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Disruptions in brain serotonin (5-hydroxytryptamine, 5-HT) signaling pathways have been associated with etiology and pathogenesis of various neuropsychiatric disorders, but specific neural mechanisms of 5-HT function are yet to be fully elucidated. Tryptophan hydroxylase 2 (TPH2) is the rate-limiting enzyme for brain 5-HT synthesis. Therefore, in this study a tamoxifen (Tam)-inducible cre-mediated conditional gene (Tph2) knockout in adult mouse brain (Tph2icKO) has been established to decipher the specific role of brain 5-HT in the regulation of behavior in adulthood.
Immunohistochemistry and high-performance liquid chromatography (HPLC) were used first to test the efficacy of Tam-inducible inactivation of Tph2 and consequential reduction of 5-HT in adult mouse brain. Tam treatment resulted in ≥90% reduction in the number of 5-HT immuno-reactive cells in the anterior raphe nuclei. HPLC revealed a significant reduction in concentration of 5-HT and its metabolite 5-hydroxyindole acetic acid (5-HIAA) in selected brain regions of Tph2icKO, indicating the effectiveness of the protocol used.
Second, standard behavioral tests were used to assess whether reduced brain 5-HT concentrations could alter anxiety-, fear- and depressive-like behavior in mice. No altered anxiety- and depressive-like behaviors were observed in Tph2icKO compared to control mice (Tph2CON) in all indices measured, but Tph2icKO mice exhibited intense and sustained freezing during context-dependent fear memory retrieval. Tph2icKO mice also exhibited locomotor hyperactivity in the aversive environments, such as the open field, and consumed more food and fluid than Tph2CON mice.
Lastly, the combined effect of maternal separation (MS) stress and adult brain 5-HT depletion on behavior was assessed in male and female mice. Here, MS stress, 5-HT depletion and their interaction elicited anxiety-like behavior in a sex-dependent manner. MS reduced exploratory behavior in both male and female mice. Reduced 5-HT enhanced anxiety in female, but not in male mice.
Furthermore, expression of genes related to the 5-HT system and emotionality (Tph2, Htr1a, Htr2a, Maoa and Avpr1a) was assessed by performing a quantitative real-time PCR. In Tph2icKO mice there was a reduction in expression of Tph2 in the raphe nuclei of both male and female mice. Interaction between MS stress and 5-HT deficiency was detected showing increased Htr2a and Maoa expression in raphe and hippocampus respectively of female mice. In male mice, MS stress and 5-HT depletion interaction effects reduced Avpr1a expression in raphe, while the expression of Htr1a, Htr2a and Maoa was differentially altered by 5-HT depletion and MS in various brain regions.
Anxiety is an affective state characterized by a sustained, long-lasting defensive response, induced by unpredictable, diffuse threat. In comparison, fear is a phasic response to predictable threat. Fear can be experimentally modeled with the help of cue conditioning. Context conditioning, in which the context serves as the best predictor of a threat due to the absence of any conditioned cues, is seen as an operationalization of sustained anxiety.
This thesis used a differential context conditioning paradigm to examine sustained attention processes in a threat context compared to a safety context for the first time. In three studies, the attention mechanisms during the processing of contextual anxiety were examined by measuring heart rate responses and steady-state-visually evoked potentials (ssVEPs). An additional focus was set on the processing of social cues (i.e. faces) and the influence of contextual information on these cues. In a last step, the correlates of sustained anxiety were compared to evoked responses by phasic fear, which was realized in a previously established paradigm combining predictable and unpredictable threat.
In the first study, a contextual stimulus was associated with an aversive loud noise, while a second context remained unpaired. This conditioning paradigm created an anxiety context (CTX+) and a safety context (CTX-). After acquisition, a social agent vs. an object was presented as a distractor in both contexts. Heart rate and cortical responses, with ssVEPs by using frequency tagging, to the contexts and the distractors were assessed. Results revealed enhanced ssVEP amplitudes for the CTX+ compared to the CTX− during acquisition and during presentation of distractor stimuli. Additionally, the heart rate was accelerated in the acquisition phase, followed by a heart rate deceleration as a psychophysiological marker of contextual anxiety.
Study 2 used the same context conditioning paradigm as Study 1. In contrast to the first study, persons with different emotional facial expressions were presented in the anxiety and safety contexts in order to compare the differential processing of these cues within periods of threat and safety. A similar anxiety response was found in the second study, although only participants who
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were aware of the contingency between contexts and aversive event showed a sensory amplification of the threat context, indicated by heart rate response and ssVEP activation. All faces irrespective of their emotional expression received increased attentional resources when presented within the anxiety context, which suggests a general hypervigilance in anxiety contexts.
In the third study, the differentiation of predictable and unpredictable threat as an operationalization of fear and anxiety was examined on a cortical and physiological level. In the predictable condition, a social cue was paired with an aversive event, while in the unpredictable condition the aversive event remained unpaired with the respective cue. A fear response to the predictable cue was found, indicated by increased oscillatory response and accelerated heart rate. Both predictable and unpredictable threat yielded increased ssVEP amplitudes evoked by the context stimuli, while the response in the unpredictable context showed longer-lasting ssVEP activation to the threat context.
To sum up, all three studies endorsed anxiety as a long-lasting defensive response. Due to the unpredictability of the aversive events, the individuals reacted with hypervigilance in the anxiety context, reflected in a facilitated processing of sensory information and an orienting response. This hypervigilance had an impact on the processing of novel cues, which appeared in the anxiety context. Considering the compared stimuli categories, the stimuli perceived in a state of anxiety received increased attentional resources, irrespective of the emotional arousal conveyed by the facial expression. Both predictable and unpredictable threat elicited sensory amplification of the contexts, while the response in the unpredictable context showed longer-lasting sensory facilitation of the threat context.