Universiẗat, Diss., 2002--Bonn.

Universiẗat, Diss., 2005--Kiel.

Ants heavily rely on olfaction for communication and orientation and ant societies are characterized by caste- and sex-specific division of labor. Olfaction plays a key role in mediating caste-specific behaviours. I investigated whether caste- and sex-specific differences in odor driven behavior are reflected in specific differences and/or adaptations in the ant olfactory system. In particular, I asked the question whether in the carpenter ant, Camponotus floridanus, the olfactory pathway exhibits structural and/or functional adaptations to processing of pheromonal and general odors. To analyze neuroanatomical specializations, the central olfactory pathway in the brain of large (major) workers, small (minor) workers, virgin queens, and males of the carpenter ant C. floridanus was investigated using fluorescent tracing, immunocytochemistry, confocal microscopy and 3D-analyzes. For physiological analyzes of processing of pheromonal and non-pheromonal odors in the first odor processing neuropil , the antennal lobe (AL), calcium imaging of olfactory projection neurons (PNs) was applied. Although different in total glomerular volumes, the numbers of olfactory glomeruli in the ALs were similar across the female worker caste and in virgin queens. Here the AL contains up to ~460 olfactory glomeruli organized in 7 distinct clusters innervated via 7 antennal sensory tracts. The AL is divided into two hemispheres regarding innervations of glomeruli by PNs with axons leaving via a dual output pathway. This pathway consists of the medial (m) and lateral (l) antenno-cerebral tract (ACT) and connects the AL with the higher integration areas in the mushroom bodies (MB) and the lateral horn (LH). M- and l-ACT PNs differ in their target areas in the MB calyx and the LH. Three additional ACTs (mediolateral - ml) project to the lateral protocerebrum only. Males had ~45% fewer glomeruli compared to females and one of the seven sensory tracts was absent. Despite a substantially smaller number of glomeruli, males possess a dual PN output pathway to the MBs. In contrast to females, however, only a small number of glomeruli were innervated by projection neurons of the m-ACT. Whereas all glomeruli in males were densely innervated by serotonergic processes, glomeruli innervated by sensory tract six lacked serotonergic innervations in the female castes. It appears that differences in general glomerular organization are subtle among the female castes, but sex-specific differences in the number, connectivity and neuromodulatory innervations of glomeruli are substantial and likely to promote differences in olfactory behavior. Calcium imaging experiments to monitor pheromonal and non-pheromonal processing in the ant AL revealed that odor responses were reproducible and comparable across individuals. Calcium responses to both odor groups were very sensitive (10-11 dilution), and patterns from both groups were partly overlapping indicating that processing of both odor classes is not spatially segregated within the AL. Intensity response patterns to the pheromone components tested (trail pheromone: nerolic acid; alarm pheromone: n-undecane), in most cases, remained invariant over a wide range of intensities (7-8 log units), whereas patterns in response to general odors (heptanal, octanol) varied across intensities. Durations of calcium responses to stimulation with the trail pheromone component nerolic acid increased with increasing odor concentration indicating that odor quality is maintained by a stable pattern (concentration invariance) and intensity is mainly encoded in the response durations of calcium activities. For n-undecane and both general odors increasing response dynamics were only monitored in very few cases. In summary, this is the first detailed structure-function analyses within the ant’s central olfactory system. The results contribute to a better understanding of important aspects of odor processing and olfactory adaptations in an insect’s central olfactory system. Furthermore, this study serves as an excellent basis for future anatomical and/or physiological experiments. / Für Ameisen spielt die olfaktorische Kommunikation und Orientierung eine zentrale Rolle hinsichtlich der Organisation des Ameisenstaates. Ob sich kasten- und geschlechtsspezifische Verhaltensunterschiede auf neuronaler Ebene und besonders im olfaktorischen System der Ameise widerspiegeln ist die zentrale Frage meiner Arbeit. Im Speziellen stellte ich die Frage, ob sich in der olfaktorischen Bahn der Rossameise Camponotus floridanus strukturelle oder funktionelle Anpassungen an die Verarbeitung von Pheromonen und generellen Düften aufzeigen lassen. Zur Analyse hinsichtlich neuroanatomischer Spezialisierungen wurde die olfaktorische Bahn im Gehirn von großen und kleinen Arbeiterinnen, Jungköniginnen und Männchen der Rossameise C. floridanus mittels Fluoreszenzmassenfärbungen, Immunzytochemie, konfokaler Laserscanningmikroskopie und 3D-Auswertung untersucht. Um die Verarbeitung von Pheromonen und generellen Düften im primären olfaktorischen Neuropil, dem Antennallobus (AL), auf physiologischer Ebene zu charakterisieren wurden olfaktorische Projektionsneurone mittels Calcium Imaging untersucht. Obwohl sich das glomeruläre Gesamtvolumen der ALs zwischen Arbeiterinnenkasten und Jungköniginnen unterscheidet, lag die Gesamtzahl der Glomeruli im AL in einem ähnlichen Bereich. Der AL besteht in allen drei weiblichen Kasten aus bis zu 460 Glomeruli, die in sieben Clustern angeordnet sind und von sieben sensorischen Eingangstrakten innerviert werden. Der AL unterteilt sich in zwei Hemispheren, deren entsprechende Glomeruli von Projektionsneuronen innverviert werden, die vom AL über die Nervenbahn des “dual output pathway” in höhere Hirnregionen projizieren. Diese Nervenbahn besteht aus dem medialen (m) und lateralen (l) Antennocerebraltrakt (ACT) und verbindet den AL mit höheren Integrationszentren wie den Pilzkörpern (MB) und dem lateralen Horn (LH). M- und l-ACT unterscheiden sich in ihren Zielregionen im MB Calyx und dem LH. Drei weitere ACTs (mediolateral – ml) projizieren ausschließlich ins laterale Protocerebrum. Männchen besitzen ca. 45% weniger Glomeruli im Vergleich zur Weibchenkaste. Ihnen fehlt weiterhin einer der sieben sensorischen Eingangstrakte vollständig. Trotz der wesentlich geringeren Anzahl an Glomeruli, besitzen auch Männchen den “dual output pathway”. Im Gegensatz zu den Weibchen ist allerdings nur eine geringe Anzahl an Glomeruli durch m-ACT Projektionsneurone innerviert. Ein weiterer Unterschied im AL von Männchen und Weibchen findet sich in den Glomeruli des sensorische Trakts Nummer sechs, die bei Weibchen keinerlei serotonerge Innervierung aufweisen während beim Männchen der gesamte AL dichte serotonerge Verzweigungen besitzt. Es zeigt sich somit, dass die kastenspezifischen Unterschiede in der allgmeinen glomerulären Organisation des AL innerhalb der Weibchenkaste nur sehr fein sind. Im Gegensatz dazu sind die geschlechtsspezifischen Unterschiede in Anzahl, Konnektivität und neuromodulatorischer Innervierung von Glomeruli zwischen Weibchen- und Männchen wesentlich ausgeprägter was Unterschiede in olfaktorisch geprägten Verhaltensweisen begünstigen könnte. Die Calcium Imaging Experimente zur Untersuchung der Verarbeitung von Pheromonen und generellen Düften im AL der Ameise zeigten, dass Duftantworten reproduzierbar und zwischen Individuen vergleichbar waren. Die Sensitivität des Calcium Signals lag für beide Duftgruppen in einem sehr niedrigen Bereich (Verdünnung 10-11). Die Antortmuster beider Duftgruppen überlappten zum Teil, was die Annahme zuläßt, dass die Verarbeitung von Pheromonen und generellen Düften keiner räumlichen Trennung innerhalb des AL unterliegt. Die Intensität der Antwortmuster auf die Pheromonkomponenten (Spurpheromon: Nerolsäure; Alarmpheromon: n-Undecan) blieben in den meisten Fällen über einen weiten Konzentrationsbereich konstant (7-8 log Einheiten). Die Dauer der Calciumantwort nach Stimulation mit Nerolsäure verlängerte sich mit steigender Duftkonzentration. Dies läßt für das Spurpheromon den Schluß zu, dass die Duftqualität in einem konstanten Duftmuster (Konzentrationsinvarianz) repräsentiert und die Duftintensität über die Dauer des Calciumsignals abgebildet wird. Da die Antwortmuster auf generelle Düfte (Heptanal, Octanol) dagegen sehr viel stärker innerhalb des getesteten Konzentrationsbereichs varrieren ließ sich für n-Undecan und die beiden generellen Düfte eine solche Dynamik nur in einigen wenigen Fällen beobachtet. Zusammenfassend ist diese Studie die erste strukturelle und funktionelle Studie des olfaktorischen Systems der Ameise. Die Ergebnisse tragen zu einem besseren Verständnis der neuronalen Adaptationen und Mechanismen hinsichtlich Duftverarbeitung im zentralen Nervensystem von Insekten bei. Außerdem liefert diese Studie eine wichtige Grundlage für zukünftige neuroanatomische und –physiologische Untersuchungen auf dem Gebiet der Neurobiologie der Insekten.

Obesity is a major health concern that is accompanied by a high risk for several disorders such as type 2 diabetes, cardiovascular disease and certain forms of cancer (Stevens et al., 2012; Lahey and Khan, 2018). Since the prevalence of obesity has nearly tripled within the last 45 years and is still on the rise (WHO, 2018), it is imperative to understand mechanisms that might underlie the emergence and maintenance of obesity to develop new prevention- and intervention strategies. The high availability of energy-rich food is one of the main contributing factors for the increasing prevalence of obesity. Hence, the mechanisms underlying eating without physiological needs come into focus. In that regard, the olfactory system plays a major role: it is equally involved in homeostatic signaling of hunger and hedonic eating (Palouzier-Paulignan et al., 2012). Given that the sense of smell is altered in obesity (for an overview see Peng et al., 2019), the overall goal of this thesis is to contribute to further understanding of the mechanisms that might underlie this phenomenon. It has been previously shown that people with obesity evaluate food odors as more pleasant (Stafford and Whittle, 2015) and show higher reactivity towards them (Proserpio et al., 2019), however, they persistently have lower olfactory function. This low function is most evident in olfactory sensitivity, i.e. picking up odors from the environment, and is therefore related to appetite and food search. Odor sensitivity evaluation is usually a lengthy procedure and is standardly performed with non-food odors. However, Stafford and Whittle (2015), revealed a different result for sensitivity to food odors: obese outperformed normal-weight participants for chocolate odor. Strikingly, further scrutiny reveals that metabolic and endocrine health factors could provide a possible explanation for divergent results of olfactory sensitivity to food and non-food odors in obesity: whereas the chocolate-study included only class 1 obese participants (BMI 30-35 kg/m2), all other studies included class 2-3 obese participants (BMI > 35 kg/m2). It is likely that obese class 2-3 participants are more affected by hormonal changes, such as higher insulin resistance and higher leptin levels than their less obese counterparts. On a recent note, it has been shown that the olfactory and endocrine systems are closely linked (Palouzier-Paulignan et al., 2012). As such there are many receptors for hunger-related hormones located in brain structures that are highly relevant for odor processing as well as for the regulation of homeostatic needs (Baly et al., 2007; Lacroix et al., 2008; Henkin, 2010). Especially, the olfactory bulbs, where olfactory information is firstly processed in the brain, have a high density of insulin and leptin receptors (Baskin et al., 1983; Thanarajah et al., 2019; Havrankova et al., 1981; Marks et al., 1990). Further, animal studies have reliably demonstrated that obesity leads to structural and functional changes in the olfactory system (Thiebaud et al., 2014; Fadool et al.2011; Riviére et al., 2016). However, brain anatomical changes in the olfactory system of humans have not been studied yet. To conclude, we firstly aimed to develop an olfactory test that is easy to administer and of short duration to apply in a complex research design, because available tests are time consuming and highly variable in duration (10-25 min). Secondly, in order to elucidate the potential link between olfactory impairments in obesity and metabolic health factors, we investigated food and non-food odor sensitivity in a wide body weight range and related it to metabolic and endocrine factors such as insulin resistance. Third, we aimed to investigate the possible relationship between obesity and brain anatomical changes in the olfactory bulbs. Study 1: In our first study, we measured olfactory sensitivity in a within-subject repeated-measures design in 20 young and healthy participants. Using the odor detection threshold subtest from the “Sniffin’ Sticks” test battery, we applied three different presentation methods: (1) gold standard, (2) shorter single staircase method and (3) ascending procedure. Compared to the gold-standard, the shorter single staircase procedure was 26% and the ascending procedure was 51% shorter in duration. Both short procedure thresholds correlated highly with the gold standard threshold. All three tests showed similar test-retest reliability. To conclude, we have developed a test that takes on average 5-7 minutes less time and is as reliable as the gold standard. Study 2: Within the second study, we focused on metabolic health parameters that might explain the relationship between odor sensitivity and obesity. We investigated food and non-food odor sensitivity in the hungry and sated state in 75 young healthy participants with normal weight, overweight and obesity in a within-subject, repeated-measures design. We assessed metabolic health status with BMI, WHR, pre- and postprandial levels of insulin, leptin, glucose, and ghrelin. We showed that odor sensitivity did not directly depend on body weight status or BMI. However, we found a strong negative mediating effect of insulin resistance as assessed by HOMA-IR score on the relationship between BMI and olfactory sensitivity for the food odor. Post-hoc regression models revealed that insulin resistance rather than obesity is responsible for this effect. To conclude, our findings indicate a strong negative association between insulin resistance and sensitivity to food odors. Study 3: In the third study, we examined neuroanatomical correlates of smell perception in obesity and its relationship with metabolic health factors. Olfactory bulb volume was assessed with magnetic resonance imaging in 67 healthy normal weight, overweight and obese participants. To examine recently proposed mechanistic explanatory models of altered smell perception in obesity, we collected parameters that are associated with metabolic health in obesity, such as insulin resistance, leptin, body fat percentage and fat mass index. We showed that in our sample, people with obesity had significantly lower olfactory bulb volume when compared to people with normal weight. Further, we found that olfactory bulb volume was negatively associated with other measures of metabolic health, especially insulin resistance, leptin, and body fat percentage. Our results imply that, similar to other diseases such as depression and Parkinson’s disease, obesity also involves a neuroanatomical change in the olfactory bulbs compared to healthy participants with normal weight. Hence, our study provides first indications that obesity is associated with brain anatomical changes in the olfactory bulbs. Conclusion The overall aim of this thesis was to shed light on the complex relationship between obesity and olfaction. Study 1 provides two easy-to-use odor threshold test procedures for clinical use or for complex research designs with limited time frames. Importantly, this thesis emphasizes the major role of metabolic health status and especially insulin resistance in the altered smell perception in obesity. Most notably, poor metabolic health mediates the relationship between obesity and olfactory sensitivity (study 2). Metabolic health parameters rather than obesity per se might be responsible for low olfactory function and should be further scrutinized in future studies. In particular, a group-design with elevated vs. normal HOMA-IR participants instead of BMI groups could provide more insights. Intriguingly, a high BMI and related metabolic health factors, such as high insulin resistance and high body fat percentage are associated with neuroanatomical changes in the olfactory system, i.e., lower olfactory bulb volume (study 3). These findings contribute to a further understanding of explanatory models introduced by Peng et al. (2019). In accordance with this metabolic and hormonal model our results support the theoretical framework that metabolic and hormonal shifts in obesity might be crucial for changes in olfactory perception. Thereby, these results provide a deeper understanding of the pathophysiological mechanisms underlying altered olfactory function in obesity. Subsequently, olfaction might represent a new target for prevention or therapy.:LIST OF ABBREVIATIONS I LIST OF FIGURES II LIST OF TABLES III I. INTRODUCTION 1 1. THE OBESITY PANDEMIC 1 2. HORMONES INVOLVED IN OBESITY AND OLFACTION 4 2.1 HORMONES IN THE REGULATION OF EATING BEHAVIOR AND OBESITY 4 2.2 HORMONES IN THE CONTEXT OF SMELL PERCEPTION 7 3. THE OLFACTORY SYSTEM 9 3.1 ANATOMY AND PHYSIOLOGY 9 3.2 MEASURING SMELL ABILITY: THREE DIMENSIONS OF OLFACTORY FUNCTION 13 3.3 THE ROLE OF OLFACTION IN THE CONTROL OF EATING BEHAVIOR 14 3.4 SMELL PERCEPTION IN OBESITY 15 4. THE LINK: WHY TARGET THE OLFACTORY SYSTEM IN OBESITY? 19 II. RATIONALE OF THE EXPERIMENTAL WORK 20 III. EXPERIMENTAL WORK 21 STUDY 1: SHORT PROCEDURE TO ASSESS ODOR DETECTION THRESHOLDS 21 STUDY 2: ODOR SENSITIVITY FOR FOOD AND NON-FOOD ODORS IN OBESITY 30 STUDY 3: BRAIN ANATOMICAL CORRELATES OF SMELL PERCEPTION IN OBESITY 47 IV. SUMMARY 60 V. REFERENCES 65 VI. APPENDIX 75 A. DECLARATION OF AUTHENTICITY 75 B. AUTHOR CONTRIBUTIONS 76 C. CURRICULUM VITAE 80 D. ACKNOWLEDGEMENTS 83

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Im Projekt wurde das Lagrangeverfahren zur Rückrechnung auf Geruchsquellen als IT-Lösung realisiert. Damit können bei Geruchsbeschwerden in Gemengelagen erstmalig Hauptverursacher ermittelt werden. Das Projekt wird als LfULG-Amtshilfe für Immissionsschutzbehörden eingesetzt. Redaktionsschluss: 31.12.2011, Stand: 30.11.2021