människans pubertetsutveckling och fertilitet