Elsevier

Experimental Neurology

Volume 297, November 2017, Pages 168-178
Experimental Neurology

Research Paper
Decreased surface expression of the δ subunit of the GABAA receptor contributes to reduced tonic inhibition in dentate granule cells in a mouse model of fragile X syndrome

https://doi.org/10.1016/j.expneurol.2017.08.008Get rights and content

Highlights

  • Tonic inhibition is reduced in the dentate gyrus in a model of Fragile X Syndrome.

  • Surface expression of GABAAR δ subunits is significantly reduced in this FXS model.

  • Labeling of the δ subunit is reduced at perisynaptic locations in this FXS model.

  • Decreased surface expression of the δ subunit limits tonic inhibition in this model.

Abstract

While numerous changes in the GABA system have been identified in models of Fragile X Syndrome (FXS), alterations in subunits of the GABAA receptors (GABAARs) that mediate tonic inhibition are particularly intriguing. Considering the key role of tonic inhibition in controlling neuronal excitability, reduced tonic inhibition could contribute to FXS-associated disorders such as hyperactivity, hypersensitivity, and increased seizure susceptibility. The current study has focused on the expression and function of the δ subunit of the GABAAR, a major subunit involved in tonic inhibition, in granule cells of the dentate gyrus in the Fmr1 knockout (KO) mouse model of FXS. Electrophysiological studies of dentate granule cells revealed a marked, nearly four-fold, decrease in tonic inhibition in the Fmr1 KO mice, as well as reduced effects of two δ subunit-preferring pharmacological agents, THIP and DS2, supporting the suggestion that δ subunit-containing GABAARs are compromised in the Fmr1 KO mice. Immunohistochemistry demonstrated a small but statistically significant decrease in δ subunit labeling in the molecular layer of the dentate gyrus in Fmr1 KO mice compared to wildtype (WT) littermates. The discrepancy between the large deficits in GABA-mediated tonic inhibition in granule cells in the Fmr1 KO mice and only modest reductions in immunolabeling of the δ subunit led to studies of surface expression of the δ subunit. Cross-linking experiments followed by Western blot analysis demonstrated a small, non-significant decrease in total δ subunit protein in the hippocampus of Fmr1 KO mice, but a four-fold decrease in surface expression of the δ subunit in these mice. No significant changes were observed in total or surface expression of the α4 subunit protein, a major partner of the δ subunit in the forebrain. Postembedding immunogold labeling for the δ subunit demonstrated a large, three-fold, decrease in the number of symmetric synapses with immunolabeling at perisynaptic locations in Fmr1 KO mice. While α4 immunogold particles were also reduced at perisynaptic locations in the Fmr1 KO mice, the labeling was increased at synaptic sites. Together these findings suggest that, in the dentate gyrus, altered surface expression of the δ subunit, rather than a decrease in δ subunit expression alone, could be limiting δ subunit-mediated tonic inhibition in this model of FXS. Finding ways to increase surface expression of the δ subunit of the GABAAR could be a novel approach to treatment of hyperexcitability-related alterations in FXS.

Introduction

Fragile X syndrome (FXS) is the most common form of inherited cognitive impairment in humans and results from loss of function of the Fmr1 gene that encodes the fragile X mental retardation protein (FMRP) (Kooy et al., 2000). This RNA-binding protein has many functions that include regulation of translation and transport of a subset of mRNAs into the dendrites and, through such functions, influences synapse development and plasticity (Bassell and Warren, 2008, Pfeiffer and Huber, 2009, for reviews). Loss of FMRP function affects multiple neurotransmitter and signaling systems, including the GABA system (D'Hulst and Kooy, 2009, Santoro et al., 2012). While numerous types of alterations in the GABA system have been reported (Braat and Kooy, 2015b, D'Hulst et al., 2009, Paluszkiewicz et al., 2011, for reviews), a reduction in GABAA receptor (GABAAR)-mediated tonic inhibition is particularly intriguing, both as a basic functional change in FXS and as a target for treatment. Tonic inhibition provides a powerful control of neuronal excitability (Brickley and Mody, 2012, Otis et al., 1991, Semyanov et al., 2004), and a decrease in tonic inhibition could contribute to the increased network excitability that is often observed in models of FXS (Gibson et al., 2008, Goncalves et al., 2013) and thus be associated with behavioral changes such as hyperactivity, hypersensitivity to sensory stimuli, and increased seizure susceptibility (Contractor et al., 2015).

Although several GABAAR subunits can be involved in tonic inhibition, the δ subunit is a critical subunit in several major brain regions, including the dentate gyrus, and expression of the δ subunit conveys special properties (Brickley and Mody, 2012). δ Subunit-containing GABAARs are located at extra- and perisynaptic sites, where they are ideally positioned to respond to ambient levels of GABA within the extracellular space or to spillover at the synapse; they have a high affinity for GABA and slow rates of desensitization; and, importantly, they are extremely sensitive to endogenous compounds such as neuroactive steroids (Saxena and Macdonald, 1994, Wei et al., 2003, Wohlfarth et al., 2002). As a result of these properties, δ subunit-containing GABAARs can be modulated continuously to help maintain the general level of excitability of neuronal networks (Belelli et al., 2009, Mtchedlishvili and Kapur, 2006, Olsen and Sieghart, 2008, Sun et al., 2004). Interestingly, the δ subunit mRNA was one of the first mRNAs to be identified as a direct target of FMRP (Miyashiro et al., 2003). If lack of FMRP leads to altered translation or transport of δ subunit mRNA or dysregulation of δ subunit expression and function, such changes could be associated with deficits in tonic inhibition and potentially constitute a key alteration in FXS.

Despite considerable interest in GABAARs in FXS, GABA-mediated tonic inhibition has been studied in only a limited number of brain regions, such as the subiculum and basolateral nucleus of the amygdala (Curia et al., 2009, Olmos-Serrano et al., 2010). Although tonic inhibition is prominent and mediated primarily by δ subunit-containing GABAARs in several major forebrain brain regions such as the dentate gyrus, alterations of δ-mediated tonic inhibition have not been studied in FXS in these regions. Altered tonic inhibition in the dentate gyrus could be quite important in FXS as this region serves as a gateway to the hippocampus and regulates the large amount of incoming information from the entorhinal cortex (Heinemann et al., 1992). Reduced control of granule cell activity in the dentate gyrus can lead to increased excitability throughout the hippocampal circuit and contribute to deficits in learning and memory as well as increased seizure susceptibility, conditions that are found in children with FXS (Berry-Kravis et al., 2010, Hagerman and Stafstrom, 2009, Musumeci et al., 1999).

The major goals of this study were to determine if tonic inhibition is impaired in dentate granule cells in the Fmr1 KO mouse model of FXS and if such changes are accompanied by alterations in the expression and localization of the δ subunit of the GABAAR in the dentate gyrus, using immunolabeling and biochemical methods. The findings suggest that decreased surface expression of the δ subunit, rather than a decrease in δ subunit expression alone, is likely limiting tonic inhibition in the dentate gyrus and add to the growing evidence for deficits in δ subunit-mediated tonic inhibition in FXS. Preliminary reports have been presented previously (Houser et al., 2014, Zhang et al., 2016).

Section snippets

Animals

Fmr1 KO male mice on a C57BL/6 background and their male wild-type (WT) littermates were used for all experiments. Fmr1 KO mice were originally obtained from Dr. William Greenough (University of Illinois at Urbana-Champaign) and a colony established at the University of California, Los Angeles. Genotypes were determined by PCR analysis of DNA extracted from tail samples. After weaning, mice were housed in groups of the same sex under standard laboratory conditions with a 12:12 h light-dark

δ Subunit labeling is slightly lower in the dentate gyrus of Fmr1 KO mice

Patterns of δ subunit immunoperoxidase labeling in the dentate gyrus were compared in Fmr1 WT and KO littermates at PN 14, 21, 28, 35 and 60. In WT animals, the normal pattern of δ subunit labeling in the hippocampus was evident as early as PN14, although the immunoreactivity appeared slightly lower at the young ages and progressively increased at older ages (Fig. 1). Labeling was highest in the molecular layer of the dentate gyrus, where the δ subunit is normally located on dendrites of

Discussion

This study provides the first evidence for decreased neuronal cell surface expression of the δ subunit of the GABAAR in a model of FXS. The major findings of the study are that 1) δ subunit-mediated tonic inhibition in dentate granule cells is substantially lower in Fmr1 KO mice than in WT littermates; 2) surface expression in the δ subunit is reduced in the dentate gyrus of the Fmr1 KO mice, as demonstrated by both biochemical and immunogold labeling studies; and 3) the substantial decrease in

Funding sources

This work was supported by the National Institutes of Health Grants HD067225 and NS075245 (to C.R.H.) and A021213 (to R.W.O.).

Acknowledgements

We thank Werner Sieghart (Medical University of Vienna, Austria) for the generous gift of GABAAR subunit antibodies.

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    1

    Authors contributed equally.

    2

    Present address: Department of Anatomy and Physiology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.

    3

    Present address: The Sainsbury Wellcome Centre for Neural Circuits and Behaviour, University College London, London, W1T 4JG, United Kingdom.

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