Increased 2-arachidonoyl-sn-glycerol levels normalize cortical responses to sound and improve behaviors in Fmr1 KO mice

Genetic reduction of MMP-9 in the Fmr1 KO mouse partially rescues prepulse inhibition of acoustic startle response

Fragile X syndrome (FXS)—caused by FMR1 gene silencing—is a severe neurodevelopmental disorder characterized by intellectual disabilities that are often comorbid with seizures, sensory hypersensitivities, anxiety, social deficits, and repetitive behaviors. Neuronal hyperexcitability is an overarching neurophysiological characteristic of FXS that may underlie FXS symptoms. About 33% of Fmr1 KO mice from our colony exhibit spontaneous seizures, a newly observed phenotype that more closely parallels seizures in FXS, compared to the audiogenic seizure phenotype in Fmr1 KO mice. In addition, we and others show that Fmr1 KO mice, like individuals with FXS, have cortical EEG gamma‐band power alterations, and at the single‐cell level, have hyperexcitable pyramidal neurons in multiple brain regions. We are using a combinatorial approach—from behavioral to EEG to single‐cell experiments—to advance FXS drug discovery. Based on our observations of altered brain expression and in vivo function of serotonin 1A receptors (5‐HT1ARs) in Fmr1 KO mice and correction of the audiogenic seizure phenotype by our novel 2‐aminotetralin‐type 5‐HT1R modulator, FPT, we are testing the hypothesis that selectively activating 5‐HT1ARs prevents seizures and corrects neurophysiological abnormalities. We evaluated the efficacy of FPT (5.6 mg/kg), a potent and efficacious 5‐HT1AR agonist, to correct EEG abnormalities in Fmr1 KO mice. We also tested the antiepileptic effects of the selective 5‐HT1AR agonist, NLX‐112 (0.25‐2.5 mg/kg), and are currently testing the effects of FPT and NLX‐112 on CA1 pyramidal neuron hyperexcitability in Fmr1 KO mice. In parallel experiments, we are evaluating the pharmacology of FPT and NLX‐112 at each of the 5‐HT G protein‐coupled receptors. Recordings from above the left somatosensory cortex showed a significantly elevated high gamma (65‐100 Hz) power ratio in Fmr1 KO mice relative to control mice at baseline (n=16, P=0.0357) and after vehicle injection (n=16, P=0.0066), a genotype difference that FPT eliminated (n=15‐16, P=0.6279). Comparisons between baseline and first injection conditions also revealed an increased delta power in Fmr1 KO mice relative to controls. Separately, NLX‐112 prevented audiogenic seizures in Fmr1 KO mice (n=10‐12, P≤0.0002), and preliminary data suggest NLX‐112 and FPT modulate CA1 pyramidal neuron activity. For example, FPT (10 µM) showed a reversible reduction of firing frequency of hippocampal CA1 neurons in Fmr1 KO mice (n=8, P<0.05). Forthcoming experiments will include evaluating the effects of NLX‐112 on cortical EEG activity in Fmr1 KO and control mice and the effects of chronic NLX‐112 and FPT on spontaneous seizures in Fmr1 KO mice. Tests of the selective 5‐HT1AR antagonist, WAY100635, will be conducted to examine a 5‐HT1AR mechanism underlying positive outcomes of NLX‐112 and FPT. At present, our convergent data suggest that 5‐HT1AR activation may ameliorate neuronal hyperexcitability, at multiple levels of analysis, in Fmr1 KO mice. Potent and selective 5‐HT1AR agonists might be pharmacotherapeutic for FXS.

Cortical layer-specific abnormalities in auditory responses in a mouse model of Fragile X Syndrome.

Background:Fragile X syndrome (FXS) is the most common single gene inherited form of mental retardation, with behaviours at the extreme of the autistic spectrum. Subjects with FXS and fragile X...

Effects of stimulus salience on touchscreen serial reversal learning in a mouse model of fragile X syndrome

P.2.013 Effects of MK-801 and medial prefrontal cortex lesions on cognitive flexibility in a novel task based on the change of interval schedules

Electroencephalographic (EEG) recordings in individuals with Fragile X Syndrome (FXS) and the mouse model of FXS ( Fmr1 KO) display cortical hyperexcitability at rest, as well as deficits in sensory-driven cortical network synchrony. A form of circuit hyperexcitability is observed in ex vivo cortical slices of Fmr1 KO mice as prolonged persistent activity, or Up, states. It is unknown if the circuit mechanisms that cause prolonged Up states contribute to FXS-relevant EEG phenotypes. Here we examined the role of endocannabinoids (eCB) in prolonged Up states in slices and resting and sensory-driven EEG phenotypes in awake Fmr1 KO mice. Bidirectional changes in eCB function are reported in the Fmr1 KO that depend on synapse type (excitatory or inhibitory). We demonstrate that pharmacological or genetic reduction of Cannabinoid Receptor 1 (CB1R) in GABAergic neurons rescues prolonged cortical Up states and deficits in sensory-driven cortical synchrony in Fmr1 KO mice. In support of these findings, recordings from Fmr1 KO cortical Layer (L) 2/3 pyramidal neurons revealed enhanced CB1R-mediated suppression of inhibitory synaptic currents. In contrast, genetic reduction of Cnr1 in glutamatergic neurons did not affect Up state duration, but deletion of Fmr1 in the same neurons was sufficient to cause long Up states. These findings support a model where loss of Fmr1 in glutamatergic neurons leads to enhanced CB1R-mediated suppression of GABAergic synaptic transmission, prolonged cortical circuit activation and reduced sensory-driven circuit synchronization. Results suggest that antagonism of CB1Rs as a therapeutic strategy to correct sensory processing deficits in FXS.

Effects of Fmr1 Gene Mutations on Sex Differences in Autism-Like Behavior and Dendritic Spine Development in Mice and Transcriptomic Studies

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.

Fragile X Syndrome (FXS) is a leading known genetic cause of intellectual disability with symptoms that include increased anxiety and social and sensory processing deficits. Recent electroencephalographic (EEG) studies in humans with FXS have identified neural oscillation deficits that include increased resting state gamma power, increased amplitude of auditory evoked potentials, and reduced phase locking of sound-evoked gamma oscillations. Similar EEG phenotypes are present in mouse models of FXS, but very little is known about the development of such abnormal responses. In the current study, we employed a 30-channel mouse multielectrode array (MEA) system to record and analyze resting and stimulus-evoked EEG signals in male P21 and P91 WT and Fmr1 KO mice. This led to several novel findings. First, P91, but not P21, Fmr1 KO mice have significantly increased resting EEG power in the low- and high-gamma frequency bands. Second, both P21 and P91 Fmr1 KO mice have markedly attenuated inter-trial phase coherence (ITPC) to spectrotemporally dynamic auditory stimuli as well as to 40 Hz and 80 Hz auditory steady-state response (ASSR) stimuli. This suggests abnormal temporal processing from early development that may lead to abnormal speech and language function in FXS. Third, we found hemispheric asymmetry of fast temporal processing in the mouse auditory cortex in WT but not Fmr1 KO mice. Together, these findings define a set of EEG phenotypes in young and adult mice that can serve as translational targets for genetic and pharmacological manipulation in phenotypic rescue studies.

Hypersensitivity to sounds is one of the prevalent symptoms in individuals with Fragile X syndrome (FXS). It manifests behaviorally early during development and is often used as a landmark for treatment efficacy. However, the physiological mechanisms and circuit-level alterations underlying this aberrant behavior remain poorly understood. Using the mouse model of FXS (Fmr1 KO), we demonstrate that functional maturation of auditory brainstem synapses is impaired in FXS. Fmr1 KO mice showed a greatly enhanced excitatory synaptic input strength in neurons of the lateral superior olive (LSO), a prominent auditory brainstem nucleus, which integrates ipsilateral excitation and contralateral inhibition to compute interaural level differences. Conversely, the glycinergic, inhibitory input properties remained unaffected. The enhanced excitation was the result of an increased number of cochlear nucleus fibers converging onto one LSO neuron, without changing individual synapse properties. Concomitantly, immunolabeling of excitatory ending markers revealed an increase in the immunolabeled area, supporting abnormally elevated excitatory input numbers. Intrinsic firing properties were only slightly enhanced. In line with the disturbed development of LSO circuitry, auditory processing was also affected in adult Fmr1 KO mice as shown with single-unit recordings of LSO neurons. These processing deficits manifested as an increase in firing rate, a broadening of the frequency response area, and a shift in the interaural level difference function of LSO neurons. Our results suggest that this aberrant synaptic development of auditory brainstem circuits might be a major underlying cause of the auditory processing deficits in FXS.SIGNIFICANCE STATEMENT Fragile X Syndrome (FXS) is the most common inheritable form of intellectual impairment, including autism. A core symptom of FXS is extreme sensitivity to loud sounds. This is one reason why individuals with FXS tend to avoid social interactions, contributing to their isolation. Here, a mouse model of FXS was used to investigate the auditory brainstem where basic sound information is first processed. Loss of the Fragile X mental retardation protein leads to excessive excitatory compared with inhibitory inputs in neurons extracting information about sound levels. Functionally, this elevated excitation results in increased firing rates, and abnormal coding of frequency and binaural sound localization cues. Imbalanced early-stage sound level processing could partially explain the auditory processing deficits in FXS.

Fragile X syndrome (FXS) is the most common form of inherited intellectual disability. In addition, there is increased incidence of anxiety, sleep irregularities, and seizure activity. The underlying cause of FXS is a loss of the fragile X mental retardation protein (FMRP) which has been shown to participate in the biosynthesis of δ subunits. Studies from both FXS patients and animal models have revealed reduced expression levels of GABAAR α4 and δ subunits with a reduced efficacy of tonic inhibition. Neurosteroids (NS) are known allosteric modulators of GABAAR channel function but recent studies from our laboratory have revealed that NS also exert persistent effects on the efficacy of tonic inhibition by increasing the PKC‐mediated phosphorylation of the α4 subunit which increases the membrane expression and boosts tonic inhibition. We have used a combination of biochemical and electrophysiological methods to assess alterations in GABAergic signaling in the hippocampus of FMRP knock‐out mouse (Fmr1 KO), a widely validated model of the human syndrome.Our preliminary studies demonstrate that Fmr1 KO mice at p21 have a decrease in phosphorylation of S443 in the α4 subunit compared to WT and an increase in the phosphorylation of β3 subunits at the S408/409 site compared to WT. We have previously showed that phosphorylation of these residues changes the trafficking of the subunits so the changes observed in Fmr1 KO mice would predictably have consequences for trafficking of these essential inhibitory subunits. However, at p48–72 Fmr1 KO mice did not exhibit any deficits in GABAAR expression levels or phosphorylation demonstrating a critical developmental deficit in GABAAR expression. We noted that there was a significant decrease in tonic inhibition in dentate gyrus granule cells in p21 Fmr1 KO mice compared to WT. A 10 min exposure to 1μM THDOC followed by 330 min wash induced a >3 fold increase in tonic current in Fmr1 KO animals which was prevented with PKC inhibition. Using a perforated multi‐electrode array we have observed in horizontal cortical‐hippocampal slices seizure like events (SLE) propagating through the dentate gyrus and into the CA3 and CA1 regions of the hippocampus of Fmr1 KO mice but in WT mice SLEs did not propagate through the dentate gyrus. We predict the increase neuronal excitability seen in Fmr1 KO mice is due to the deficits in tonic inhibition.We have observed that THDOC treatment effectively reversed tonic current deficits in Fmr1 KO mice. Future studies will focus on ameliorating behavioral deficits in Fmr1 KO mice with synthetic neuroactive steroids that display improved pharmacokinetic properties.Support or Funding InformationThis work was supported by grants from the National Institutes of Health (NIH)‐National Institute of Alcoholism and Alcohol Abuse grant AA017938 (P.A.D), NIH‐National Institute of Mental Health grant, MH097446, and DOD, AR140209 (PAD & SJM), NIH‐National Institute of Neurological Disorders and Stroke grant NS051195, NS056359, NS081735, NS080064, NS087662 (SJM), the Simons Foundation #206026 (S.J.M.).

Effects of chronic inhibition of phosphodiesterase-4D on behavior and regional rates of cerebral protein synthesis in a mouse model of fragile X syndrome

Effects of chronic immobilization stress on anxiety-like behavior and basolateral amygdala morphology in Fmr1 knockout mice

Fragile X syndrome (FXS) is an inherited form of intellectual disability and autism. Among other symptoms, FXS patients demonstrate abnormalities in sensory processing and communication. Clinical, behavioral, and electrophysiological studies consistently show auditory hypersensitivity in humans with FXS. Consistent with observations in humans, the Fmr1 KO mouse model of FXS also shows evidence of altered auditory processing and communication deficiencies. A well-known and commonly used phenotype in pre-clinical studies of FXS is audiogenic seizures. In addition, increased acoustic startle response is seen in the Fmr1 KO mice. In vivo electrophysiological recordings indicate hyper-excitable responses, broader frequency tuning, and abnormal spectrotemporal processing in primary auditory cortex of Fmr1 KO mice. Thus, auditory hyper-excitability is a robust, reliable, and translatable biomarker in Fmr1 KO mice. Abnormal auditory evoked responses have been used as outcome measures to test therapeutics in FXS patients. Given that similarly abnormal responses are present in Fmr1 KO mice suggests that cellular mechanisms can be addressed. Sensory cortical deficits are relatively more tractable from a mechanistic perspective than more complex social behaviors that are typically studied in autism and FXS. The focus of this review is to bring together clinical, functional, and structural studies in humans with electrophysiological and behavioral studies in mice to make the case that auditory hypersensitivity provides a unique opportunity to integrate molecular, cellular, circuit level studies with behavioral outcomes in the search for therapeutics for FXS and other autism spectrum disorders.