Molecular Psychiatry (2006) 11, 1106–1112 & 2006 Nature Publishing Group All rights reserved 1359-4184/06 $30.00 www.nature.com/mp ORIGINAL ARTICLE Serotonin transporter gene variation impacts innate fear processing: acoustic startle response and emotional startle B Brocke1, D Armbruster1,JMu¨ller1, T Hensch1, CP Jacob2, K-P Lesch2, C Kirschbaum3 and A Strobel1 1Personality and Individual Differences, Institute of Psychology II, Dresden University of Technology, Dresden, Germany; 2Molecular and Clinical Psychobiology, Department of Psychiatry and Psychotherapy, University of Wu¨rzburg, Wu¨rzburg, Germany and 3Institute of Psychology I, Dresden University of Technology, Dresden, Germany Anxiety-related behaviors are closely linked to neural circuits relaying fear-specific informa- tion to the amygdala. Many of these circuits, like those underlying processing of innate fear, are remarkably well understood. Recent imaging studies have contributed to this knowledge by discriminating more detailed corticoamygdalar associations mediating processing fear and anxiety. However, little is known about the underlying molecular mechanisms. We used the acoustic startle paradigm to investigate the impact of molecular genetic variation of serotonergic function on the acoustic startle response and its fear potentiation. Startle magnitudes to noise bursts as measured with the eye blink response were recorded in 66 healthy volunteers under four conditions: presenting unpleasant and pleasant affective pictures as well as neutral pictures, and presenting the startle stimulus without additional stimuli as a baseline. Subjects were genotyped for functional polymorphism in the transcriptional control region of the serotonin transporter gene (5-hydroxytryptamine transporter gene-linked region: 5-HTTLPR). Analyses of variance revealed a significant effect of 5-HTTLPR on overall startle responses across conditions. Carriers of the short (s) allele exhibited stronger startle responses than l/l homozygotes. However, we could not confirm our hypothesis of enhanced fear potentiation of the startle in s allele carriers. In conclusion, the results provide first evidence that the startle response is sensitive to genetic variation in the serotonin pathway. Despite some issues remaining to be resolved, the startle paradigm may provide a valuable endophenotype of fear processing and underlying serotonergic influences. Molecular Psychiatry (2006) 11, 1106–1112. doi:10.1038/sj.mp.4001908; published online 10 October 2006 Keywords: acoustic startle response; amygdala; 5-HTTLPR; fear; serotonergic system Introduction or in primitive cortex that are directly activated by primary reinforcement and are assumed to be compar- Anxiety is associated with a spectrum of behaviors, able in all mammalian species.1,2 Most of the including temperamental traits, conditioned and behaviors associated with the withdrawal system, innate fear, anxiety disorders and depression. Anxiety that is, anxiety-related behaviors, are closely linked to disorders account for an essential part of all the neural circuits relaying fear-specific information to conditions that psychotherapists and psychiatrists are the amygdala.3 However, most of the current know- consulted for. It is thus important that we understand ledge about the neuronal substrates and molecular as much as possible how anxiety-related neuro- mechanisms of fear processing is based on animal circuits work. research. The advent of the genetic variance approach Basic emotions such as fear and anxiety can be seen and genomic imaging has rendered it possible to as prototypes of primitive systems that developed to elucidate molecular genetic factors and neuronal ensure the survival of organisms. These prototypical circuitries underlying fear processing also in healthy systems involve approach to stimuli to enhance or humans. maintain species or withdrawal from dangerous A recent functional imaging study has underscored events. The survival systems comprise neural struc- the importance of the amygdala for processing fear tures and their connections within subcortical areas and anxiety in humans.4 A series of subsequent imaging studies successively discriminated function- Correspondence: Dr B Brocke, Personality and Individual ally divergent components of a more extensive Differences, Institute of Psychology II, Dresden University of amygdala-relayed corticolimbic circuit for fear pro- Technology, Dresden 01062, Germany. cessing in humans.5–7 More specifically, using E-mail: [email protected] 7 Received 6 January 2006; revised 31 August 2006; accepted 1 functional connectivity analyses, Pezawas et al. September 2006; published online 10 October 2006 identified two distinct regions within the perigenual 5-HTTLPR and acoustic startle response B Brocke et al 1107 anterior cingulate cortex (pACC), which are function- reticularis pontis caudalis (PnC) and spinal moto- ally connected with the amygdala: the rostral and neurons, the excitation of which gives rise to the caudal subgenual ACC, which were positively and acoustic startle response (ASR). Because the startling negatively correlated with amygdala activity, respec- stimuli of the ASR are aversive and able to induce a tively. Furthermore, both regions showed strong state of fear or anxiety,11 the ASR can be regarded as a positive connectivity with each other, suggesting that means to study innate fear responses. they form a feedback loop. Furthermore, the ASR can be potentiated by With regard to the molecular mechanisms under- presenting the acoustic startle stimulus in the lying fear processing, the efficacy of serotonergic presence of a cue that had previously been paired compounds in the treatment of depression and most with an unconditioned fear stimulus. There is con- anxiety disorders suggests serotonergic neurotrans- verging evidence that this fear-potentiated startle mission being one of the most important molecular (FPS) is crucially modulated by the amygdala.12 In factors modulating anxiety-related behavior.8 In line models of this amygdala modulation of the FPS, fear with this assumption, the results of the mentioned stimuli proceed from sense receptor systems to the genomic imaging studies suggest that carriers of the sensory thalamus and then to the amygdala complex short (s) allele of a functional polymorphism in the and to the sensory cortex, which also gives rise to a transcriptional control region of the serotonin trans- projection to the amygdala.13 From the amygdala, porter gene (5-hydroxytryptamine transporter gene- there are three basic efferent connections: projections linked region: 5-HTTLPR),9 which impairs serotonin from the central amygdala to the lateral hypothalamic reuptake, exhibit greater amygdala activity in re- area (autonomic system), projections to the midbrain sponse to fearful or threatening facial expressions central gray (freezing and escape behavior) and to the than individuals homozygous for the long allele (l/l).4 PnC, which modulates the startle reflex.1,14 It has been This finding suggests that increased anxiety in shown that lesions of the amygdala block the carriers of the 5-HTTLPR s allele9 might reflect a expression of FPS.12 Hence, FPS is a valid tool for hyper-activity or responsiveness of their amygdalae. the study of amygdala-modulated conditioned fear However, recent studies5–7,10 have offered further responses. insights into this hyper-activity revealing that the 5- Concerning the role of serotonin in the modulation HTTLPR s allele has strong impact on the corticolim- of ASR and FPS, there is evidence that in rats bic feedback circuit described above. Carriers of the systemic administration of agonists of the serotonin 5-HTTLPR s allele showed a significant reduction of 1A receptor [5-hydroxytryptamine (5-HT1A) receptor] amygdala–pACC functional connectivity as compared augments ASR amplitudes15–18 and reduces or even to l/l homozygotes. More specifically, the findings blocks FPS.16,19 In contrast, evidence from human suggest that the amygdala overactivation in s allele studies is scattered and in part contradictory: while in carriers reflect a failure of downregulation of the one study, the selective serotonin reuptake inhibitor amygdala response. As a consequence, the anxiety (SSRI) citalopram increased the ASR,20 other studies proneness of s allele carriers might not be seen as a employing the SSRIs fluvoxamine21 or sertraline22 simple overactivity of the amygdala complex, but as a did not find evidence for altered ASR amplitudes. basic dysfunction in fear processing. Unfortunately, emotional startle modulation was not In the light of these findings, differential amygdala examined in these studies. In an emotional startle reactivity can be regarded as a most revealing paradigm, citalopram abolished FPS on unpleasant endophenotype for the study of genetic variation of pictures, but did not alter the ASR on neutral the serotonin system. However, (1) the particular pictures.23 In two further studies, tryptophan supple- nature of the underlying molecular mechanisms is not mentation, which stimulates synthesis of serotonin well understood up to now and (2) this endopheno- was found to reduce the ASR, but not to significantly type presently is restricted to imaging results without affect FPS in healthy women,24 whereas tryptophan including those parts of amygdala-relayed circuits, depletion resulted in an overall increase of the ASR which are related to clearcut and valid behavioral across emotional conditions in a group