Impaired GABAergic inhibition in the hippocampus of Fmr1 knockout mice
Introduction
Fragile X syndrome is the most common form of inherited intellectual disability with a prevalence of approximately 1 in 5000 (Coffee et al., 2009). In addition to cognitive impairment, patients are characterized by typical facial dysmorphic features, macroorchidism, and several behavioral problems including hyperactivity, enhanced fear and social anxiety, aggression and autistic-like behavior (Abbeduto et al., 2014, Hagerman, 2002, Loesch et al., 2007, Santos et al., 2014, Smith et al., 2012). Moreover, 20% of patients suffer from epileptic seizures (Musumeci et al., 1999). The disease is typically caused by a CGG repeat expansion in the 5′ untranslated region of the fragile X mental retardation 1 (FMR1) gene (Verkerk et al., 1991). Expansion of this repeat above the threshold of 200 units induces hypermethylation of the repeat itself and a CpG island in the associated promoter region, resulting in transcriptional silencing and, consequently, absence of the encoded fragile X mental retardation protein (FMRP) (Pieretti et al., 1991). FMRP is an RNA binding protein that interacts with many neuronal mRNAs and is thought to be involved in the regulation of mRNA transport, translation and stability (Bassell and Warren, 2008, De Rubeis and Bagni, 2010).
Studies in animal models of fragile X syndrome have proven to be essential for unraveling the molecular mechanisms underlying the disease and led to the identification of potential therapeutic targets (Bagni et al., 2012, Braat and Kooy, 2014, Darnell and Klann, 2013, Heulens and Kooy, 2011, Wijetunge et al., 2012). Exaggerated group 1 metabotropic glutamate receptor (mGluR) signaling (Bear et al., 2004) in parallel with impaired GABAergic signaling (Braat and Kooy, 2015a, Braat and Kooy, 2015b) are among the targets identified, suggesting the clinical consequences of the absence of FMRP are at least in part due to a disturbance of the inhibition/excitation balance (Contractor et al., 2015).
Several studies have revealed brain region-specific deficits in the inhibitory GABAergic system of Fmr1 knockout mice (Adusei et al., 2010, D'Antuono et al., 2003, D'Hulst et al., 2006, El Idrissi et al., 2005, Gantois et al., 2006, Kratovac and Corbin, 2013, Vislay et al., 2013). Underexpression was confirmed in patients (D'Hulst et al., 2015). Gamma aminobutyric acid type A (GABAA) receptors are the principle receptors mediating fast inhibition in the central nervous system (Olsen and Sieghart, 2008). These are heteropentameric ligand-gated chloride channels, assembled as a nonrandom combination of the 19 known subunits [α(1–6), β(1–3), γ(1–3), δ, ε, ρ(1–3), θ and π] with further increased variability by alternative splicing (Farrant and Nusser, 2005, Huntsman et al., 1998, Jin et al., 2004, Piton et al., 2013). The resulting distinct subtypes have an unique developmental, regional and (sub)cellular expression pattern, distinct physiological properties and sensitivities to GABA and allosteric modulators (Farrant and Nusser, 2005)(D'Hulst et al., 2009). Interestingly, GABAA receptors are involved in processes such as anxiety, epilepsy, insomnia, depression and learning and memory (Rudolph and Knoflach, 2011), all implicated in fragile X syndrome (D'Hulst and Kooy, 2007).
Of the many open questions around GABA-ergic dysfunction in fragile X syndrome we focused in this study on the functional consequences of the absence of Fmrp on GABAA receptor-mediated phasic inhibition.
Section snippets
Mouse breeding and genotyping
Male Fmr1 knockout mice and wild-type littermates were generated by crossing females heterozygous for the Fmr1 mutation (B6.129P2-Fmr1tm1Cgr/Ant or Fmr1 KO2 backcrossed for more than 20 generations to C57BL/6 J) and C57BL/6 J wild-type males (Charles River, Wilmington, MA, USA). Genotypes were determined by PCR on DNA isolated from tail biopsies (Bakker et al., 1994; Mientjes et al., 2006). All animals were housed in mixed genotype groups of approximately 5 littermates in standard mouse cages
Stimulus-evoked GABAA-ergic inhibition of Fmr1 knockout mice is markedly reduced
We examined functional properties of GABAA-ergic inhibition in the hippocampus of Fmr1 knockout mice using whole-cell voltage clamp recordings of CA1 pyramidal neurons. As shown in Fig. 1A, Fmr1 knockout mice had significantly reduced eIPSC amplitudes in response to stimulation intensities varying from 10 to 100 μA (p = 0.013, RM-ANOVA). An average reduction of about 30% indicated a deteriorated inhibition in Fmr1 knockout mice.
To investigate whether the decreased inhibition was a consequence
Discussion
Proper brain function depends on a correct balance between excitatory and inhibitory signaling (Gatto and Broadie, 2010, Yizhar et al., 2011). Recent studies support the hypothesis that this balance is disturbed in animal models of fragile X syndrome and underlies the cognitive impairment and behavioral abnormalities (Contractor et al., 2015, Gibson et al., 2008, Paluszkiewicz et al., 2011a)(Braat and Kooy, 2015a, Braat and Kooy, 2015b). Deficits of hippocampus-dependent functions are of
Acknowledgements
This work was funded by FWO (G.0D76.14), VIB, Queen Elisabeth Foundation Belgium (FMRE, to C.B), FRAXA, interdisciplinary research grants from KU Leuven (IDO/06/004 and GOA 12/008), BOF-NOI from Antwerp University (to L.R.), the Agency for Innovation by Science and Technology, Flanders (IWT 101164 to S.B.),Telethon and Associazione Italiana Sindrome X Fragile (to C.B.).
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2022, Cell ReportsCitation Excerpt :Importantly, FMRP regulation of GABAARs and tonic inhibition is an interplay of multiple mechanisms, which, in addition to protein-protein interactions reported here, include translational regulation and changes in receptor surface expression. This is evident in reduced expression levels of the GABAAR δ subunit in several brain areas, including the dentate gyrus (Adusei et al., 2010; D'Hulst et al., 2009; D'Hulst et al., 2006; D'Hulst et al., 2015; Olmos-Serrano et al., 2010; Sabanov et al., 2017; Vien et al., 2015; Zhang et al., 2017), and reduced surface levels of the δ subunit reported in GCs (Zhang et al., 2017). In addition, reduced ambient GABA levels due to decreased glutamic acid decarboxylase (GAD) or reduced GABA release have been observed in Fmr1 KO mice (Braat et al., 2015; Davidovic et al., 2011; Olmos-Serrano et al., 2010).
Spontaneous seizures in adult Fmr1 knockout mice: FVB.129P2-Pde6b<sup>+</sup> Tyr<sup>c-ch</sup> Fmr1<sup>tm1Cgr</sup>/J
2022, Epilepsy ResearchCitation Excerpt :Neuroanatomical perturbations in Fmr1 knockout mice include increased dendritic spine density and increased immature spines in pyramidal cells (Ivanco and Greenough, 2002; Liu et al., 2011; Weiler and Greenough, 1999). Also observed in the brains of these mice are increases in the intrinsic activity of glutamate NMDA receptors, decreases in expression of GABAA receptor α-, β-, and δ-subunits, and compromised ion channel functionality, especially potassium and calcium channels, indicating altered electrical properties of brain cells (Contractor et al., 2015; D'Hulst et al., 2006; Deng and Klyachko, 2016; Sabanov et al., 2017; Zhang et al., 2017, 2014). For example, increased membrane excitability or electrical circuit dysfunctions have been reported in the somatosensory cortex (Zhang et al., 2016), the auditory cortex (Lovelace et al., 2020), and the hippocampus (Boone et al., 2018; Luque et al., 2017) of Fmr1 knockout mice.
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These authors contributed equally to this work.