5-HTT deficiency affects neuroplasticity and increases stress sensitivity resulting in altered spatial learning performance in the Morris water maze but not in the Barnes maze.

The purpose of this study was to evaluate whether spatial hippocampus-dependent learning is affected by the serotonergic system and stress. Therefore, 5-HTT knockout (-/-), heterozygous (+/-) and wildtype (+/+) mice were subjected to the Barnes maze (BM) and the Morris water maze (WM), the latter being discussed as more aversive. Additionally, immediate early gene (IEG) expression, hippocampal adult neurogenesis (aN), and blood plasma corticosterone were analyzed. While the performance of 5-HTT-/- mice in the BM was undistinguishable from both other genotypes, they performed worse in the WM. However, in the course of the repeated WM trials 5-HTT-/- mice advanced to wildtype level. The experience of a single trial of either the WM or the BM resulted in increased plasma corticosterone levels in all genotypes. After several trials 5-HTT-/- mice exhibited higher corticosterone concentrations compared with both other genotypes in both tests. Corticosterone levels were highest in 5-HTT-/- mice tested in the WM indicating greater aversiveness of the WM and a greater stress sensitivity of 5-HTT deficient mice. Quantitative immunohistochemistry in the hippocampus revealed increased cell counts positive for the IEG products cFos and Arc as well as for proliferation marker Ki67 and immature neuron marker NeuroD in 5-HTT-/- mice compared to 5-HTT+/+ mice, irrespective of the test. Most differences were found in the suprapyramidal blade of the dentate gyrus of the septal hippocampus. Ki67-immunohistochemistry revealed a genotype x environment interaction with 5-HTT genotype differences in naïve controls and WM experience exclusively yielding more Ki67-positive cells in 5-HTT+/+ mice. Moreover, in 5-HTT-/- mice we demonstrate that learning performance correlates with the extent of aN. Overall, higher baseline IEG expression and increased an in the hippocampus of 5-HTT-/- mice together with increased stress sensitivity may constitute the neurobiological correlate of raised alertness, possibly impeding optimal learning performance in the more stressful WM.

Unexpected effects of early-life adversity and social enrichment on the anxiety profile of mice varying in serotonin transporter genotype.

Developmental mechanisms that shape behaviour are under environmental as well as genetic influence, commonly referred to as gene-by-environment interaction (GxE). Here, we compared the role of different early environments - adverse, standard, and enriched - for the modulation of the anxiety profile in mice varying in serotonin transporter (5-HTT) genotype. Early-life adversity was simulated by exposing lactating 5-HTT +/- dams to soiled bedding of unfamiliar males (UMB), signalling the danger of infanticide. An enriched early environment was established by communal nesting (CN). 5-HTT +/- females of a third group were housed under standard nesting conditions (SN) of individual nesting. The offspring (5-HTT +/+, 5-HTT +/-, and 5-HTT -/-) were analyzed for anxiety-like and exploratory behaviour in a battery of tests. The main findings were: (1) Maternal care was reduced in UMB compared to CN dams. (2) There was no significant variation in state anxiety levels between UMB, SN, and CN offspring. (3) UMB offspring showed significantly lower levels of trait anxiety compared to CN offspring, while SN offspring were intermediate. (4) There was a significant main effect of genotype, with highest levels of state and trait anxiety in 5-HTT -/- mice. The findings corroborate that anxiety profiles in mice can be affected by both early environmental conditions and 5-HTT genotype. Notably, state and trait anxiety of an individual can independently be affected by the early environment.

To attack, or not to attack? The role of serotonin transporter genotype in the display of maternal aggression.

Aggressive behavior in males has been intensively investigated regarding the influence of the brain serotonergic system. Despite some inconsistencies, a general conclusion is that low levels of serotonin (5-HT) are associated with high levels of male aggression. The role of the serotonergic system for female aggression is less well researched. Female mice rarely show intraspecific aggressive behavior, except during lactation, when they may exhibit intense aggression towards intruders to protect their pups. The aim of the present study was to investigate the impact of 5-HT transporter (5-HTT) inactivation on maternal aggression in mice. Therefore, lactating homozygous and heterozygous 5-HTT knockout as well as wildtype mice were confronted with male intruders in their home cages. Homozygous 5-HTT knockout dams, which exhibit highest levels of extracellular 5-HT in the brain, were significantly less prone to initiate offensive aggression than wildtype controls. Moreover, they showed longer latencies to attack the intruder, attacked less often and displayed an overall lower frequency of offensive aggressive behavior patterns than wildtype dams. Heterozygous 5-HTT knockout mothers generally showed intermediate levels of aggressive behavior. Thus, our data indicate that higher extracellular including synaptic levels of 5-HT are associated with lower intensity of aggressive behavior in lactating mice, adding support to the inhibitory role of 5-HT in aggression also in females.

Social defeat: impact on fear extinction and amygdala-prefrontal cortical theta synchrony in 5-HTT deficient mice.

Emotions, such as fear and anxiety, can be modulated by both environmental and genetic factors. One genetic factor is for example the genetically encoded variation of the serotonin transporter (5-HTT) expression. In this context, the 5-HTT plays a key role in the regulation of central 5-HT neurotransmission, which is critically involved in the physiological regulation of emotions including fear and anxiety. However, a systematic study which examines the combined influence of environmental and genetic factors on fear-related behavior and the underlying neurophysiological basis is missing. Therefore, in this study we used the 5-HTT-deficient mouse model for studying emotional dysregulation to evaluate consequences of genotype specific disruption of 5-HTT function and repeated social defeat for fear-related behaviors and corresponding neurophysiological activities in the lateral amygdala (LA) and infralimbic region of the medial prefrontal cortex (mPFC) in male 5-HTT wild-type (+/+), homo- (-/-) and heterozygous (+/-) mice. Naive males and experienced losers (generated in a resident-intruder paradigm) of all three genotypes, unilaterally equipped with recording electrodes in LA and mPFC, underwent a Pavlovian fear conditioning. Fear memory and extinction of conditioned fear was examined while recording neuronal activity simultaneously with fear-related behavior. Compared to naive 5-HTT+/+ and +/- mice, 5-HTT-/- mice showed impaired recall of extinction. In addition, 5-HTT-/- and +/- experienced losers showed delayed extinction learning and impaired recall of extinction. Impaired behavioral responses were accompanied by increased theta synchronization between the LA and mPFC during extinction learning in 5-HTT-/- and +/- losers. Furthermore, impaired extinction recall was accompanied with increased theta synchronization in 5-HTT-/- naive and in 5-HTT-/- and +/- loser mice. In conclusion, extinction learning and memory of conditioned fear can be modulated by both the 5-HTT gene activity and social experiences in adulthood, accompanied by corresponding alterations of the theta activity in the amygdala-prefrontal cortex network.

Living in a dangerous world decreases maternal care: a study in serotonin transporter knockout mice.

Adverse early experiences can profoundly influence the adult behavioral profile. When pregnant and lactating mice are confronted with soiled bedding of unfamiliar males (UMB), these stimuli signal the danger of infanticide and thus simulate a "dangerous world". In a previous study, offspring of UMB treated mothers were shown to display increased anxiety-like behavior and reduced exploratory locomotion as adults, compared to mice treated with neutral bedding (NB, "safe environment"). The aim of this study was to elucidate the mechanisms conveying these effects of living in a "dangerous world" to offspring behavior. We hypothesized the mother to be the major link and focused on the influence of UMB on maternal stress hormones and behavior. Thus, we investigated fecal corticosterone metabolites (CM) and maternal care of pregnant and lactating mice either treated with NB or UMB. The offspring were subsequently tested for their anxiety-like and exploratory behavior. Mothers treated with UMB showed a significantly higher increase of fecal CM following the initial treatment, than NB treated mothers, indicating that the odor cues of potentially infanticidal males represented an ethologically relevant stimulus. Whereas the hormonal stress response habituated, living in a "dangerous world" led to a distinct and consistent reduction of maternal care behavior, particularly concerning the duration of licking and grooming the pups. Surprisingly, we could not confirm our former findings of altered phenotypes in the offspring of UMB treated mothers. In summary, we hypothesize that the frequently described effects of early life adversity on the offspring's behavioral profile are mediated primarily by maternal care in altricial rodents.

The winner and loser effect, serotonin transporter genotype, and the display of offensive aggression.

Aggressive behaviour results from a complex interplay between genetic and environmental factors. Key modulators of aggression include the serotonergic system on the molecular level and experience in prior aggressive contests as an environmental factor. The aim of this study was to elucidate the effects of fighting experience on the display of offensive aggressive behaviour in adult male mice varying in serotonin transporter (5-HTT) genotype. 5-HTT +/+, 5-HTT +/- and 5-HTT -/- mice were given either a winning or a losing experience on each of three consecutive days and were subsequently observed for their offensive aggressive behaviour as residents against a docile intruder from the C3H strain in a resident-intruder paradigm. The main findings were: There was no significant difference between the amount of offensive aggressive behaviour displayed by the genotypes. Winners showed more engagement with the intruder, attacked him faster and exhibited overall higher aggression scores than losers. There was no significant genotype × social experience interaction: winning and losing had a similar effect on offensive aggressive behaviour in all three 5-HTT genotypes. We conclude that social experience in terms of having been a winner or having been a loser rather than the 5-HTT genotype determines the behaviour towards a docile intruder.

Away game or home match: the influence of venue and serotonin transporter genotype on the display of offensive aggression.

Aggression can be modulated by both genetic and environmental factors. Here, we analyse how the serotonin transporter (5-HTT) genotype and the environmental situation in which a contest takes place shape the display of offensive aggression. Therefore, male wildtype, heterozygous, and homozygous 5-HTT knockout mice, which are known to differ in inborn levels of anxiety, were confronted three times with a docile opponent in one of three environmental situations: own territory, opponent's territory or neutral area. The main findings were: The frequency of approaching the contestant in order to gather information about him depended significantly on the venue but not on the genotype with lowest frequencies in the opponent's territory. The decision how quickly to attack the opponent was significantly influenced by the 5-HTT genotype but not by the venue: Homozygous 5-HTT knockout mice showed longest latencies. The sum of offensive aggression was significantly influenced by the 5-HTT genotype, the environmental situation, and a genotype by environment interaction. It is likely that, due to their varying genetic predisposition for anxiety, mice of the three genotypes were differentially affected by the aversiveness of the respective venue and the opponent's behaviour, which influenced their decision to display offensive aggression. As a consequence, the amount of aggression shown by homozygous 5-HTT knockout mice was influenced by the venue and the opponent's behaviour, whereas heterozygotes reacted only to the venue. Strikingly, wildtypes behaved always the same way, irrespective of venue and opponent.

Consequences of serotonin transporter genotype and early adversity on behavioral profile - pathology or adaptation?

This review focuses on how behavioral profile is shaped by early adversity in individuals with varying serotonin transporter (5-HTT) genotype. In a recent study on 5-HTT knockout mice Heiming et al. (2009) simulated a 'dangerous environment' by confronting pregnant and lactating females with odor cues of unfamiliar males, indicating the risk of infant killing. Growing up in a dangerous environment induced increased anxiety-related behavior and decreased exploratory locomotion in the offspring, the effects being most pronounced in mice lacking 5-HTT expression. We argue that these alterations in behavioral profile represent adaptive maternal effects that help the individuals to cope with adversity. In principle, such effects of adversity on behavioral profile should not automatically be regarded as pathological. Rather and in accordance with modern evolutionary theory they may represent adaptations, although individuals with 5-HTT genotype induced susceptibility to adversity may be at risk of developing pathologies.

Social status and day-to-day behaviour of male serotonin transporter knockout mice.

Humans differing in the amount of serotonin transporter (5-HTT) are known to be differentially prone to neuropsychiatric disorders. Genetically modified mice eliciting abrogated transporter function display a number of corresponding phenotypic changes in behavioural tests. However, a characterisation of the effects of serotonergic malfunction on the day-to-day life is still missing. Yet, this is precisely what an animal model is needed for in order to be meaningful for translation into human anxiety disorders. Homozygous 5-HTT knockout mice, heterozygous 5-HTT mice, and wild-type controls were housed in groups of males of the same genotype in spacious and richly structured cages. This enriched environment allowed the animals to show a wide variety of spontaneous behavioural patterns quantified by a trained experimenter. Additionally the mice could emigrate from the cages through a tunnel and a water basin. The results revealed unaltered daily behaviour in heterozygous mice. In knockouts, however, reduced locomotion, increased socio-positive behaviour, and reduced aggressive behaviour were observed. Nevertheless, all groups showed a significant amount of aggressive behaviour and there were no differences regarding the establishment of dominance relationships, emigration, and the number of animals remaining in their groups. In a second step, pairs of heterozygous and wild-type males and pairs of knockout and wild-type males were brought together in order to assess their ability to obtain a dominant social position in a direct encounter. Heterozygous mice did not differ from wild-type mice but knockout mice were significantly inferior in obtaining the dominant position. In addition to confirming multiple effects of abolished 5-HTT function in a real life situation, this study supports the central role of the 5-HTT in the control of social interactions.

Modulation of behavioural profile and stress response by 5-HTT genotype and social experience in adulthood.

Behavioural profiles can be shaped by genotype and environmental factors during early phases of life. The aim of this study was to investigate whether anxiety-like behaviour, exploration and adrenocortical stress responses can be modulated by genotype and social experiences in adulthood. Male mice lacking the serotonin transporter gene which is under scrutiny for anxiety disorders were compared with heterozygous and wildtype controls. Concerning social experiences, the males of all three genotypes were provided with a winner or a loser experience in a resident-intruder paradigm on three consecutive days. Anxiety-like behaviour and exploration were recorded in the dark-light, elevated plus-maze and open-field test. To non-invasively assess adrenocortical activity, corticosterone metabolites were determined from feces. The main findings were: Repeated social experience, irrespective of winning or losing, elevated levels of anxiety-like behaviour and decreased exploration. In losers a distinct effect of genotype occurred, with homozygous knockout males showing more anxiety-like behaviour and less exploration than the other genotypes. In winners no genotype-dependent variation was found. Genotypes did not differ in basal stress hormone secretion. There was, however, a main effect of social experience with higher activation of the stress hormone system in losers than in winners. This effect was strongest in the heterozygous genotype. In conclusion, our data show that anxiety circuits retain their plasticity throughout adulthood and can be shaped by genotype and social experiences during this phase of life. Moreover, responsiveness towards negative life experiences is influenced significantly by the 5-HTT genotype.

Living in a dangerous world: the shaping of behavioral profile by early environment and 5-HTT genotype.

Anxiety and anxiety disorders are influenced by both, environmental and genetic factors. One genetic factor under scrutiny for anxiety disorders is the genetically encoded variation of the serotonin transporter (5-HTT). The aim of this study was to elucidate the effects of a threatening environment during early phases of life on anxiety-like (ANX) and exploratory behavior (EXP) in adult mice, varying in serotonin transporter (5-HTT) genotype. For this purpose, pregnant and lactating 5-HTT +/- dams were repeatedly exposed to olfactory cues of unfamiliar adult males by introducing small amounts of soiled bedding to their home cage. These stimuli signal the danger of infanticide and simulate a threatening environment. Control females were treated with neutral bedding. The offspring (5-HTT +/+, +/-, -/-) were examined for their ANX and EXP. The main results were: (1) a main effect of genotype existed, with 5-HTT -/- showing higher levels of ANX and lower levels of EXP than 5-HTT +/- and wildtypes. (2) When mothers had lived in a threatening environment, their offspring showed increased ANX and reduced EXP compared to controls. (3) These effects were most pronounced in 5-HTT -/- mice. By applying a new ecologically relevant paradigm we conclude: If 5-HTT +/- mothers live in a threatening environment during pregnancy and lactation, their offspring behavioral profile will, in principle, be shaped in an adaptive way preparing the young for an adverse environment. This process is, however, modulated by 5-HTT genotype, bearing the risk that individuals with impaired serotonergic neurotransmission (5-HTT -/-) will develop an exaggerated, potentially pathological level of anxiety from gene x environment interactions.