Targeting 5‐HT1A Receptors to Correct Neuronal Hyperexcitability in Fmr1 Knockout Mice

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

BackgroundIndividuals with Fragile X syndrome (FXS) and autism spectrum disorder (ASD) exhibit an array of symptoms, including sociability deficits, increased anxiety, hyperactivity, and sensory hyperexcitability. It is unclear how endocannabinoid (eCB) modulation can be targeted to alleviate neurophysiological abnormalities in FXS as behavioral research reveals benefits to inhibiting cannabinoid (CB) receptor activation and increasing endocannabinoid ligand levels. Here, we hypothesize that enhancement of 2-arachidonoyl-sn-glycerol (2-AG) in Fragile X mental retardation 1 gene knock-out (Fmr1 KO) mice may reduce cortical hyperexcitability and behavioral abnormalities observed in FXS.MethodsTo test whether an increase in 2-AG levels normalized cortical responses in a mouse model of FXS, animals were subjected to electroencephalography (EEG) recording and behavioral assessment following treatment with JZL-184, an irreversible inhibitor of monoacylglycerol lipase (MAGL). Assessment of 2-AG was performed using lipidomic analysis in conjunction with various doses and time points post-administration of JZL-184. Baseline electrocortical activity and evoked responses to sound stimuli were measured using a 30-channel multielectrode array (MEA) in adult male mice before, 4 h, and 1 day post-intraperitoneal injection of JZL-184 or vehicle. Behavior assessment was done using the open field and elevated plus maze 4 h post-treatment.ResultsLipidomic analysis showed that 8 mg/kg JZL-184 significantly increased the levels of 2-AG in the auditory cortex of both Fmr1 KO and WT mice 4 h post-treatment compared to vehicle controls. EEG recordings revealed a reduction in the abnormally enhanced baseline gamma-band power in Fmr1 KO mice and significantly improved evoked synchronization to auditory stimuli in the gamma-band range post-JZL-184 treatment. JZL-184 treatment also ameliorated anxiety-like and hyperactivity phenotypes in Fmr1 KO mice.ConclusionsOverall, these results indicate that increasing 2-AG levels may serve as a potential therapeutic approach to normalize cortical responses and improve behavioral outcomes in FXS and possibly other ASDs.

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.

STEP inhibition reverses behavioral, electrophysiologic, and synaptic abnormalities in Fmr1 KO mice

Synaptic NMDA receptor-mediated currents in anterior piriform cortex are reduced in the adult fragile X mouse

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.).

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

Reduced perineuronal net expression in Fmr1 KO mice auditory cortex and amygdala is linked to impaired fear-associated memory.

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.

Progranulin is an FMRP target that influences macroorchidism but not behaviour in a mouse model of Fragile X Syndrome

Activation of 5-HT7 Serotonin Receptors Reverses Metabotropic Glutamate Receptor-Mediated Synaptic Plasticity in Wild-Type and Fmr1 Knockout Mice, a Model of Fragile X Syndrome

Acute and Repeated Administration of NLX-101, a Selective Serotonin-1A Receptor Biased Agonist, Reduces Audiogenic Seizures in Developing Fmr1 Knockout Mice

ER stress-induced modulation of neural activity and seizure susceptibility is impaired in a fragile X syndrome mouse model

Objective To explore the possible role of Nav1.1 in the pathogenesis of increased susceptibility to epileptic seizures in FMR1 knockout (FMR1 KO) mice. Methods FVB strain FMR1 KO mice and wild type (WT) controls at ages of 2 and 4 weeks old were chosen; immunohistochemistry was used to determine the expression of Nav1.1 in different brain regions (striate cortex, temporal cortex, piriform cortex, hippocampus CA1, CA3 and dentate gyrus), and Western blotting was used to determine the Nav1.1 level in the cerebral acustici cortex and hippocampus. Results The mean optical density of Nav1.1 was significantly increased in the striate cortex, temporal acustici cortex, piriform cortex, regions of CA1 and dentate gyrus in FMR1 KO mice at ages of 2 and 4 weeks (2 weeks: 0.058±0.006, 0.054±0.006, 0.130±0.015, 0.090±0.009 and 0.142±0.010; 4 weeks: 0.066±0.007, 0.060±0.007, 0.159±0.018, 0.102±0.015 and 0.192±0.025) as compared with the age-matched WT mice (2 weeks: 0.049±0.007, 0.046±0.007, 0.118±0.012, 0.080±0.009 and 0.133±0.010; 4 weeks: 0.051±0.007, 0.048±0.005, 0.127±0.012, 0.089±0.012 and 0.175±0.024, P<0.05). The levels of Nav1.1 in the cerebral cortex and hippocampus in FMR1 KO mice at ages of 2 and 4 weeks (2 weeks: 0.635±0.082 and 0.954±0.111; 4 weeks: 0.819±0.064 and 1.145±0.159) were also significantly increased as compared with the age-matched WT mice (2 weeks: 0.382±0.025 and 0.555±0.056; 4 weeks: 0.550±0.040 and 0.847±0.127,P<0.05). Conclusion Increased expression of Nav1.1 might play an important role in the increased susceptibility to epileptic seizures in FMR1 KO mice. Key words: Fragile X syndrome; Nav1.1; Epilepsy; Susceptibility

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