Optogenetic examination of salt taste in mice

This thesis describes a series of experiments designed to evaluate the hypothesis that Type I taste receptor cells play a critical role in the detection and transduction of sodium taste via of epithelial sodium channels (ENaCs). Experiment 1 validated the function of a simple and affordable behavioral apparatus (hardware and software) for testing taste preference and taste aversion in mice. Experiment 2 demonstrated a pharmacological method for rapid induction of salt appetite in mice. Experiment 3 showed that optogenetic stimulation of Type I taste receptor cells (TRCs) in transgenic mice could drive consumption of tap water under conditions of salt hunger. The fourth and final experiment assessed whether conditioned taste aversions to sodium would generalize to optogenetic stimulation of Type I taste receptor cells in transgenic mice, with inconclusive results

Cloning TRPC1 to enhance Calcium signal in a model of bitter taste transduction.

The sense of taste is used to evaluate the quality of food. In particular, animals detect at least five basic tastes: sweet, bitter, salty, umami and sour. Each one is associated to a given food’s property, for instance bitter taste has been thought to be necessary to detect toxins. Bitter taste transduction starts with the binding of bitter tastant with one or more bitter taste receptors (TAS2Rs). They are G protein-coupled receptors (GPCRs) and the binding with tastants leads to the activation of G protein α-gustducin. In particular, βγ subunits activate phospholipase PLCβ2. This event causes the production of inositol 1,4,5-thriphosphate (IP3) and release of Ca2+ from internal stores. The elevation of Ca2+ activates transient receptor potential channel M5 (TRPM5) and this allows the depolarization of the cell. The elevation of [Ca2+]i is the studied event to understand the behavior of taste receptors in presence of different tastants. Indeed, although some receptors give a strong signal with one tastant, the same receptors could give a low signal with other molecules, or some receptors may give low signal per se. Unfortunately TRPM5 does not allow Ca2+ entry and it could not be used in our experiments. Hence, since the mechanism of activation by elevation of intracellular Ca2+ is common between TRPM5 and TRPC1, another TRP channel, and since the latter is permeable also for Ca2+ , further increasing the [Ca2+]i , we chose to clone TRPC1 in a cellular model of bitter taste transduction, trying to enhance the calcium signal. We started from an extract of fetal human brain to isolate the cDNA of TRPC1, then we included the cDNA in an expression vector and transiently transfected HEK293 cells, with both TRPC1 and a TAS2R. These cells already stably express a chimeric G-protein α subunit G16gust44, involved in the signal pathway of taste. Thanks to the fluorescence given from Fluo-4 when it binds Ca2+, we studied the Ca2+ signals obtaining different profiles in presence or not of TRPC1. For example, in cells expressing TRPC1 and TAS2R43 or TAS2R14 the signal was higher than in cells transfected with TAS2R and mock (plasmid without any insert, used as negative control). On the contrary, with TAS2R10 we had the opposite result, with a higher signal in cells expressing only the receptor. Improving Ca2+ signal could be possible to deorphanize receptors whose tastants are still not known just because of low signal, or to find other molecules that activate a given TAS2R and so extent the range of activators of that receptor. Moreover, it could be possible to study inhibitors in those receptors that have strong signal with a tastant and low signal with other(s) substance(s)

Processing at Primary Chemosensory Neurons

Chemosensory perception involves the detection of chemical compounds. In animals, there are 2 chemical senses: taste, and olfaction. The two are related in that they utilize ligand-gated receptors, expressed in primary sensory neurons, to detect chemical stimuli from the surrounding environment. However, the processing of these inputs is quite different in the two systems, leading to divergent roles for olfaction and taste in sensory perception. This dissertation highlights some of these differences, by looking at processing of ethologically relevant stimuli at the very peripheral receptor neurons. The work is divided into 2 parts: water sensing by the mammalian taste system, and CO₂ sensing by the Drosophila olfactory system. In Chapter 1, I talk about water sensing in the mammalian taste system. Initiation of drinking behavior relies on peripheral water detection. It is likely that this detection is mediated, at least in part, by the taste system. Here, I have shown that acid-sensing taste receptor cells (TRCs) that were previously suggested as the sensors for sour taste, also respond to water. This response is mediated by a bicarbonate-dependent molecular mechanism, likely involving the Carbonic Anhydrase enzyme family. Furthermore, optogenetic stimulation of the acid-sensing TRCs in thirsty animals induces robust licking responses towards the light source, even in the absence of water. Conversely, thirsty animals lacking functional acid-sensing TRCs show compromised discrimination between water and non-aqueous fluids. Taken together, this work reveals the cellular mechanism of water detection by the mammalian taste system. In chapter 2, I talk about CO₂ sensing in the fruit fly. The Drosophila olfactory system responds to most odors with the activation of a large subset of its olfactory receptors (ORs). This broad activation is a consequence of the ORs having affinity to multiple chemical compounds. In contrast, a small number of odors, like CO₂, elicit responses in only single ORs. These ORs are, in contrast to most ORs, very narrowly tuned, generally responding only to that one odor. It has been assumed up until now that the specificity of these unique ORs is inherited by the olfactory receptor neurons (ORNs) they are expressed in, and even in the projection neurons (PNs), that the ORNs synapse onto. I show here that CO₂, though it activates only a single OR, the GR63a/GR21a hetero-dimer complex, actually activates multiple ORN axon terminals. This activation is due to lateral excitatory connections between axon terminals of the GR63a/GR21a expressing ORNs, and at least 4 other ORN types. Focusing on one of these ORNs, Ab1B, I show the lateral connections bypass the ORN cell bodies, only driving responses at the axon terminals. Consequently, Ab1B ORN axon terminals receive 2 sources of excitatory input, a feed-forward excitation from its endogenous OR, and a lateral excitation from GR63a/GR21a. This effectively divides the ORN into 2 compartments, distinct in their odor tuning. Finally, I show that lateral excitation is a general feature of the ORN circuit by silencing the feed-forward input of another ORN class, Ab1A. The Ab1A cell body is completely silent, but the axon terminals retain odor responses from lateral excitatory inputs. Thus, there is a lateral flow of odor information between multiple ORNs of the Drosophila olfactory system.</p

A2BR Adenosine Receptor Modulates Sweet Taste in Circumvallate Taste Buds

In response to taste stimulation, taste buds release ATP, which activates ionotropic ATP receptors (P2X2/P2X3) on taste nerves as well as metabotropic (P2Y) purinergic receptors on taste bud cells. The action of the extracellular ATP is terminated by ectonucleotidases, ultimately generating adenosine, which itself can activate one or more G-protein coupled adenosine receptors: A1, A2A, A2B, and A3. Here we investigated the expression of adenosine receptors in mouse taste buds at both the nucleotide and protein expression levels. Of the adenosine receptors, only A2B receptor (A2BR) is expressed specifically in taste epithelia. Further, A2BR is expressed abundantly only in a subset of taste bud cells of posterior (circumvallate, foliate), but not anterior (fungiform, palate) taste fields in mice. Analysis of double-labeled tissue indicates that A2BR occurs on Type II taste bud cells that also express Gα14, which is present only in sweet-sensitive taste cells of the foliate and circumvallate papillae. Glossopharyngeal nerve recordings from A2BR knockout mice show significantly reduced responses to both sucrose and synthetic sweeteners, but normal responses to tastants representing other qualities. Thus, our study identified a novel regulator of sweet taste, the A2BR, which functions to potentiate sweet responses in posterior lingual taste fields

Concentration Coding in the Accessory Olfactory System

Understanding how sensory systems encode stimuli is a fundamental question of neuroscience. The role of every sensory system is to encode information about the identity and quantity of stimuli in the environment. Primary sensory neurons in the periphery are faced with the task of representing all relevant information for further processing by downstream circuits, ultimately leading to detection, classification and potential response. However, environmental variability potentially alters stimulus properties in non-relevant ways. Here, we address these problems using the mouse accessory olfactory system: AOS) as a model. The AOS is an independent olfactory system possessed by most terrestrial vertebrates, although not humans, and is specialized to detect social cues. It mediates behaviors such as reproduction, aggression, and individual identification. Non-volatile compounds found in urine, including sulfated steroids, are the main source of AOS stimuli. Vomeronasal sensory neurons: VSNs), the primary sensory neurons of the AOS, are located in the base of the nasal cavity, and they detect the identity and quantity of stimuli. However, like other sensory cues, urine is subject to environmental modulation through mechanisms such as evaporation and dilution that affect the concentrations of ligands in non-biologically relevant ways. Ideally, the AOS represents stimuli in ways that are stable across condition. In the scope of this thesis, I explore how the AOS represents concentration at the levels of the individual neuron, the circuit and the whole animal. Using extracellular recordings of explanted tissue, we characterized how VSNs encode stimuli. VSNs fired predominantly in trains of action potentials with similar structure during spontaneous and stimulus-driven activity. Using pharmacological and genetic tools, we demonstrated that the signal transduction cascade influences the structure of both spontaneous and stimulus-driven activity. Then, we explored the representation of concentration of sulfated steroids by VSNs and the circuit mechanisms by which the AOS can represent concentration information in a manner invariant to environmental uncertainties. We identified ratio-coding as a means for stable concentration representation. The ratio of the concentrations of non-volatile ligands found in urine will not change following urine evaporation or dilution, while the individual concentrations will. This property allows for both insensitivity to changes in absolute concentration and sensitivity to changes in relative concentration. Using extracellular recording and computational modeling, we have demonstrated that VSN activity can be used to robustly encode concentration using ratios. Finally, we attempted to develop a novel behavioral assay to investigate how mice detect AOS stimuli

Intravital microscopic interrogation of peripheral taste sensation

Intravital microscopy is a powerful tool in neuroscience but has not been adapted to the taste sensory organ due to anatomical constraint. Here we developed an imaging window to facilitate microscopic access to the murine tongue in vivo. Real-time two-photon microscopy allowed the visualization of three-dimensional microanatomy of the intact tongue mucosa and functional activity of taste cells in response to topically administered tastants in live mice. Video microscopy also showed the calcium activity of taste cells elicited by small-sized tastants in the blood circulation. Molecular kinetic analysis suggested that intravascular taste sensation takes place at the microvilli on the apical side of taste cells after diffusion of the molecules through the pericellular capillaries and tight junctions in the taste bud. Our results demonstrate the capabilities and utilities of the new tool for taste research in vivo

Involvement of the insular cortex in regulating glucocorticoid effects on memory consolidation of inhibitory avoidance training

Glucocorticoids are known to enhance the consolidation of memory of emotionally arousing experiences by acting upon a network of interconnected brain regions. Although animal studies typically do not consider the insular cortex (IC) to be part of this network, the present findings indicate that the IC is importantly involved in regulating glucocorticoid effects on memory consolidation of emotionally arousing inhibitory avoidance training. The specific glucocorticoid receptor (GR) agonist RU 28362 (3 or 10 ng in 0.5 mu l) infused bilaterally into the IC of male SpragueDawley rats immediately after one-trial inhibitory avoidance training dose-dependently enhanced 48 h retention performance. Moreover, training on the inhibitory avoidance task increased neuronal activity of the IC, as assessed by an increased number of cells expressing immunoreactivity for phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2). However, systemic administration of a memory-enhancing dose of corticosterone (1 mg/kg) after inhibitory avoidance training rapidly reduced the number of pERK1/2-positive cells in the IC, suggesting that glucocorticoid administration reduces overall neuronal activity of the IC. To investigate which components of the inhibitory avoidance training experience were influenced by the intra-IC glucocorticoid administration, in the last experiment rats were trained on a modified inhibitory avoidance task in which context exposure and footshock training occur on two sequential days. RU 28362 administration into the IC enhanced later retention when infused immediately after either the context or footshock training. Thus, these findings indicate that the IC mediates glucocorticoid effects on the consolidation of memory of different components of inhibitory avoidance training and suggest that the IC might be an important element of the rodent brain network involved in emotional regulation of learning and memory.</p

Modulation of Fat Taste by Diet and Hormones

The prevalence of obesity worldwide continues to rise despite efforts to reverse the trend. While many factors contribute to the onset and maintenance of obesity, caloric intake and dietary composition have been shown be primary contributors. The oral cavity is one of the first systems to encounter food and determine its hedonic value. As the gateway to ingestion, the taste system plays a unique role in the initial decisions surrounding the control of food intake. Nutrients like carbohydrates, protein, minerals, and fat all have dedicated systems to allow their recognition at this outermost site of the enteric nervous system. Recent research has shown this system to have a high degree of plasticity, where it may tune itself to the nutritional needs of an organism. The work in this dissertation examined how circulating hormones and dietary changes alter fatty acid detection in the oral cavity thereby altering fat intake. Firstly, we examined the role high dietary fat intake has on fatty acid taste responses. We concluded that high dietary fat intake significantly increases inward currents elicited by linoleic acid (LA) in taste cells, these changes are dependent on the type of dietary fatty acids consumed, and only occur in a subset of fatty acid responding taste cells that are not thought to be the classical receptor cells of the taste bud. Additionally, to better understand physiological factors modulating fat taste sensing, we examined the effects of the orexigenic hormone ghrelin in the taste system. Through a conditioned taste aversion assay, systemic Ghrl-/- male mice exhibit diminished fat taste sensitivity compared to wild type (WT) mice with corresponding decreased calcium responses to fatty acids in taste cells. Lastly, ghrelin receptor (GHSR) agonists increased calcium responses to taste cells in WT mice. These data suggest that ghrelin plays a modulatory role in fat taste sensitivity. To further examine these effects using Ghsr-/- mice we observed Ghsr-/- females consume significantly less high fat diet than their WT counterparts. Ghsr-/- females also showed a significant reduction in fatty acid detection via a conditioned taste aversion assay with no threshold changes observed in males. Collectively, these studies demonstrate that the taste system is plastic and is modulated by diet, circulating hormone levels, and sex to selectively alter food intake

Effet du neuropeptide sNPF sur le comportement de l'abeille domestique (Apis mellifera)

Chez les vertébrés, le neuropeptide Y (NPY) joue un rôle crucial dans la survie individuelle en modulant à la fois les comportements liés à la nourriture et au stress. Des niveaux élevés de NPY corrèlent avec une augmentation de la faim provoquant une ingestion plus importante et réduisent la sensibilité aux stimuli stressants. Chez les invertébrés, deux homologues indépendants de NPY ont été identifiés : le neuropeptide F (NPF) et le neuropeptide F court (sNPF). Chez l'abeille domestique (Apis mellifera), npf et snpf ainsi que leur peptides respectifs NPF et sNPF ont été identifiés or seul sNPF possède un récepteur, suggérant un rôle fonctionnel de ce neuropeptide chez cet insecte. Nous avons étudié l'impact de sNPF sur une multitude de comportements comprenant l'ingestion de nourriture de bonne et mauvaise qualité, les réponses appétitives et aversives, les apprentissages et la mémoire appétitifs et aversifs. Nos résultats révèlent qu'une élévation artificielle des niveaux de sNPF via une application topique chez les butineuses augmente la prise alimentaire de nourriture bonne et mauvaise qualité. De plus, en utilisant une variété de tests pour étudier les réponses sensorielles, nous avons montré que sNPF a un rôle clé dans la modulation des réponses appétitives, mais cet effet est absent pour les réponses aversives. Les abeilles nourries et traitées avec du sNPF augmentent leur réponse au saccharose et aux stimuli olfactifs appétitifs, de façon similaire aux abeilles affamées. En adéquation avec les derniers résultats, des enregistrements in vivo multi photoniques de l'activité neuronale du lobe antennaire, le premier centre olfactif dans le cerveau de l'abeille, montrent une baisse des réponses aux odeurs appétitives chez les abeilles nourries qui est rétablie par le traitement avec le sNPF au même niveau que les abeilles affamées. Par ailleurs, l'effet modulatoire du sNPF était totalement absent sur les réponses aversives contrairement à ce qui a été observé chez la drosophile et les vertébrés, indiquant que chez les abeilles, sNPF n'augmente pas la tolérance aux stimuli stressants. Etant donné l'amplification causée par le traitement sNPF sur la réponse au saccharose, nous avons étudié si cet effet se retrouvait dans des protocoles d'apprentissage pour lesquels les abeilles étaient entraînées à discriminer un stimulus récompensé par du saccharose d'un autre qui ne l'est pas. Nous avons étudié l'effet du sNPF sur les apprentissages et mémoires appétitifs visuels et olfactifs. Dans le premier cas, des abeilles en semi libre vol ont été entraînées à discriminer deux couleurs dans un labyrinthe en Y après une application topique de sNPF. Dans le second cas, des abeilles en contention ont été entraînées à discriminer deux odeurs après une application topique de sNPF via le conditionnement du réflexe d'extension du proboscis. En parallèle, nous avons étudié les effets du sNPF sur l'apprentissage aversif gustatif pour lequel les abeilles en contention apprennent l'association entre une stimulation gustative de l'antenne avec un choc électrique après une application topique de sNPF. Nos résultats montrent une nette amélioration de l'apprentissage et mémoire appétitifs visuels et des tendances allant dans le même sens dans le cas de l'apprentissage appétitif olfactifs. A l'inverse, aucun effet n'a été observé quant à l'apprentissage et la mémoire aversifs gustatifs, ce qui est cohérent avec l'absence d'effet de sNPF sur les réponses sensorielles aversives. Ce travail de thèse a montré que le sNPF affecte plusieurs modalités de comportements (ingestion, gustation, olfaction, vision, apprentissage, mémoire) et les processus neuronaux (lobe antennaire) liés aux comportements appétitifs, mais non aversifs, chez l'abeille. Par conséquent, ce travail fournit de nouvelles perspectives pour étudier les processus d'ingestion et le comportement alimentaire des abeilles.Neuropeptide Y (NPY) signalling plays a crucial role for individual survival in vertebrates as it mediates both food- and stress-related behaviours. High NPY level correlates with increased hunger and leads to a larger food intake while it also reduces sensitivity to stressful stimuli. In invertebrates, two independent homologs of NPY have been identified: the neuropeptide F (NPF) and the short neuropeptide F (sNPF). In honey bees (Apis mellifera), both NPF and sNPF have been reported but only sNPF was found to have a dedicated receptor sNPFR, thus indicating that sNPF/sNPFR provides a functional signalling pathway in this insect. We thus studied the impact of sNPF on multiple behavioural components, including food-related behaviours such as ingestion of palatable and unpalatable food, appetitive and aversive responsiveness, and appetitive and aversive associative learning and memory retention. Our results show that increasing artificially sNPF levels in honey bee foragers via topical exposure, increases significantly their consumption of both palatable and unpalatable food. In addition, using various responsiveness tests, we showed that sNPF is a key player in the modulation of appetitive but not aversive responsiveness. Fed foragers treated with sNPF exhibited a significant increase in their responsiveness to sucrose solutions and to appetitive olfactory stimuli, matching the levels of starved bees. In agreement with this last finding, in vivo multiphoton recordings of neural activity in the antennal lobe, the primary olfactory centre of the bee brain, showed a decreased responsiveness to appetitive odours in fed bees, which was rescued by treatment with sNPF to the level exhibited by starved bees. Interestingly, the modulatory effect of sNPF was totally absent in responsiveness to aversive stimuli contrarily to what has been observed in vertebrates and flies, thus indicating that in bees, sNPF dos not increase tolerance to stressors. Given the enhancing effect of sNPF on appetitive responsiveness, we next studied if this effect translates to different appetitive learning protocols in which bees are trained to discriminate a stimulus that is rewarded with a sucrose solution from another that is not. We studied the effect of sNPF on both appetitive visual and olfactory learning and memory retention. In the first case, free-flying bees were trained to discriminate two colors in a Y-maze following topical increase of sNPF. In the second case, harnessed bees were trained to discriminate two odorants following topical application of sNPF, using the conditioning of the proboscis extension reflex. In parallel, we studied the effect of sNPF for aversive gustatory learning in which harnessed bees learning the association of antennal taste with electric shock, following topical application of sNPF. Our results revealed a clear improvement of appetitive color learning and retention and a mitigated tendency in the same direction in the case of appetitive olfactory learning. On the contrary, no effect was observed in the case of the aversive gustatory learning and retention, consistently with the lack of effect of sNPF on aversive responsiveness. To sum up, this work showed that sNPF affects multiple appetitive behavioural modules (ingestion, gustation, olfaction, vision, learning, memory) and central processing (antennal lobe activity) in the honey bee while being dispensable for aversive ones. It provides therefore a rich and multifaceted view of the effects of this neuropeptide on the behaviour of a social insect and opens new research perspective to study ingestion processes and appetitive behaviour in bees

Cochlea – A Physiological Description of a Finely Structured Sense Organ

The whole inner ear or the cochlea, responsible for hearing perception, represents a unique sense organ, including the organ of Corti and the inner ear endo- and perilymph. The fluid homeostasis of the lymph spaces with its parameters volume, concentration, osmolarity and pressure, as well as the finely aligned hair cell receptors, their supporting cells and structures embedded in these unique fluid spaces, corresponds to the specific necessities for adequate response to continuous stimulation and the outstanding discrimination capacity of the hearing system. The manuscript gives an overview and describes the structural characteristics and distinct physiological hearing qualities of the cochlea in comparison with the other human receptor cells and sense organs