Comprehensive Analysis of Ultrasonic Vocalizations in a Mouse Model of Fragile X Syndrome Reveals Limited, Call Type Specific Deficits

Fragile X syndrome (FXS) is a well-recognized form of inherited mental retardation, caused by a mutation in the fragile X mental retardation 1 (Fmr1) gene. The gene is located on the long arm of the X chromosome and encodes fragile X mental retardation protein (FMRP). Absence of FMRP in fragile X patients as well as in Fmr1 knockout (KO) mice results, among other changes, in abnormal dendritic spine formation and altered synaptic plasticity in the neocortex and hippocampus. Clinical features of FXS include cognitive impairment, anxiety, abnormal social interaction, mental retardation, motor coordination and speech articulation deficits. Mouse pups generate ultrasonic vocalizations (USVs) when isolated from their mothers. Whether those social ultrasonic vocalizations are deficient in mouse models of FXS is unknown. Here we compared isolation-induced USVs generated by pups of Fmr1-KO mice with those of their wild type (WT) littermates. Though the total number of calls was not significantly different between genotypes, a detailed analysis of 10 different categories of calls revealed that loss of Fmr1 expression in mice causes limited and call-type specific deficits in ultrasonic vocalization: the carrier frequency of flat calls was higher, the percentage of downward calls was lower and that the frequency range of complex calls was wider in Fmr1-KO mice compared to their WT littermates.     

Fmr1 knockout mice are impaired in a leverpress escape/avoidance task

Fragile X syndrome (FXS) is the most common form of inherited mental retardation (MR). FXS is typically caused by a mutation of the Fmr1 gene (Verkerk et al. 1991, Cell 65, 905-914). To better understand the role of Fmr1 and its gene product fragile X mental-retardation protein (FMRP) in central nervous system function, researchers have turned to the use of animal model systems to generate an Fmr1 knockout (KO) mouse that is deficient in FMRP (Bakker et al. 1994, Cell 78, 23-33). Unfortunately, a number of studies have found no consistent, robust learning and memory impairment in the Fmr1 KO mice. We conducted a study to assess the performance of Fmr1 KO and wildtype (WT) animals in a leverpress escape/avoidance paradigm. Fmr1 KO and WT littermates were studied in four daily 1-h sessions. The Fmr1 KO mice performed fewer avoidance and total responses than WT mice. The KO animals were not simply deficient in avoidance, but a within-factor ANOVA revealed that they did not acquire the leverpress response to any appreciable degree. Observation during the sessions indicated that the Fmr1 KO animals clearly responded to the shock, eliminating an obvious sensory explanation for the deficit. The fact that other studies have found that the KO mice displayed increased exploratory and locomotor activity compared with WT controls argues against a motoric deficit. Future studies will attempt to delineate the nature of the behavioral deficit as well as attempt to rescue the response with glutamatergic or dopaminergic agents.     

Deficits in Tactile Learning in a Mouse Model of Fragile X Syndrome

The fragile X mental retardation 1 mutant mouse (Fmr1 KO) recapitulates several of the neurologic deficits associated with Fragile X syndrome (FXS). As tactile hypersensitivity is a hallmark of FXS, we examined the sensory representation of individual whiskers in somatosensory barrel cortex of Fmr1 KO and wild-type (WT) mice and compared their performance in a whisker-dependent learning paradigm, the gap cross assay. Fmr1 KO mice exhibited elevated responses to stimulation of individual whiskers as measured by optical imaging of intrinsic signals. In the gap cross task, initial performance of Fmr1 KO mice was indistinguishable from WT controls. However, while WT mice improved significantly with experience at all gap distances, Fmr1 KO mice displayed significant and specific deficits in improvement at longer distances which rely solely on tactile information from whiskers. Thus, Fmr1 KO mice possess altered cortical responses to sensory input that correlates with a deficit in tactile learning.     

Reduction of BDNF expression in Fmr1 knockout mice worsens cognitive deficits but improves hyperactivity and sensorimotor deficits

Fragile X syndrome (FXS) is a common cause of inherited intellectual disability and a well-characterized form of autism spectrum disorder. As brain-derived neurotrophic factor (BDNF) is implicated in the pathophysiology of FXS we examined the effects of reduced BDNF expression on the behavioral phenotype of an animal model of FXS, Fmr1 knockout (KO) mice, crossed with mice carrying a deletion of one copy of the Bdnf gene (Bdnf(+/-)). Fmr1 KO mice showed age-dependent alterations in hippocampal BDNF expression that declined after the age of 4 months compared to wild-type controls. Mild deficits in water maze learning in Bdnf(+/-) and Fmr1 KO mice were exaggerated and contextual fear learning significantly impaired in double transgenics. Reduced BDNF expression did not alter basal nociceptive responses or central hypersensitivity in Fmr1 KO mice. Paradoxically, the locomotor hyperactivity and deficits in sensorimotor learning and startle responses characteristic of Fmr1 KO mice were ameliorated by reducing BNDF, suggesting changes in simultaneously and in parallel working hippocampus-dependent and striatum-dependent systems. Furthermore, the obesity normally seen in Bdnf(+/-) mice was eliminated by the absence of fragile X mental retardation protein 1 (FMRP). Reduced BDNF decreased the survival of newborn cells in the ventral part of the hippocampus both in the presence and absence of FMRP. Since a short neurite phenotype characteristic of newborn cells lacking FMRP was not found in cells derived from double mutant mice, changes in neuronal maturation likely contributed to the behavioral phenotype. Our results show that the absence of FMRP modifies the diverse effects of BDNF on the FXS phenotype.     

The effect of an mGluR5 inhibitor on procedural memory and avoidance discrimination impairments in Fmr1 KO mice

Fragile X syndrome (FXS) is the most common inherited form of intellectual disability. Patients with FXS do not only suffer from cognitive problems, but also from abnormalities/deficits in procedural memory formation. It has been proposed that a lack of fragile X mental retardation protein (FMRP) leads to altered long-term plasticity by deregulation of various translational processes at the synapses, and that part of these impairments might be rescued by the inhibition of type I metabotropic glutamate receptors (mGluRs). We recently developed the Erasmus Ladder, which allows us to test, without any invasive approaches, simultaneously, both procedural memory formation and avoidance behavior during unperturbed and perturbed locomotion in mice. Here, we investigated the impact of a potent and selective mGluR5 inhibitor (Fenobam) on the behavior of Fmr1 KO mice during the Erasmus Ladder task. Fmr1 KO mice showed deficits in associative motor learning as well as avoidance behavior, both of which were rescued by intraperitoneal administration of Fenobam. While the Fmr1 KO mice did benefit from the treatment, control littermates suffered from a significant negative side effect in that their motor learning skills, but not their avoidance behavior, were significantly affected. On the basis of these studies in the FXS animal model, it may be worthwhile to investigate the effects of mGluR inhibitors on both the cognitive functions and procedural skills in FXS patients. However, the use of mGluR inhibitors appears to be strongly contraindicated in healthy controls or non-FXS patients with intellectual disability.     

A direct role for FMRP in activity-dependent dendritic mRNA transport links filopodial-spine morphogenesis to fragile X syndrome

The function of local protein synthesis in synaptic plasticity and its dysregulation in fragile X syndrome (FXS) is well studied, however the contribution of regulated mRNA transport to this function remains unclear. We report a function for the fragile X mental retardation protein (FMRP) in the rapid, activity-regulated transport of mRNAs important for synaptogenesis and plasticity. mRNAs were deficient in glutamatergic signaling-induced dendritic localization in neurons from Fmr1 KO mice, and single mRNA particle dynamics in live neurons revealed diminished kinesis. Motor-dependent translocation of FMRP and cognate mRNAs involved the C terminus of FMRP and kinesin light chain, and KO brain showed reduced kinesin-associated mRNAs. Acute suppression of FMRP and target mRNA transport in WT neurons resulted in altered filopodia-spine morphology that mimicked the FXS phenotype. These findings highlight a mechanism for stimulus-induced dendritic mRNA transport and link its impairment in a mouse model of FXS to altered developmental morphologic plasticity.     

Lithium treatment alleviates impaired cognition in a mouse model of fragile X syndrome

Fragile X syndrome (FXS) is caused by suppressed expression of fragile X mental retardation protein (FMRP), which results in intellectual disability accompanied by many variably manifested characteristics, such as hyperactivity, seizures and autistic‐like behaviors. Treatment of mice that lack FMRP, Fmr1 knockout (KO) mice, with lithium has been reported to ameliorate locomotor hyperactivity, prevent hypersensitivity to audiogenic seizures, improve passive avoidance behavior and attenuate sociability deficits. To focus on the defining characteristic of FXS, which is cognitive impairment, we tested if lithium treatment ameliorated impairments in four cognitive tasks in Fmr1 KO mice, tested if the response to lithium differed in adolescent and adult mice and tested if therapeutic effects persisted after discontinuation of lithium administration. Fmr1 KO mice displayed impaired cognition in the novel object detection task, temporal ordering for objects task and coordinate and categorical spatial processing tasks. Chronic lithium treatment of adolescent (from 4 to 8 weeks of age) and adult (from 8 to 12 weeks of age) mice abolished cognitive impairments in all four cognitive tasks. Cognitive deficits returned after lithium treatment was discontinued for 4 weeks. These results show that Fmr1 KO mice exhibit severe impairments in these cognitive tasks, that lithium is equally effective in normalizing cognition in these tasks whether it is administered to young or adult mice and that lithium administration must be continued for the cognitive improvements to be sustained. These findings provide further evidence that lithium administration may be beneficial for individuals with FXS .     

Male and female Fmr1 knockout mice on C57 albino background exhibit spatial learning and memory impairments

Impaired spatial learning is a prominent deficit in fragile X syndrome (FXS). Previous studies using the Fmr1 knockout (KO) mouse model of FXS have not consistently reported a deficit in spatial learning. Fmr1 KO mice bred onto an albino C57BL/6J‐Tyr^c‐Brd background showed significant deficits in several primary measures of performance during place navigation and probe trials in the Morris water maze. Fmr1 KO mice were also impaired during a serial reversal version of the water maze task. We examined fear conditioning as an additional cognitive screen. Knockout mice exhibited contextual memory deficits when trained with unsignaled shocks; however, deficits were not found in a separate group of KO mice trained with signaled shocks. No potentially confounding genotypic differences in locomotor activity were observed. A decreased anxiety‐like profile was apparent in the open field, as others have noted, and also in the platform test. Also as previously reported, startle reactivity to loud auditory stimuli was decreased, prepulse inhibition and social interaction increased in KO mice. Female Fmr1 KO mice were tested along with male KO mice in all assays, except for social interaction. The female and male KO exhibited very similar impairments indicating that sex does not generally drive the behavioral symptoms of the disorder. Our results suggest that procedural factors, such as the use of albino mice, may help to reliably detect spatial learning and memory impairments in both sexes of Fmr1 KO mice, making it more useful for understanding FXS and a platform for evaluating potential therapeutics.     

Increased Training Intensity Induces Proper Membrane Localization of Actin Remodeling Proteins in the Hippocampus Preventing Cognitive Deficits: Implications for Fragile X Syndrome

Behavioral intervention therapy has proven beneficial in the treatment of autism and intellectual disabilities (ID), raising the possibility of certain changes in molecular mechanisms activated by these interventions that may promote learning. Fragile X syndrome (FXS) is a neurodevelopmental disorder characterized by autistic features and intellectual disability and can serve as a model to examine mechanisms that promote learning. FXS results from mutations in the fragile X mental retardation 1 gene (Fmr1) that prevents expression of the Fmr1 protein (FMRP), a messenger RNA (mRNA) translation regulator at synapses. Among many other functions, FMRP organizes a complex with the actin cytoskeleton-regulating small Rho GTPase Rac1. As in humans, Fmr1 KO mice lacking FMRP display autistic-like behaviors and deformities of actin-rich synaptic structures in addition to impaired hippocampal learning and synaptic plasticity. These features have been previously linked to proper function of actin remodeling proteins that includes Rac1. An important step in Rac1 activation and function is its translocation to the membrane, where it can influence synaptic actin cytoskeleton remodeling during hippocampus-dependent learning. Herein, we report that Fmr1 KO mouse hippocampus exhibits increased levels of membrane-bound Rac1, which may prevent proper learning-induced synaptic changes. We also determine that increasing training intensity during fear conditioning (FC) training restores contextual memory in Fmr1 KO mice and reduces membrane-bound Rac1 in Fmr1 KO hippocampus. Increased training intensity also results in normalized long-term potentiation in hippocampal slices taken from Fmr1 KO mice. These results point to interventional treatments providing new therapeutic options for FXS-related cognitive dysfunction.     

Excessive Astrocyte-Derived Neurotrophin-3 Contributes to the Abnormal Neuronal Dendritic Development in a Mouse Model of Fragile X Syndrome

Fragile X syndrome (FXS) is a form of inherited mental retardation in humans that results from expansion of a CGG repeat in the Fmr1 gene. Recent studies suggest a role of astrocytes in neuronal development. However, the mechanisms involved in the regulation process of astrocytes from FXS remain unclear. In this study, we found that astrocytes derived from a Fragile X model, the Fmr1 knockout (KO) mouse which lacks FMRP expression, inhibited the proper elaboration of dendritic processes of neurons in vitro. Furthermore, astrocytic conditioned medium (ACM) from KO astrocytes inhibited proper dendritic growth of both wild-type (WT) and KO neurons. Inducing expression of FMRP by transfection of FMRP vectors in KO astrocytes restored dendritic morphology and levels of synaptic proteins. Further experiments revealed elevated levels of the neurotrophin-3 (NT-3) in KO ACM and the prefrontal cortex of Fmr1 KO mice. However, the levels of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), and ciliary neurotrophic factor (CNTF) were normal. FMRP has multiple RNA–binding motifs and is involved in translational regulation. RNA–binding protein immunoprecipitation (RIP) showed the NT-3 mRNA interacted with FMRP in WT astrocytes. Addition of high concentrations of exogenous NT-3 to culture medium reduced the dendrites of neurons and synaptic protein levels, whereas these measures were ameliorated by neutralizing antibody to NT-3 or knockdown of NT-3 expression in KO astrocytes through short hairpin RNAs (shRNAs). Prefrontal cortex microinjection of WT astrocytes or NT-3 shRNA infected KO astrocytes rescued the deficit of trace fear memory in KO mice, concomitantly decreased the NT-3 levels in the prefrontal cortex. This study indicates that excessive NT-3 from astrocytes contributes to the abnormal neuronal dendritic development and that astrocytes could be a potential therapeutic target for FXS.     

A comparative study of the performance of individuals with fragile X syndrome and Fmr1 knockout mice on Hebb-Williams mazes

Fragile X syndrome (FXS) is the most prevalent form of heritable mental retardation. It arises from a mutation in the FMR1 gene on the X chromosome that interferes with expression of fragile X mental retardation protein (FMRP) and leads to a wide range of behavioural and cognitive deficits. Previous studies have shown a deficit in basic visual perceptual processing as well as spatial abilities in FXS. How such a deficit may impact spatial navigation remains unknown. The current study extended previous research by evaluating spatial learning and memory using both virtual and physical versions of Hebb-Williams mazes, which allows for testing of humans and animals under comparable conditions. We compared the performance of individuals affected by FXS to typically developing individuals of equivalent mental age as well as the performance of Fmr1 knockout mice to wild-type control mice on the same maze problems. In human participants, performance of the comparison group improved across trials, showing expected significant decreases in both errors and latency. In contrast, the performance of the fragile X group remained at similar levels across trials. Although wild-type control mice made significantly fewer errors than the Fmr1 knockout mice, latencies were not statistically different between the groups. These findings suggest that affected humans and mice show similar spatial learning deficits attributable to the lack of FMRP. The implications of these data are discussed including the notion that Hebb-Williams mazes may represent a useful tool to examine the impact of pharmacological interventions on mitigating or reversing the symptoms associated with FXS.     

Impaired social contacts with familiar anesthetized conspecific in CA3‐restricted brain‐derived neurotrophic factor knockout mice

Familiarity is conveyed by social cues and determines behaviors toward conspecifics. Here, we characterize a novel assay for social behaviors in mice—contacts with anesthetized conspecific—which eliminates reciprocal interactions, including intermale aggression and shows behaviors that are independent of the demonstrator's activity. During the initial 10 minutes (phase‐1), the wild‐type (WT) subjects contacted the anesthetized conspecifics vigorously regardless of familiarity. During the subsequent 80 minutes (phase‐2), however, they contacted more with familiar than unfamiliar conspecifics. We then applied this test to highly aggressive mice with a hippocampal CA3‐restricted knockout (KO) of brain‐derived neurotrophic factor (BDNF), in which aggression may mask other social behaviors. The KO mice showed less preference for contacting familiar conspecifics than did WT mice during phase‐2 but no differences during phase‐1. Among nonsocial behaviors, WT mice also spent less time eating in the presence of familiar than with unfamiliar conspecifics, which was not seen in KO mice. In addition, KO mice exhibited reduced pain sensitization. Altogether, these findings suggest that CA3‐specific deletion of BDNF results in deficits in circuits that process social cues from familiar conspecifics as well as pain and may underlie empathy‐like behaviors.     

Longitudinal in vivo developmental changes of metabolites in the hippocampus of Fmr1 knockout mice

Fragile X syndrome (FXS) is the most common form of inherited mental retardation and is studied in the Fmr1 knockout (KO) mouse, which models both the anatomical and behavioral changes observed in FXS patients. In vitro studies have shown many alterations in synaptic plasticity and increased density of immature dendritic spines in the hippocampus, a region involved in learning and memory. In this study, magnetic resonance imaging (MRI) and ¹H magnetic resonance spectroscopy (MRS) were used to determine in vivo longitudinal changes in volume and metabolites in the hippocampus during the critical period of early myelination and synaptogenesis at post‐natal days (PND) 18, 21, and 30 in Fmr1 KO mice compared with wild‐type (WT) controls. MRI demonstrated an increase in volume of the hippocampus in the Fmr1 KO mouse compared with controls. MRS revealed significant developmental changes in the ratios of hippocampal metabolites N‐acetylaspartate (NAA), myo‐inositol (Ins), and taurine to total creatine (tCr) in Fmr1 KO mice compared with WT controls. Ins was decreased at PND 30, and taurine was increased at all ages studied in Fmr1 KO mice compared with controls. An imbalance of brain metabolites in the hippocampus of Fmr1 KO mice during the critical developmental period of synaptogenesis and early myelination could have long‐lasting effects that adversely affect brain development and contribute to ongoing alterations in brain function.     

Programming social behavior by the maternal fragile X protein

The developing fetus and neonate are highly sensitive to maternal environment. Besides the well‐documented effects of maternal stress, nutrition and infections, maternal mutations, by altering the fetal, perinatal and/or early postnatal environment, can impact the behavior of genetically normal offspring. Mutation/premutation in the X‐linked FMR1 (encoding the translational regulator FMRP) in females, although primarily responsible for causing fragile X syndrome (FXS) in their children, may also elicit such maternal effects. We showed that a deficit in maternal FMRP in mice results in hyperactivity in the genetically normal offspring. To test if maternal FMRP has a broader intergenerational effect, we measured social behavior, a core dimension of neurodevelopmental disorders, in offspring of FMRP‐deficient dams. We found that male offspring of Fmr1^+/? mothers, independent of their own Fmr1 genotype, exhibit increased approach and reduced avoidance toward conspecific strangers, reminiscent of ‘indiscriminate friendliness’ or the lack of stranger anxiety, diagnosed in neglected children and in patients with Asperger's and Williams syndrome. Furthermore, social interaction failed to activate mesolimbic/amygdala regions, encoding social aversion, in these mice, providing a neurobiological basis for the behavioral abnormality. This work identifies a novel role for FMRP that extends its function beyond the well‐established genetic function into intergenerational non‐genetic inheritance/programming of social behavior and the corresponding neuronal circuit. As FXS premutation and some psychiatric conditions that can be associated with reduced FMRP expression are more prevalent in mothers than full FMR1 mutation, our findings potentially broaden the significance of FMRP‐dependent programming of social behavior beyond the FXS population.     

脆性X综合征致病机理与治疗

脆性X综合征(fragile X syndrome, FXS)是最常见的遗传性智力障碍疾病,主要是由于X染色体上脆性X智力低下基因1(fragile X-mental retardation gene 1, FMR1)5’端非翻译区CGG三核苷酸的重复扩增及其相邻部位CpG岛的异常甲基化而导致其编码产物脆性X智力低下蛋白(fragile X mental retardation protein, FMRP)的缺失引起。目前,基因诊断已成为FXS诊断的金标准,但临床治疗仍缺乏特异性。本文首先介绍了FMRP的结构与功能,剖析了FXS的致病机制,然后阐述了FXS中与FMRP表达相关的信号转导途径,深入探讨并总结了靶向干预FXS中信号通路、基因编辑逆转FMR1沉默以及靶向降解FXS异常表达蛋白的治疗策略。     

Changes in Sensitivity of Reward and Motor Behavior to Dopaminergic,Glutamatergic, and Cholinergic Drugs in a Mouse Model of Fragile X Syndrome

Fragile X syndrome (FXS) is a leading cause of intellectual disability. FXS is caused by loss of function of the FMR1 gene, and mice in which Fmr1 has been inactivated have been used extensively as a preclinical model for FXS. We investigated the behavioral pharmacology of drugs acting through dopaminergic, glutamatergic, and cholinergic systems in fragile X (Fmr1 ^-/Y) mice with intracranial self-stimulation (ICSS) and locomotor activity measurements. We also measured brain expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine biosynthesis. Fmr1 ^-/Y mice were more sensitive than wild type mice to the rewarding effects of cocaine, but less sensitive to its locomotor stimulating effects. Anhedonic but not motor depressant effects of the atypical neuroleptic, aripiprazole, were reduced in Fmr1 ^-/Y mice. The mGluR5-selective antagonist, 6-methyl-2-(phenylethynyl)pyridine (MPEP), was more rewarding and the preferential M1 antagonist, trihexyphenidyl, was less rewarding in Fmr1 ^-/Y than wild type mice. Motor stimulation by MPEP was unchanged, but stimulation by trihexyphenidyl was markedly increased, in Fmr1 ^-/Y mice. Numbers of midbrain TH+ neurons in the ventral tegmental area were unchanged, but were lower in the substantia nigra of Fmr1 ^-/Y mice, although no changes in TH levels were found in their forebrain targets. The data are discussed in the context of known changes in the synaptic physiology and pharmacology of limbic motor systems in the Fmr1 ^-/Y mouse model. Preclinical findings suggest that drugs acting through multiple neurotransmitter systems may be necessary to fully address abnormal behaviors in individuals with FXS.