Research report
Gabrb3 gene deficient mice exhibit impaired social and exploratory behaviors, deficits in non-selective attention and hypoplasia of cerebellar vermal lobules: A potential model of autism spectrum disorder

https://doi.org/10.1016/j.bbr.2007.09.009Get rights and content

Abstract

Objective

GABAA receptors play an important regulatory role in the developmental events leading to the formation of complex neuronal networks and to the behaviors they govern. The primary aim of this study was to assess whether gabrb3 gene deficient (gabrb3−/−) mice exhibit abnormal social behavior, a core deficit associated with autism spectrum disorder.

Methods

Social and exploratory behaviors along with non-selective attention were assessed in gabrb3−/−, littermates (gabrb3+/+) and progenitor strains, C57BL/6J and 129/SvJ. In addition, semi-quantitative assessments of the size of cerebellar vermal lobules were performed on gabrb3+/+ and gabrb3−/− mice.

Results

Relative to controls, gabrb3−/− mice exhibited significant deficits in activities related to social behavior including sociability, social novelty and nesting. In addition, gabrb3−/− mice also exhibited differences in exploratory behavior compared to controls, as well as reductions in the frequency and duration of rearing episodes, suggested as being an index of non-selective attention. Gabrb3−/− mice also displayed significant hypoplasia of the cerebellar vermis compared to gabrb3+/+ mice.

Conclusions

The observed behavioral deficits, especially regarding social behaviors, strengthens the face validity of the gabrb3 gene deficient mouse as being a model of autism spectrum disorder.

Introduction

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder characterized by impaired social behaviors, stereotypical or restrictive behavioral patterns, and deficits in language and communication [31]. In addition to the three core deficits, a wide continuum of behavioral phenotypes have also been reported in association with ASD, including cognitive impairment, hyperactivity, epilepsy, motor deficits, attentional deficits, hypotonia and sleep disturbances [2], [11], [47], [74]. To date, no single region of the brain or pathophysiological mechanism has yet been identified as being the cause of ASD. However, the cerebellum, frontal cortex, hippocampus, amygdala and the cerebello-thalamo-cortical circuit have all been implicated in ASD [68]. The neural underpinnings of ASD are also poorly understood, although, there is evidence for a strong genetic component [60], even though no candidate gene has been verified to date. Nevertheless, one part of the genome stands out, chromosomal region 15q11-q13, which has been strongly implicated in ASD via linkage and association studies [50], [51], [56], [70]. Furthermore, maternal duplications of this region remain one of the most common cytogenetic abnormalities found in cases of idiopathic ASD, accounting for about 1–2% of the cases reported [22]. In addition, deletion of this region results in either Angelman syndrome or Prader-Willi syndrome depending from which parent the deletion was inherited. Both syndromes often meet the diagnostic criteria for ASD as defined by DSM-IV [61], [65], [69], [73]. Within this chromosomal region there exists a gene cluster of GABAA receptor subunit genes: GABRB3, GABRA5, and GABRG3, encoding for the GABAA receptor subunits β3, α5, and γ3, respectively.

GABAA receptors are heterooligomeric GABA-gated chloride channels constructed from eight classes of subunits exhibiting varying amino acid sequence homologies (α1–6, β1–4, γ1–3, δ, ρ1–2, π, ɛ and θ) that produce multiple GABAA receptor isoforms with various GABA sensitivities and associated pharmacologies [14], [58]. These subunits exhibit unique regional and temporal distribution within the central nervous system. During development GABAA receptors play a role in proliferation, migration, and differentiation of precursor cells that orchestrate the development of the embryonic brain [9]. A developmental deficiency in any of these roles would adversely effect the temporal ordering of neurogenesis and synaptogenesis, thereby affecting maturation of circuits that are later involved in complex behaviors.

The high prevalence rate of ASD, 1 out of 150 births [20] has prompted an urgency to develop animal models as a fundamental step toward comprehending the complex molecular underpinnings associated with this disorder. A suitable animal model should meet three fundamental criteria (1) face validity: behavioral characteristics should mirror those present in the human disorder; (2) construct validity: similarities in the underlying etiology should exist between the human disorder and the animal model; and (3) predictive validity: the outcome of a treatment regime applied to the animal model should reflect the likely impact on humans with the disorder. While no model can be expected to replicate the full complexity of the human behavioral phenotype, an animal model that exhibits specific behavioral and morphological characteristics typically associated with ASD would be invaluable.

Numerous studies, employing a diverse set of approaches including autoradiographic, molecular biological, and genetics, provide overwhelming support for the role of GABAergic mechanisms in the etiology of ASD [13], [17], [38], [70]. In light of these observations, mice that have a targeted disruption of the mouse equivalent of the human GABRB3 gene, which encodes the β3 subunit of the GABAA receptor, can be argued to possess construct validity in reference to ASD. In addition, gabrb3 gene disrupted (gabrb3−/−) mice exhibit numerous behavioral abnormalities, including many often reported in association with ASD [26], [27], [39]. In this present study we sought to further extend the face validity inherent to the gabrb3−/− mouse relative to behaviors associated with ASD. Given the primacy of its deficit in ASD, special emphasis was placed on the assessment of social behavior. In addition, we assessed other behaviors often impaired in ASD, including exploratory behavior and non-selective attention. Lastly, as the cerebellar vermis has been cited as being abnormal in ASD [64] we performed a simple morphological assessment of this region in gabrb3−/− and gabrb3+/+ mice. In addition, this region has been reported to be crucial to the consolidation of aversely motivated contextual memory, an elementary form of spatial learning [66] and a feature previously found to be disrupted in gabrb3−/− mice [27]. The cerebellum has also been reported to be involved in exploratory behaviors [62], shifting attention [1] and spatial orientation [41], features likely to impact the behaviors being assessed within the current study. In light of the overwhelming evidence implicating the GABAergic system in ASD and the numerous parallels between the ASD phenotype and the phenotype observed in gabrb3−/− mice, this timely study provides a crucial connection between the disruption of the gabrb3 gene and the impairment of social behavior, a key diagnostic component of the ASD phenotype. Insights gained from the current efforts aid in bridging gaps in our understanding of the interconnectiveness between genetics, development and behavioral outcome.

Section snippets

Mice

All mice used in this study were male, consistent with the 4:1 male–female ratio prevalent in autism [29]. Gabrb3 gene knockout mice (gabrb3−/−) and wild-type littermates gabrb3+/+ were produced at the University of Pittsburgh and the Veterans Affairs Palo Alto Health Care System. Techniques used to disrupt the gabrb3 gene have been previously described [39]. C57BL/6J and 129/SvJ mice were obtained from Jackson Laboratory (Davis, CA) at 6–8 weeks of age. All animal protocols conformed to the

Social behavior

A repeated measures ANOVA of the baseline locomotor activity of the four genotypes, gabrb3−/− (n = 6), gabrb3+/+ (n = 6), C57Bl/6J (n = 6) and 129/SvJ (n = 6) mice indicated a significant effect of genotype (F3,20 = 9.55, p < 0.001), with gabrb3−/− mice displaying a significantly higher degree of ambulatory activity than control mice (Table 1), in agreement with previous reports of hyperactivity in gabrb3−/− mice [39]. In addition, following a significant ANOVA (F11,60 = 11.95, p < 0.001), gabrb3−/− mice were

Discussion

The GABRB3 gene is vital to proper brain development and to mature brain function. The consequences of disrupting this gene have been demonstrated in mice, which exhibit numerous abnormalities, many of which have been observed in association with the neurodevelopmental disorders Angelman syndrome and ASD [26], [27], [39]. The present study provides a crucial addition to this body of work by demonstrating that gabrb3−/− mice exhibit deficits in social behavior, a core feature of ASD. In

Acknowledgments

This research was supported by a grant to Timothy M. DeLorey, Ph.D. from the National Institute of Mental Health, MH065393 and a grant to Gregg E. Homanics, Ph.D. from the National Institute on Alcohol Abuse and Alcoholism, AA10422. We would like to acknowledge Thomas Fjallstam and Carolyn Ferguson for expert technical support.

References (79)

  • H. Hara

    Autism and epilepsy: a retrospective follow-up study

    Brain Dev

    (2007)
  • N.S. Harris et al.

    Neuroanatomic contributions to slowed orienting of attention in children with autism.

    Brain Res Cogn Brain Res

    (1999)
  • E. Hashemi et al.

    Gabrb3 gene deficient mice exhibit increased risk assessment behavior, hypotonia and expansion of the plexus of locus coeruleus dendrites.

    Brain Res

    (2007)
  • H. Isumi et al.

    Differential development of the human cerebellar vermis: immunohistochemical and morphometrical evaluation

    Brain Dev

    (1997)
  • C.C. Joyal et al.

    Effects of midline and lateral cerebellar lesions on motor coordination and spatial orientation

    Brain Res

    (1996)
  • J.W. Koo et al.

    Hypoplasia of spiral and Scarpa's ganglion cells in GABA(A) receptor beta(3) subunit knockout mice

    Hear Res

    (2002)
  • M.D. Krasowski et al.

    A deficit of functional GABA(A) receptors in neurons of beta 3 subunit knockout mice

    Neurosci Lett

    (1998)
  • R. Lalonde et al.

    The cerebellum and learning processes in animals.

    Brain Res Brain Res Rev

    (1990)
  • H.C. Leiner et al.

    Cognitive and language functions of the human cerebellum

    Trends Neurosci

    (1993)
  • N. Lijam et al.

    Social interaction and sensorimotor gating abnormalities in mice lacking Dvl1

    Cell

    (1997)
  • A.M. Persico et al.

    Searching for ways out of the autism maze: genetic, epigenetic and environmental clues

    Trends Neurosci

    (2006)
  • L. Petrosini et al.

    Watch how to do it! New advances in learning by observation

    Brain Res Brain Res Rev

    (2003)
  • K. Pierce et al.

    Evidence for a cerebellar role in reduced exploration and stereotyped behavior in autism

    Biol Psychiatry

    (2001)
  • Y. Shao et al.

    Fine mapping of autistic disorder to chromosome 15q11-q13 by use of phenotypic subtypes

    Am J Hum Genet

    (2003)
  • S.T. Sinkkonen et al.

    Mouse models of Angelman syndrome, a neurodevelopmental disorder, display different brain regional GABA(A) receptor alterations

    Neurosci Lett

    (2003)
  • S. Steffenburg et al.

    Autism in Angelman syndrome: a population-based study

    Pediatr Neurol

    (1996)
  • E.J. Van Bockstaele

    Morphological substrates underlying opioid, epinephrine and gamma-aminobutyric acid inhibitory actions in the rat locus coeruleus

    Brain Res Bull

    (1998)
  • J.P. Wisor et al.

    Sleep states and sleep electroencephalographic spectral power in mice lacking the beta 3 subunit of the GABA(A) receptor

    Brain Res

    (2002)
  • G. Allen et al.

    Attentional activation of the cerebellum independent of motor involvement

    Science

    (1997)
  • G. Allen et al.

    Attention function and dysfunction in autism

    Front Biosci

    (2001)
  • J. Altman

    Morphological and behavioral markers of environmentally induced retardation of brain development: an animal model

    Environ Health Perspect

    (1987)
  • A.J. Ayres et al.

    Hyper-responsivity to touch and vestibular stimuli as a predictor of positive response to sensory integration procedures by autistic children

    Am J Occup Ther

    (1980)
  • J.L. Barker et al.

    GABAergic cells and signals in CNS development

    Perspect Dev Neurobiol

    (1998)
  • C. Barthelemy et al.

    Validation of the Revised Behavior Summarized Evaluation Scale

    J Autism Dev Disord

    (1997)
  • G.J. Blatt et al.

    Density and distribution of hippocampal neurotransmitter receptors in autism: an autoradiographic study

    J Autism Dev Disord

    (2001)
  • T.P. Bonnert et al.

    theta, a novel gamma-aminobutyric acid type A receptor subunit

    Proc Natl Acad Sci USA

    (1999)
  • H.S. Bracha et al.

    An automated electronic method for quantifying spinning (circling) in children with autistic disorder

    J Neuropsychiatry Clin Neurosci

    (1995)
  • J. Broida et al.

    Mice: progesterone and the regulation of strain differences in pregnancy-induced nest building

    Behav Neurosci

    (1983)
  • L. Burd et al.

    15-year follow-up of a boy with pyridoxine (vitamin B6)-dependent seizures with autism, breath holding, and severe mental retardation

    J Child Neurol

    (2000)
  • Cited by (171)

    • Sediment quality assessment combining chemical and biological (non)target analysis

      2021, Aquatic Toxicology
      Citation Excerpt :

      The Gamma-aminobutyric acid receptor subunit beta-3 (GABRB3) related to cell differentiation was also the highest enriched gene in HC2 (unique DEGs in embryos exposed to SEJ) associated gene ontology (Table 4). The crucial role of GABRB3 controls the differentiation of embryonic brain precursor cells, and when a mutation occurs in GABRB3, it can lead to behavioral disorders such as the autism spectrum (DeLorey et al., 2008; Warrier et al., 2013). Therefore, it is possible that some cyclic hydrocarbons, mono-aromatic hydrocarbons and heterogeneous hydrocarbons (N-, O-, S-) in SEJ can be associated with neurological and neuronal interruption.

    View all citing articles on Scopus
    View full text