The loss of fragile X mental retardation (FMR1) gene function causes fragile X syndrome (FXS), a common mental retardation syndrome. Anxiety and abnormal social behaviors are prominent features of FXS in humans. To better understand the role of FMR1 in these behaviors, we analyzed anxiety-related and social behaviors in Fmr1 knockout (KO) mice. In the mirrored chamber test, Fmr1 KO mice showed greater aversion to the central mirrored chamber than wild-type (WT) littermates, suggesting increased anxiety-like responses to reflected images of mice. Fmr1 KO mice exhibited abnormal social interactions in a tube test of social dominance, winning fewer matches than WT littermates. In a partition test, Fmr1 KO mice had normal levels of social interest and social recognition. However, during direct interaction tests, Fmr1 KO mice showed significant increases in sniffing behaviors. We further tested the influence of environmental familiarity on the social responses of Fmr1 KO mice to unfamiliar partners. In unfamiliar partitioned cages, Fmr1 KO mice did not differ from WT mice in investigation of unfamiliar partners. However, in familiar partitioned cages, Fmr1 KO mice showed less investigation of a newly introduced partner during the first 5 min and more investigation during the last 5 min of a 20-min partition test, behaviors consistent with initial social anxiety followed by enhanced social investigation. Our findings indicate that the loss of Fmr1 gene function results in altered anxiety and social behavior in mice and demonstrate that the Fmr1 KO mouse is a relevant animal model for the abnormal social responses seen in FXS.

Scientific AbstractFragile X syndrome (FXS) is the most common inherited form of intellectual disability in humans. In addition to cognitive impairment, patients may exhibit hyperactivity, attention deficits, social difficulties and anxiety, and autistic-like behaviors. The degree to which patients display these behaviors varies considerably and is influenced by family history, suggesting that genetic modifiers play a role in the expression of behaviors in FXS. Several studies have examined behavior in a mouse model of FXS in which the Fmr1 gene has been ablated. Most of those studies were done in Fmr1 knockout mice on a pure C57BL/6 or FVB strain background. To gain a better understanding of the effects of genetic background on behaviors resulting from the loss of Fmr1 gene expression, we generated F1 hybrid lines from female Fmr1 heterozygous mice on a pure C57BL/6J background bred with male Fmr1 wild-type mice of various background strains (A/J, DBA/2J, FVB/NJ, 129S1/SvImJ and CD-1). Male Fmr1 knockout and wild-type littermates from each line were examined in an extensive behavioral test battery. Results clearly indicate that multiple behavioral responses are dependent on genetic background, including autistic-like traits that are present on limited genetic backgrounds. This approach has allowed us to identify improved models for different behavioral symptoms present in FXS including autistic-like traits. Keywordsfragile X syndrome; autism; genetic; behavior; animal model; mouse model IntroductionFragile X syndrome (FXS) is widely acknowledged as the most common form of inherited intellectual disability (ID). The prevalence of FXS is estimated to be 1/4000 males and 1/8000 females, which accounts for approximately one-third of all X-linked ID cases (Sherman, 2002). The FXS phenotype is often described as clinically indistinct due to the broad spectrum of involvement of the various physical, cognitive, and behavioral abnormalities associated with the syndrome. What is often less appreciated, but which may be more critical to the overall quality of life, is the fact that individuals with FXS have several other behavioral abnormalities including: attention deficit, hyperactivity/ hyperkinesis, anxiety, depression, irritability, mania, obsessive-compulsive behavior, aggression, and self-injurious behavior (Hagerman, 2002). FXS patients are also hypersensitive to many different sensory stimuli. Interestingly, a number of autistic-like * Correspondence: Richard Paylor, rpaylor@bcm.edu, NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript behaviors have been associated with FXS, including poor eye contact, tactile defensiveness, hand biting, hand flapping, and perseveration of speech and behavior (Hagerman, 2002). An estimated 21-50% of individuals with FXS meet diagnostic criteria for autistic disorder, displaying social difficulty, communication problems and perseverative or repetitive behaviors (Moss & Howlin, 2009). Like many complex genetic syndromes, there is significant...

Dopamine (DA) and serotonin (5HT) are reported to serve important roles in aggression in a wide variety of animals. Previous investigations of 5HT function in adult Drosophila behavior have relied on pharmacological manipulations, or on combinations of genetic tools that simultaneously target both DA and 5HT neurons. Here, we generated a transgenic line that allows selective, direct manipulation of serotonergic neurons and asked whether DA and 5HT have separable effects on aggression. Quantitative morphological examination demonstrated that our newly generated tryptophan hydroxylase (TRH)-Gal4 driver line was highly selective for 5HT-containing neurons. This line was used in conjunction with already available Gal4 driver lines that target DA or both DA and 5HT neurons to acutely alter the function of aminergic systems. First, we showed that acute impairment of DA and 5HT neurotransmission using expression of a temperature sensitive form of dynamin completely abolished mid- and high-level aggression. These flies did not escalate fights beyond brief low-intensity interactions and therefore did not yield dominance relationships. We showed next that manipulation of either 5HT or DA neurotransmission failed to duplicate this phenotype. Selective disruption of 5HT neurotransmission yielded flies that fought, but with reduced ability to escalate fights, leading to fewer dominance relationships. Acute activation of 5HT neurons using temperature sensitive dTrpA1 channel expression, in contrast, resulted in flies that escalated fights faster and that fought at higher intensities. Finally, acute disruption of DA neurotransmission produced hyperactive flies that moved faster than controls, and rarely engaged in any social interactions. By separately manipulating 5HT- and DA- neuron systems, we collected evidence demonstrating a direct role for 5HT in the escalation of aggression in Drosophila.

Monoamines, including dopamine (DA), have been linked to aggression in various species. However, the precise role or roles served by the amine in aggression have been difficult to define because dopaminergic systems influence many behaviors, and all can be altered by changing the function of dopaminergic neurons. In the fruit fly, with the powerful genetic tools available, small subsets of brain cells can be reliably manipulated, offering enormous advantages for exploration of how and where amine neurons fit into the circuits involved with aggression. By combining the GAL4/upstream activating sequence (UAS) binary system with the Flippase (FLP) recombination technique, we were able to restrict the numbers of targeted DA neurons down to a single-cell level. To explore the function of these individual dopaminergic neurons, we inactivated them with the tetanus toxin light chain, a genetically encoded inhibitor of neurotransmitter release, or activated them with dTrpA1, a temperature-sensitive cation channel. We found two sets of dopaminergic neurons that modulate aggression, one from the T1 cluster and another from the PPM3 cluster. Both activation and inactivation of these neurons resulted in an increase in aggression. We demonstrate that the presynaptic terminals of the identified T1 and PPM3 dopaminergic neurons project to different parts of the central complex, overlapping with the receptor fields of DD2R and DopR DA receptor subtypes, respectively. These data suggest that the two types of dopaminergic neurons may influence aggression through interactions in the central complex region of the brain involving two different DA receptor subtypes.A ggression is an innate behavior commonly used to obtain resources such as territories, mates, or food. Although some features of aggression are species-specific, broad similarities exist across species in the behavioral patterns and neurochemical systems involved (1, 2). Monoamines such as dopamine (DA) and serotonin have been linked to aggression in many species (3). Elaboration of the neural pathways that control aggression, however, has proven difficult because monoaminergic neurons also regulate other behaviors (1, 2). The pharmacological and genetic manipulations usually used for altering amine neuron function influence most of the neurons within a given monoaminergic population and thereby cause many behavioral changes. This makes it difficult to pinpoint the relationship between any particular amine-induced behavioral phenotype and an associated neuronal pathway.The fruit fly offers enormous advantages in these regards because existing powerful genetic methods permit manipulation of small subsets of brain cells for exploration of the neural mechanisms of behavior (4-6). In the Drosophila model of aggression (7,8), males fight and form stable hierarchical relationships. Using this model, we have shown that acute shutdown of serotonergic neurons yielded flies that could initiate fights but showed a reduced ability to escalate aggression. Activation of serotonergic ne...

SUMMARY Monoamine serotonin (5HT) has been linked to aggression for many years across species [1–3]. However, elaboration of the neurochemical pathways that govern aggression has proven difficult because monoaminergic neurons also regulate other behaviors [4, 5]. There are about 100 serotonergic neurons in the Drosophila nervous system and they influence sleep [6], circadian rhythms [7], memory [8, 9] and courtship [10]. In the Drosophila model of aggression [11] the acute shut down of the entire serotonergic system yields flies that fight less, while induced activation of 5HT neurons promotes aggression [12]. Using intersectional genetics we restricted the population of 5HT neurons that can be reproducibly manipulated to identify those that modulate aggression. Although similar approaches were used recently to find aggression-modulating dopaminergic [13] and FruM –positive peptidergic [14] neurons, the downstream anatomical targets of the neurons that make up aggression-controlling circuits remain poorly understood. Here we identified a symmetrical pair of serotonergic PLP neurons that are necessary for the proper escalation of aggression. Silencing these neurons reduced, and activating them increased aggression in male flies. GFP reconstitution across synaptic partners (GRASP) [15] analyses suggests that 5HT-PLP neurons form contacts with 5HT1A receptor - expressing neurons in two distinct anatomical regions of the brain. Activation of these 5HT1A receptor-expressing neurons, in turn, caused reductions in aggression. Our studies, therefore, suggest that aggression may be held in check, at least in part, by inhibitory input from 5HT1A receptor-bearing neurons, which can be released by activation of the 5HT-PLP neurons.