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The slowly activating vacuolar SV/TPC1 channel is ubiquitously expressed in plants and provides a large cation conductance in the vacuolar membrane. Thereby, monovalent (K+, Na+) and in principle also divalent cations, such as Ca2+, can pass through the channel. The SV/TPC1 channel is activated upon membrane depolarization and cytosolic Ca2+ but inhibited by luminal calcium. With respect to the latter, two luminal Ca2+ binding sites (site 1 Asp240/Asp454/Glu528, site 2 Glu239/Asp240/Glu457) were identified to coordinate luminal Ca2+. In this work, the characteristics of the SV/TPC1 channels in terms of regulation and function were further elucidated, focusing on the TPC1s of Arabidopsis thaliana and Vicia faba. For electrophysiological analysis of the role of distinct pore residues for channel gating and luminal Ca2+ sensing, TPC1 channel variants were generated by site-directed mutagenesis and transiently expressed as eGFP/eYFP-fusion constructs in Arabidopsis thaliana mesophyll protoplasts of the TPC1 loss-of-function mutant attpc1-2.
1. As visualized by confocal fluorescence laser-scanning microscopy, all AtTPC1 (WT, E605A/Q, D606N, D607N, E605A/D606N, E605Q/D606N/D607N, E457N/E605A/D606N) and VfTPC1 channel variants (WT, N458E/A607E/ N608D) were correctly targeted to the vacuole membrane.
2. Patch-clamp studies revealed that removal of one of the negative charges at position Glu605 or Asp606 was already sufficient to promote voltage-dependent channel activation with higher voltage sensitivity. The combined neutralization of these residues (E605A/D606N), however, was required to additionally reduce the luminal Ca2+ sensitivity of the AtTPC1 channel, leading to hyperactive AtTPC1 channels. Thus, the residues Glu605/Asp606 are functionally coupled with the voltage sensor of AtTPC1 channel, thereby modulating channel gating, and form a novel luminal Ca2+ sensing site 3 in AtTPC1 at the luminal entrance of the ion transport pathway.
3. Interestingly, this novel luminal Ca2+ sensing site 3 (Glu605/Asp606) and Glu457 from the luminal Ca2+ sensing site 2 of the luminal Ca2+-sensitive AtTPC1 channel were neutralized by either asparagine or alanine in the TPC1 channel from Vicia faba and many other Fabaceae. Moreover, the VfTPC1 was validated to be a hyperactive TPC1 channel with higher tolerance to luminal Ca2+ loads which was in contrast to the AtTPC1 channel features. As a result, VfTPC1 but not AtTPC1 conferred the hyperexcitability of vacuoles. When AtTPC1 was mutated for the three VfTPC1-homologous polymorphic site residues, the AtTPC1 triple mutant (E457N/E605A/D606N) gained VfTPC1-like characteristics. However, when VfTPC1 was mutated for the three AtTPC1-homologous polymorphic site residues, the VfTPC1 triple mutant (N458E/A607E/N608D) still sustained VfTPC1-WT-like features. These findings indicate that the hyperactivity of VfTPC1 is achieved in part by the loss of negatively charged amino acids at positions that - as part of the luminal Ca2+ sensing sites 2 and 3 – are homologous to AtTPC1-Glu457/Glu605/Asp606 and are likely stabilized by other unknown residues or domains.
4.The luminal polymorphic pore residues (Glu605/Asp606 in AtTPC1) apparently do not contribute to the unitary conductance of TPC1. Under symmetrical K+ conditions, a single channel conductance of about 80 pS was determined for AtTPC1 wild type and the AtTPC1 double mutant E605A/D606A. This is in line with the three-fold higher unitary conductance of VfTPC1 (232 pS), which harbors neutral luminal pore residues at the homologous sites to AtTPC1.
In conclusion, by studying TPC1 channel from Arabidopsis thaliana and Vicia faba, the present thesis provides evidence that the natural TPC1 channel variants exhibit differences in voltage gating, luminal Ca2+ sensitivity and luminal Ca2+ binding sites.
Neuste Studien haben ergeben, dass Asc-1 Knock-out Mäuse aufgrund einer verminderten intrazellulären Glycinkonzentration in synaptischen Boutons im Gehirn, einen Hyperekplexie-ähnlichen Phänotyp entwickeln. Aufgrund nicht vollständig geklärter Ursachen für die Entstehung des Krankheitsbildes der Hyperekplexie beim Menschen, wurde eine Kohorte von 51 Patienten zusammengetragen, um vor dem Hintergrund der Forschungsergebnisse zu Asc-1 im Tiermodell, das kodierende Gen beim Menschen SLC7A10 als mögliches Kandidatengen auf Sequenzalterationen zu untersuchen. Hierfür wurde aus Vollblut der an Hyperekplexie erkrankten Patienten genomische DNA isoliert, um mittels PCR und anschließendem Screening der Sequenzen, Mutationen innerhalb funktionell wichtiger Bereiche des Gens zu eruieren. Neben weiteren Sequenzunterschieden, die meist in Introns gefunden wurden, wurde die codierende Mutation G307R innerhalb von Exon 7 identifiziert, die letztendlich der Grund für eine Versuchsreihe war, um zu hinterfragen, ob dieser Aminosäureaustausch in der Proteinsequenz funktionelle Konsequenzen zur Folge hat. HEK293-Zellen wurden mit dem zuvor hergestellten Klon G307R transfiziert, um über Biotinylierung, immuncytochemische Färbungen und funktionelle Untersuchungen die Aktivität des Transporters zu beurteilen. Hier zeigte sich ein Funktionsverlust von über 95 %, bei uneingeschränkter Oberflächenexpression. ASC-1 bestätigt sich damit als neue Ursache in der Ausprägung von Hyperekplexie. Ferner können Zusammenhänge mit geistiger Retardierung und eingeschränkter neuronaler Plastizität bestehen.
The phytohormone auxin performs important functions in the initiation of plant tissues and organs, as well as in the control of root growth in conjunction with external stimuli such as gravity, water and nutrient availability. These functions are based primarily on the auxin-dependent regulation of cell division and elongation. Important for the latter is the control of the cell turgor by the vacuole. As storage for nutrients, metabolites and toxins, vacuoles are of vital importance. Vacuolar stored metabolites and ions are exchanged across the vacuolar membrane with the cytoplasm via active transport processes as well as passively through ion channels. In their function as second messenger, calcium ions are important regulators but also subject to vacuolar transport processes. Changes in the cytosolic calcium concentration not only act locally, but are also associated with signal transduction over longer distances. In this work, electrophysiological methods were combined with imaging techniques to gain insights into the interaction between cytosolic calcium signals, vacuolar transport processes and auxin physiology in the intact plant organism.
Calcium signals are involved in the regulation of vacuolar ion channels and transporters. In order to investigate this in the intact organism, intracellular microelectrode measurements were performed in the model system of bulging Arabidopsis thaliana root hairs. By means of the two-electrode voltage-clamp technique, it could be confirmed that the vacuolar membrane is the limiting electrical resistance during intravacuolar measurements and thus measured ion currents actually represent only the currents across the vacuolar membrane. The already known time-dependent decrease of vacuolar conductivity during intravacuolar experiments could be further correlated with an impalement-related, transient increase of the cytosolic calcium concentration. Intravacuolar voltage-clamp experiments in root hair cells of calcium reporter plants confirmed this relationship between vacuolar conductivity and the cytosolic calcium concentration.
However, the vacuole is not just a recipient of cytosolic calcium signals. Since the vacuole represents the largest intracellular calcium reservoir, it has long been argued that it is also involved in the generation of such signals. This could be confirmed in intact root hair cells. Changes in the vacuolar membrane potential affected the cytosolic calcium concentration in these cells. While depolarizing potentials led to an increase of the cytosolic calcium concentration, hyperpolarization of the vacuolar membrane caused the opposite. Thermodynamic considerations of passive and active calcium transport across the vacuolar membrane suggested that the results described herein reflect the behaviour of vacuolar H+/Ca2+ exchangers whose activity is determined by the proton motive force.
In addition, cytosolic calcium has been shown to be a key regulator of a rapid auxin-induced signaling pathway that regulates polar transport of the hormone.
In the same model system of bulging root hairs it could be shown that the external application of auxin results in a very fast, auxin concentration- and pH-dependent depolarization of the plasma membrane potential. Synchronous with the depolarization of the plasma membrane potential, transient calcium signals were recorded in the cytosol. These were caused by an auxin-activated influx of calcium ions through the ion channel CNGC14. Experiments on loss-of-function mutants as well as pharmacological experiments showed that the auxin-induced activation of the calcium channel requires auxin-perception by the F-box proteins of the TIR1/AFB family.
Investigations of auxin-dependent depolarization as well as the auxin-induced influx of protons into epidermal root cells of loss-of-function mutants showed that the secondary active uptake of auxin by the high-affinity transport protein AUX1 is responsible for the rapid depolarization
Not only the cytosolic calcium signals correlated with CNGC14 function, but also the AUX1-mediated depolarization of root hairs. An unchanged expression of AUX1 in the cngc14 loss-of-function mutant suggested that the activity of AUX1 must be post-translationally regulated. This hypothesis was supported by experiments in which treatment with the calcium channel blocker lanthanum led to inactivation of AUX1 in the wild type.
The cytosolic loading of individual epidermal root cells with auxin resulted in the spread of lateral and acropetal calcium waves. These correlated with a shift of the auxin gradient at the root apex and thus supported a hypothetical calcium-dependent regulation of polar auxin transport. A model for a rapid, auxin-induced and calcium-dependent signaling pathway is presented and its importance for gravitropic root growth is discussed. Since AUX1-mediated depolarization varied with external phosphate concentration, the importance of this rapid signaling pathway is also discussed for the adaptation of root hair growth to an inadequate availability of phosphate.
Humans use their eyes not only as visual input devices to perceive the environment, but also as an action tool in order to generate intended effects in their environment. For instance, glances are used to direct someone else's attention to a place of interest, indicating that gaze control is an important part of social communication. Previous research on gaze control in a social context mainly focused on the gaze recipient by asking how humans respond to perceived gaze (gaze cueing). So far, this perspective has hardly considered the actor’s point of view by neglecting to investigate what mental processes are involved when actors decide to perform an eye movement to trigger a gaze response in another person. Furthermore, eye movements are also used to affect the non-social environment, for instance when unlocking the smartphone with the help of the eyes. This and other observations demonstrate the necessity to consider gaze control in contexts other than social communication whilst at the same time focusing on commonalities and differences inherent to the nature of a social (vs. non-social) action context. Thus, the present work explores the cognitive mechanisms that control such goal-oriented eye movements in both social and non-social contexts.
The experiments presented throughout this work are built on pre-established paradigms from both the oculomotor research domain and from basic cognitive psychology. These paradigms are based on the principle of ideomotor action control, which provides an explanatory framework for understanding how goal-oriented, intentional actions come into being. The ideomotor idea suggests that humans acquire associations between their actions and the resulting effects, which can be accessed in a bi-directional manner: Actions can trigger anticipations of their effects, but the anticipated resulting effects can also trigger the associated actions. According to ideomotor theory, action generation involves the mental anticipation of the intended effect (i.e., the action goal) to activate the associated motor pattern. The present experiments involve situations where participants control the gaze of a virtual face via their eye movements. The triggered gaze responses of the virtual face are consistent to the participant’s eye movements, representing visual action effects. Experimental situations are varied with respect to determinants of action-effect learning (e.g., contingency, contiguity, action mode during acquisition) in order to unravel the underlying dynamics of oculomotor control in these situations. In addition to faces, conditions involving changes in non-social objects were included to address the question of whether mechanisms underlying gaze control differ for social versus non-social context situations.
The results of the present work can be summarized into three major findings. 1. My data suggest that humans indeed acquire bi-directional associations between their eye movements and the subsequently perceived gaze response of another person, which in turn affect oculomotor action control via the anticipation of the intended effects. The observed results show for the first time that eye movements in a gaze-interaction scenario are represented in terms of their gaze response in others. This observation is in line with the ideomotor theory of action control. 2. The present series of experiments confirms and extends pioneering results of Huestegge and Kreutzfeldt (2012) with respect to the significant influence of action effects in gaze control. I have shown that the results of Huestegge and Kreutzfeldt (2012) can be replicated across different contexts with different stimulus material given that the perceived action effects were sufficiently salient. 3. Furthermore, I could show that mechanisms of gaze control in a social gaze-interaction context do not appear to be qualitatively different from those in a non-social context.
All in all, the results support recent theoretical claims emphasizing the role of anticipation-based action control in social interaction. Moreover, my results suggest that anticipation-based gaze control in a social context is based on the same general psychological mechanisms as ideomotor gaze control, and thus should be considered as an integral part rather than as a special form of ideomotor gaze control.
Mutationen im Glycintransporter 2 (GlyT2) stellen die präsynaptische Komponente der neurologischen Erkrankung Hyperekplexie oder Startle Disease dar. Der neuronale Na+/Cl- -abhängige GlyT2 ist für das Recycling von Glycin verantwortlich und bildet an inhibitorischen glycinergen Synapsen die Hauptquelle des freigesetzten Transmitters. Dominante, rezessive und zusammengesetzte heterozygote Mutationen wurden bereits identifiziert, von denen die meisten zu einer beeinträchtigten Glycinaufnahme führen. In dieser Arbeit konnten wir eine neue pathogene Mutation innerhalb des neuronalen Glycintransporter-2-Gens (SLC6A5, OMIM604159) in einer Familie identifizieren, in der beide Elternteile heterozygote Träger waren. Ein homozygotes Kind litt an schweren neuromotorischen Defiziten, wohingegen Heterozygote keine Symptome aufwiesen. Die neue rezessive Mutation c.1286C>T erzeugte einen missense Aminosäureaustausch von Prolin gegen Leucin an Position 429 (pP429L) in der Transmembrandomäne 5. Wir haben die GlyT2P429L-Variante mittels Homologiemodellierung, immuncytochemischer Färbungen, Western Blot Analysen, Biotinylierung und funktioneller Glycinaufnahmetests charakterisiert. Der mutierte GlyT2 zeigte beim Proteintransport durch verschiedene intrazelluläre Kompartimente zur Zelloberfläche keine Defizite. Die gesamte Proteinexpression war jedoch signifikant verringert. Obwohl GlyT2P429L an der Zelloberfläche vorhanden ist, zeigte er einen Verlust der Proteinfunktion. Die Co-Expression der Mutante mit dem Wildtyp-Protein, die die Situation der Eltern widerspiegelte, hatte keinen Einfluss auf die Transporterfunktion und erklärte somit ihren nicht symptomatischen Phänotyp. Wenn jedoch die Mutante im Vergleich zum Wildtyp-Protein im Überschuss exprimiert wurde, war die Glycinaufnahme signifikant verringert. Die Strukturanalyse ergab, dass der eingeführte Leucinrest an Position 429 zu Konformationsänderungen in der α-Helix 5 führt, die in unmittelbarer Nähe zur Natriumbindungsstelle des Transporters lokalisiert sind. Dies deutet darauf hin, dass die Zugangsmechanismen des GlyT2 gestört sein könnten und einen vollständigen Verlust der Transportaktivität verursachen. Unsere Ergebnisse belegen, dass P429 in GlyT2 ein strukturell wichtiger Aminosäurerest ist, der eine wichtige funktionelle Rolle beim Glycintransport spielt.
Das Sprouty-related, EVH1 domain containing protein 2 (SPRED2) ist ein
inhibitorisches, downstream von Ras wirkendes Protein des MAP-Kinase Signalwegs,
welches entscheidenden Einfluss auf die Regulation von Proliferation, Expression von
Proteinen und der zellulären Homöostase hat. Der kardiale Phänotyp von SPRED2-
defizienten Mäusen zeigt nicht nur eine deutliche linksventrikuläre Hypertrophie,
sondern auch eine erhöhte Fibrosierung des Herzgewebes. Zellulär wird die SPRED2-
Defizienz durch die Akkumulation von vesikulären Strukturen innerhalb der Zelle,
sowie eine markant erhöhte Anzahl von Vesikeln entlang der longitudinalen Reihen
der Mitochondrien gekennzeichnet.
Ziel dieser Arbeit war es, den Charakter dieser vesikulären Strukturen näher zu
beleuchten und festzustellen, in welchem Zusammenhang die subzellulär veränderte
Architektur mit der Hypertrophie der SPRED2-defizienten Tiere steht. Um diese
Fragestellung zu beantworten, wurde zunächst nach einem vesikulären
Degradationsmechanismus gesucht, der in SPRED2-/--Cardiomyocyten betroffen sein
könnte. Die Macroautophagie, im folgenden Autophagie bezeichnet, ist ein solcher
Degradationsmechanismus, bei dem selektiv langlebige Proteine und Zellorganellen
abgebaut werden. Es konnten signifikante Veränderung der Protein-Level an
Schlüsselpositionen der Autophagie identifiziert werden. Das Ubiquitin-aktivierende
(E1) Enzym Homolog Atg7 sowie die Cystein-Protease Atg4B zeigen sich im SPRED2-
KO deutlich reduziert. Ebenso Atg16L, das als essentieller Bestandteil des Atg5-
Atg12-Atg16-Konjugationssystems bei der Konjugation von MAPLC3-II an das
Phospholipid Phosphatidylethanolamin beteiligt ist. Die Autophagie-Rate als
Verhältnis von konjugiertem zu unkonjugiertem MAPLC3 ist ebenfalls reduziert. Die
Akkumulation der autophagischen Vesikel zeigt sich kongruent zu dem erhöhten
Protein-Level der autophagischen Cargo-Rezeptoren SQSTM1 und NBR1, sowie des
lysosomalen Markers CathepsinD. Außer der verringerten Autophagie-Rate zeigt sich
in Einklang mit der Fibrosierung des Herzgewebes eine erhöht aktive Caspase-3 als
Marker für Apoptose. Um die mitochondriale Integrität näher zu beleuchten, wurde die
Menge an reaktiven Sauerstoffspezies (ROS) in Wildtyp und SPRED2-KO untersucht.
Hierbei zeigte sich eine erhöhte Menge an ROS im KO, was ein Hinweis auf eine
Beeinträchtigung der Mitochondrien darstellt.
Letztlich wurde die Hypothese überprüft, ob ein gestörter Transport der Vesikel
durch eine Beeinträchtigung der Motorproteine Dynein und Kinesin vorliegt. In der Tat
zeigte sich die Aktivität der Dynein-ATPase verringert in der Abwesenheit von
SPRED2. Diese Beobachtung wird durch die erhöhten Mengen des vSNARE-Proteins
VTI1b unterstützt, was letztlich die Akkumulation der autophagischen Vesikel mit einer
verringerten Fähigkeit zur Membranfusion und dem ineffizienteren Transport der
Vesikel in Einklang bringt.
Da die gesamten Experimente in einem globalen SPRED2-KO System
durchgeführt wurden, können eventuelle Auswirkungen der beeinflussten hormonellen
Situation der SPRED2-KO Tiere auf den Herzphänotyp nicht final ausgeschlossen
werden. Um die genaue Wirkung einer SPRED2-Defizienz auf das Herzgewebe und
das Herz als Organ zu untersuchen, wurde im Rahmen dieser Arbeit eine SPRED2-
defiziente knockout Mauslinie mit konditionalem Potential generiert, die eine
gesteuerte Deletion von SPRED2 im Herzgewebe erlaubt.
Einleitung:
TRPC-Kanäle spielen eine wichtige Rolle in der Pathologie der Herzinsuffizienz und kardialen Hypertrophie. Diese Effekte werden unter anderem über den Calcineurin-NFAT-Signalweg vermittelt. Ein wichtiger Interaktionspartner und Regulator von TRPC-Kanälen ist das Protein FKBP52. Mittels eines Yeast Two-Hybrid Systems wurde in einer kardialen cDNA library eine Interaktion zwischen einem C-terminalen Fragment von TRPC3 (AS 742-848), welches außerhalb der bekannten FKBP-Bindungsdomäne (AS 703-714) liegt, und FKBP52 beobachtet. Da dies eine weitere Bindungsstelle in FKBP52 vermuten ließ, erzeugten wir ein Fragment von FKBP52, welches FKBP52s genannt wurde und dem die funktionell relevante PPIase I-Domäne mit der bekannten Bindungsstelle fehlt. Eine erste Co-IP zwischen diesem Fragment und TRPC3 war erfolgreich.
Ziel:
Die Bestimmung, ob die Anwesenheit des verkürzten FKBP52 in vivo die Komplexbildung aus TRPC3 bzw. TRPC4 und dem Wildtyp-FKBP52 unterdrückt. Zusätzlich, ob FKBP52s die Interaktion zwischen TRPC3 bzw. TRPC4 und Calcineurin in vivo unterbricht und damit die Aktivierung des Calcineurin-NFAT-Signalweges hemmt.
Methoden:
Co-Immunopräzipitationen (Co-IP) wurden mit HEK-293-Zellen durchgeführt, die mit cDNA transfiziert wurden, welche Gene für TRPC3, TRPC4, Calcineurin A und FKBP52s enthielt. Zur Bestimmung der nukleären Translokation von NFATc1 mittels Fluoreszenzmikroskopie wurden HEK-293-Zellen mit TRPC3, TRPC4, GFP-NFATc1 ± FKBP52s transfiziert. Die statistische Analyse erfolgte mit einer One-Way ANOVA.
Ergebnisse:
In dieser Arbeit konnte gezeigt werden, dass FKBP52 sowohl mit TRPC3 als auch mit TRPC4 interagiert. Ebenso wurde festgestellt, dass FKBP52 auch ohne seine katalytische PPIase I-Domäne Bindungen mit TRPC3 bzw. TRPC4 eingeht. Dieses FKBP52-Konstrukt nimmt ebenso an der Komplexbildung mit TRPC3 bzw. TRPC4 und Calcineurin teil. Des Weiteren ließ sich für TRPC3 zeigen, dass unter Stimulation mit Carbachol (GPCR-Agonist) bei Anwesenheit dieses gekürzten FKBP52 eine signifikant geringere Aktivierung und Wanderung des Transkriptionsfaktors NFAT in den Nucleus erfolgte.
Schlussfolgerung:
FKBP52 spielt daher eine wichtige Rolle in dieser Signalkaskade, indem es entscheidend an der Aktivierung von Calcineurin und dessen Rekrutierung zum TRPC-Kanalkomplex beteiligt ist und damit auch an der Aktivierung des Calcineurin-NFAT-Signalweges.
The present dissertation aims to shed light on different mechanisms of socio-emotional feedback in social decision-making situations. The objective is to evaluate emotional facial expressions as feedback stimuli, i.e., responses of interaction partners to certain social decisions. In addition to human faces, artificial emojis are also examined due to their relevance for modern digital communication. Previous research on the influence of emotional feedback suggests that a person's behavior can be effectively reinforced by rewarding stimuli. In the context of this dissertation, the differences in the feedback processing of human photographs and emojis, but also the evaluation of socially expected versus socially unexpected feedback were examined in detail in four studies. In addition to behavioral data, we used the electroencephalogram (EEG) in all studies to investigate neural correlates of social decision-making and emotional feedback.
As the central paradigm, all studies were based on a modified ultimatum game. The game is structured as follows: there is a so-called proposer who holds a specific amount of money (e.g., 10 cents) and offers the responder a certain amount (e.g., 3 cents). The responder then decides whether to accept or reject the offer. In the version of the ultimatum game presented here, different types of proposers are introduced. After the participants have accepted or rejected in the role of the responder, the different proposers react to the participant’s decision with specific emotional facial expressions. Different feedback patterns are used for the individual experiments conducted in the course of this dissertation.
In the first study, we investigated the influence of emotional feedback on decision-making in the modified version of the ultimatum game. We were able to show that a proposer who responds to the acceptance of an offer with a smiling face achieves more accepted offers overall than a control proposer who responds to both accepted and rejected offers with a neutral facial expression. Consequently, the smile served as a positive reinforcement. Similarly, a sad expression in response to a rejected offer also resulted in higher acceptance rates as compared to the control identity, which could be considered an expression of compassion for that proposer. On a neuronal level, we could show that there are differences between simply looking at negative emotional stimuli (i.e., sad and angry faces) and their appearance as feedback stimuli after rejected offers in the modified ultimatum game. The so-called feedback-related negativity was reduced (i.e., more positive) when negative emotions appeared as feedback from the proposers. We argued that these findings might show that the participants wanted to punish the proposers by rejecting an offer for its unfairness and therefore the negative feedback met their expectations. The altered processing of negative emotional facial expressions in the ultimatum game could therefore indicate that the punishment is interpreted as successful. This includes the expectation that the interaction partner will change his behavior in the future and eventually make fairer offers.
In the second study we wanted to show that smiling and sad emojis as feedback stimuli in the modified ultimatum game can also lead to increased acceptance rates. Contrary to our assumptions, this effect could not be observed. At the neural level as well, the findings did not correspond to our assumptions and differed strongly from those of the first study. One finding, however, was that the neural P3 component showed how the use of emojis as feedback stimuli particularly characterizes certain types of proposers. This is supported by the fact that the P3 is increased for the proposer who rewards an acceptance with a smile as well as for the proposer who reacts to rejection with a sad emoji compared to the neutral control proposer.
The third study examined the discrepancy between the findings of the first and second study. Accordingly, both humans and emojis representing the different proposers were presented in the ultimatum game. In addition, emojis were selected that showed a higher similarity to known emojis from common messenger services compared to the second study. We were able to replicate that the proposers in the ultimatum game, who reward an acceptance of the offer with a smile, led to an increased acceptance rate compared to the neutral control proposers. This difference is independent of whether the proposers are represented by emojis or human faces. With regard to the neural correlates, we were able to demonstrate that emojis and human faces differ strongly in their neural processing. Emojis showed stronger activation than human faces in the face-processing N170 component, the feedback-related negativity and the P3 component. We concluded that the results of the N170 and feedback-related negativity could indicate a signal for missing social information of emojis compared to faces. The increased P3 amplitude for emojis might imply that emojis appear unexpectedly as reward stimuli in a social decision task compared to human faces.
The last study of this project dealt with socially unexpected feedback. In comparison to the first three studies, new proposer identities were implemented. In particular, the focus was on a proposer who reacted to the rejection of an offer unexpectedly with a smile and to the acceptance with a neutral facial expression. According to the results, participants approach this unexpected smile through increased rejection, although it is accompanied by financial loss. In addition, as reported in studies one and three, we were able to show that proposers who respond to the acceptance of an offer with a smiling face and thus meet the expectations of the participants have higher offer acceptance rates than the control proposer. At the neuronal level, especially the feedback from the socially unexpected proposer led to an increased P3 amplitude, which indicates that smiling after rejection is attributed a special subjective importance.
The experiments provide new insights into the social influence through emotional feedback and the processing of relevant social cues. Due to the conceptual similarity of the studies, it was possible to differentiate between stable findings and potentially stimulus-dependent deviations, thus creating a well-founded contribution to the current research. Therefore, the novel paradigm presented here, and the knowledge gained from it could also play an important role in the future for clinical questions dealing with limited social competencies.
Gazes are of central relevance for people. They are crucial for navigating the world and communicating with others. Nevertheless, research in recent years shows that many findings from experimental research on gaze behavior cannot be transferred from the laboratory to everyday behavior. For example, the frequency with which conspecifics are looked at is considerably higher in experimental contexts than what can be observed in daily behavior. In short: findings from laboratories cannot be generalized into general statements. This thesis is dedicated to this matter. The dissertation describes and documents the current state of research on social attention through a literature review, including a meta-analysis on the /gaze cueing/ paradigm and an empirical study on the robustness of gaze following behavior. In addition, virtual reality was used in one of the first studies in this research field. Virtual reality has the potential to significantly improve the transferability of experimental laboratory studies to everyday behavior. This is because the technology enables a high degree of experimental control in naturalistic research designs. As such, it has the potential to transform empirical research in the same way that the introduction of computers to psychological research did some 50 years ago. The general literature review on social attention is extended to the classic /gaze cueing/ paradigm through a systematic review of publications and a meta-analytic evaluation (Study 1). The cumulative evidence supported the findings of primary studies: Covert spatial attention is directed by faces. However, the experimental factors included do not explain the surprisingly large variance in the published results. Thus, there seem to be further, not well-understood variables influencing these social processes. Moreover, classic /gaze cueing/ studies have limited ecological validity. This is discussed as a central reason for the lack of generalisability. Ecological validity describes the correspondence between experimental factors and realistic situations. A stimulus or an experimental design can have high and low ecological validity on different dimensions and have different influences on behavior. Empirical research on gaze following behavior showed that the /gaze cueing/ effect also occurs with contextually embedded stimuli (Study 2). The contextual integration of the directional cue contrasted classical /gaze cueing/ studies, which usually show heads in isolation. The research results can thus be transferred /within/ laboratory studies to higher ecologically valid research paradigms. However, research shows that the lack of ecological validity in experimental designs significantly limits the transferability of experimental findings to complex situations /outside/ the laboratory. This seems to be particularly the case when social interactions and norms are investigated. However, ecological validity is also often limited in these studies for other factors, such as contextual embedding /of participants/, free exploration behavior (and, thus, attentional control), or multimodality. In a first study, such high ecological validity was achieved for these factors with virtual reality, which could not be achieved in the laboratory so far (Study 3). Notably, the observed fixation patterns showed differences even under /most similar/ conditions in the laboratory and natural environments. Interestingly, these were similar to findings also derived from comparisons of eye movement in the laboratory and field investigations. These findings, which previously came from hardly comparable groups, were thus confirmed by the present Study 3 (which did not have this limitation). Overall, /virtual reality/ is a new technical approach to contemporary social attention research that pushes the boundaries of previous experimental research. The traditional trade-off between ecological validity and experimental control thus becomes obsolete, and laboratory studies can closely inherit an excellent approximation of reality. Finally, the present work describes and discusses the possibilities of this technology and its practical implementation. Within this context, the extent to which this development can still guarantee a constructive classification of different laboratory tests in the future is examined.
In deafness, which is caused by the malfunctioning of the inner ear, an implantation of a cochlear implant (CI) is able to restore hearing. The CI is a neural prosthesis that is located within the cochlea. It replaces the function of the inner hair cells by direct electrical stimulation of the auditory nerve fibers. The CI enables many deaf or severe hearing-impaired people to achieve a good speech perception. Nevertheless, there is a lot of potential for further improvements. Compared to normal-hearing listeners rate pitch discrimination is much worse. Rate pitch discrimination is the ability to distinguish the pitch of two stimuli with two different pulse rates. This ability is important for enjoying music as well as speech perception (in noise). Further, the small dynamic range in electrical hearing (compared to normal-hearing listeners) and therefore the small intensity resolution limits the performance of CI users. Both, rate pitch coding and dynamic range were investigated in this doctoral thesis.
For the first issue, a pitch discrimination task was designed to determine the just-noticeable-difference (JND) in pitch with 200 and 400 pps as reference. Additionally to the default biphasic pulse (single pulse) the experiment was performed with double pulses. The double pulse consists out of two biphasic pulses directly after each other and a small interpulse interval (IPI) in between. Three different IPIs (15, 50, and 150 µs) were tested. The statistical analysis of JNDs revealed no significant effects between stimulation with single-pulse or double-pulse trains.
A follow-up study investigated an alternating pulse train consisting of single and double pulses. To investigate if the 400 pps alternating pulse train is comparable in pitch with the 400 pps single-pulse train, a pairwise pitch comparison test was conducted. The alternating pulse train was compared with single-pulse trains at 200, 300 and 400 pps. The results showed that the alternating pulse train is for most subjects similar in pitch with the 200 pps single-pulse train. Therefore, pitch perception seemed to be dominated by the double pulses within the pulse train.
Accordingly, double pulses with different amplitudes were tested. Based on the facilitation effect, a larger neuronal response was expected by stimulating with two pulses with a short IPI within the temporal facilitation range. In other studies, this effect was shown to be maximal in CIs of the manufacturer Cochlear, with first pulse amplitudes set at or slightly below the electrically evoked compound action potential (ECAP) threshold. The second pulse amplitude did not influence the facilitation effect and therefore could be choose at will. Similarly, this effect was tested in this thesis with CIs of the manufacturer MED-EL. Nevertheless, to achieve a proper signal-to-noise ratio, technical issues had to be addressed like a high noise floor, resulting in incorrect determination of the ECAP threshold. After solving this issues, the maximum facilitation effect was around the ECAP threshold as in the previous study with Cochlear. For future studies this effect could be used in a modified double pulse rate pitch experiment with the first pulse amplitude at ECAP threshold and the second pulse amplitude variable to set the most comfortable loudness level (MCL).
The last study within this thesis investigated the loudness perception at two different loudness levels and the resulting dynamic range for different interphase-gaps (IPG). A larger IPG can reduce the amplitude at same loudness level to save battery power. However, it was unknown if the IPG has an influence on the dynamic range. Different IPGs (10 and 30 µs) were compared with the default IPG (2.1 µs) in a loudness matching experiment. The experiment was performed at the most comfortable loudness level (MCL) of the subject and the amplitude of half the dynamic range (50%-ADR). An upper dynamic range was calculated from the results of MCL and 50%-ADR (therefore not the whole dynamic range was covered). As expected from previous studies a larger IPG resulted in smaller amplitudes. However, the observed effect was larger at MCL than at 50%-ADR which resulted in a smaller upper dynamic range. This is the first time a decrease of this dynamic range was shown.
Adapting defensive behavior to the characteristics of a threatening situation is a fundamental function of the brain. Particularly, threat imminence plays a major role for the organization of defensive responses. Acute threat prompts phasic physiological responses, which are usually associated with an intense feeling of fear. In contrast, diffuse and potentially threatening situations elicit a sustained state of anxious apprehension. Detection of the threatening stimulus defines the key event in this framework, initiating the transition from potential to acute threat. Consequently, attention to threat is crucial for supporting defensive behavior. The functions of attention are finely tuned to the characteristics of a threatening situation. Potential threat is associated with hypervigilance, in order to facilitate threat detection. Once a threatening stimulus has been identified, attention is selectively focused on the source of danger. Even though the concepts of selective attention and hypervigilance to threat are well established, evidence for their neural correlates remain scarce. Therefore, a major goal of this thesis is to elucidate the neural correlates of selective attention to acute threat and hypervigilance during potential threat. A second aim of this thesis is to provide a mechanistic account for the interaction of fear and anxiety. While contemporary models view fear and anxiety as mutually exclusive, recent findings for the neural networks of fear and anxiety suggest potential interactions. In four studies, aversive cue conditioning was used to induce acute threat, while context conditioning served as a laboratory model of potential threat. To quantify neural correlates of selective attention and hypervigilance, steady-state visual evoked potentials (ssVEPs) were measured as an index of visuocortical responding. Study 1 compared visuocortical responses to acute and potential threat for high versus low trait-anxious individuals. All individuals demonstrated enhanced electrocortical responses to the central cue in the acute threat condition, suggesting evidence for the neural correlate of selective attention. However, only low anxious individuals revealed facilitated processing of the contexts in the potential threat condition, reflecting a neural correlate of hypervigilance. High anxious individuals did not discriminate among contexts. These findings contribute to the notion of aberrational processing of potential threat for high anxious individuals. Study 2 and 3 realized orthogonal combinations of cue and context conditioning to investigate potential interactions of fear and anxiety. In contrast to Study 1 and 2, Study 3 used verbal instructions to induce potentially threatening contexts. Besides ssVEPs, threat ratings and skin conductance responses (SCRs) were recorded as efferent indices of defensive responding. None of these studies found further evidence for the neural correlates of hypervigilance and selective attention. However, results for ratings and SCRs revealed additive effects of fear and anxiety, suggesting that fear and anxiety are not mutually exclusive, but interact linearly to organize and facilitate defensive behavior. Study 4 tested ssVEPs to more ecologically valid forms of context conditioning, using flickering video stimuli of virtual offices to establish context representations. Contrary to expectations, results revealed decreased visuocortical responses during sustained presentations of anxiety compared to neutral contexts. A disruption of ssVEP signals eventually suggests interferences by continuously changing video streams which are enhanced as a function of motivational relevance. In summary, this thesis provided evidence for the neural correlates of attention only for isolated forms of fear and anxiety, but not for their interaction. In contrast, an additive interaction model of fear and anxiety for measures of defensive responding offers a new perspective on the topography of defensive behavior.
Im Rahmen des interdisziplinären Promotionsschwerpunkts Resilienzfaktoren der Schmerzverarbeitung des evangelischen Studienwerks in Zusammenarbeit mit der Julius-Maximilians-Universität Würzburg und der Otto-Friedrich-Universität Bamberg untersuche ich in diesem Promotionsprojekt den Einfluss von Sicherheit auf die Schmerzverarbeitung. Es ist bekannt, dass die Schmerzverarbeitung durch Emotionen moduliert werden kann. Man geht davon aus, dass negative Emotionen den Schmerz in der Regel verstärken, während positive Emotionen zu einer Schmerzreduktion führen. Frühere Studien fanden heraus, dass die Erwartung eines aversiven Ereignisses zu Bedrohung und stärkeren Schmerzen führt. Es stellt sich die Frage, ob das Gegenteil von Bedrohung, nämlich Sicherheit, zu einer Verringerung der Schmerzen führen kann. Um diese Hypothese zu untersuchen, habe ich drei Experimente an gesunden ProbandInnen durchgeführt.
In dieser Arbeit wurde der Einfluss sozialer Stresserfahrung sowie des 5-Htt-Genotyps auf die neuronale Morphologie bestimmter Hirnregionen anhand eines Mausmodells untersucht. Es wurde in mit Golgi-Cox gefärbten Gehirnen der 5-HTT-KO-Linie in der lateralen Amygdala (LA) die Apikal- und Basaldendriten pyramidenzellähnlicher Neurone und die Apikaldendriten der Pyramidenzellen der Cornu ammonis (CA)3-Region des Hippocampus mithilfe des Neurolucidasystems rekonstruiert und die so gewonnenen Daten anschließend statistisch ausgewertet.
Die erzielten Ergebnisse belegen, dass vor allem die Erfahrung von sozialem Verteidigungsstress aber auch der 5-Htt-Genotyp (WT, HET, KO) im Mausmodell signifikanten Einfluss auf die Morphologie der Neurone der LA und der CA3-Region besitzen. Um die in dieser Arbeit mit allen drei 5-Htt-Genotypen erzielten Ergebnisse der LA-Neurone besser mit den Ergebnissen von Nietzer und Bonn (nur WT, KO) vergleichen zu können (Nietzer et al., 2011), wurden die von mir erhobenen Daten nicht nur in einem 3er-Vergleich, sondern auch einem 2er-Vergleich (WT vs. KO) statistisch analysiert. Untersuchungen der LA-Neurone aller drei 5-Htt-Genotypen zeigen, dass sozialer Stress zu einer Zunahme der Komplexität der Dendritenbäume durch längere und auch stärker verzweigte Dendriten vor allem in der Gruppe der WT-Mäuse führt. HET- und KO-Mäuse zeigten keinen entsprechenden Stress-Effekt. Darüber hinaus zeigten sich deutliche Genotypeffekte. Unabhängig vom Stresserleben besitzen HET-Mäuse längere Dendriten als WT-Mäuse sowie eine höhere Spinedichte als WT- und KO-Mäuse. Die Hypothese, die in der Arbeit von Nietzer et al. aufgestellt wurde, dass eine vollständige 5-HTT-Defizienz zu mehr Spines führt, ließ sich hier weder durch den 3er- noch durch den 2er-Vergleich replizieren. Die Pyramidenzellen der CA3-Region, die in dieser Studie zum ersten Mal analysiert wurden, zeigen in Bezug auf die durch den Stress ausgelösten Veränderungen ein im Vergleich zu den LA-Neuronen entgegengesetzten Effekt. Der soziale Stress führt hier zu einer Dendritenatrophie in der WT-Gruppe mit kürzeren und weniger komplexen Dendriten. Außerdem führte er zu einer geringeren Spinedichte bei den HET-Mäusen. Es zeigten sich klare Genotypeffekte, unabhängig von der Stresserfahrung, mit einer reduzierten Spinedichte der KO-Mäuse gegenüber den WT-Mäusen und einer nur in den Kontrollen detektierten, reduzierten Spinedichte der KO-Mäuse im Vergleich zu den WT- und HET-Mäusen. Sowohl in der LA als auch in der CA3-Region lassen sich Kompensationsmechanismen des 5-HTT-Defizits der HET-Tiere vermuten, über die die KO-Tiere nicht verfügen.
Die in LA und CA3 gezeigten gegensätzlichen Auswirkungen des sozialen Stresses weisen auf die unterschiedlichen Funktionen dieser beiden Regionen im Furchtkreislauf und/oder bei der Verarbeitung von Stress hin. Darüber hinaus deutet diese Arbeit darauf hin, dass Arbeiten mit ähnlichen Untersuchungsmethoden und sogar gleichem Untersuchungsmaterial unterschiedliche Ergebnisse liefern können.