GABA_A receptors are heterooligomeric GABA-gated chloride channels constructed from eight classes of subunits exhibiting varying amino acid sequence homologies (α_1–6, β_1–4, γ_1–3, δ, ρ _1–2, π, ε and θ) that produce multiple GABA_A 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 GABA_A 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.

Gabrb3^-/- mice clearly exhibited social deficits, as indicated by a significant reduction in overall social engagement in both sociability and social novelty testing, however, some aspects of social curiosity appear to remain intact. While gabrb3^-/- mice spent less time in the proximity of unfamiliar mice than did their control counterparts (see Fig. 4), we were able to discern from closely examining the direct interactive contact that gabrb3^-/- mice exhibited towards either of the two Stranger mice in the social novelty test, that there was a preference for the novel mouse over the now familiar mouse (compare Figure 3 with Figure 4). The discrepancy between the two measurements can be partially explained by noting that the automated scoring method collects data on total time spent in all three chambers including the central/neutral chamber, whereas the observer scoring method only collects data pertaining to direct social engagement (nose touching or sniffing within 2 cm of the unfamiliar mouse) as opposed to just being in the social proximity of the unfamiliar mouse. If one examines the total amount of direct social engagement that each mouse genotype exhibits, one easily discerns that gabrb3^-/- mice displayed an attenuated level of interaction towards unfamiliar mice in general, compared to controls (Figure 5). We also observed differences in the nesting behavior of gabrb3^-/- mice, a behavioral feature associated with abnormalities in social behavior [52] and that has likewise been observed in other mouse models linked to ASD, including Mecp2 gene mutation mice, a model of Rett syndrome and in mice deficient in the Dvl1 gene [46, 48, 52].

As a rodent's social behavior is highly dependent on olfactory cues, one must considered whether olfaction differences between gabrb3^-/- and gabrb3^+/+ may have contributed to the observed differences in social behavior. Indeed, gabrb3^-/- mice have been reported to exhibit subtle differences in olfactory discrimination, however, gabrb3^-/- mice were found to exhibit better odor discrimination than gabrb3^+/+ mice in certain tasks [57]. We also noted that gabrb3^-/- mice were as adept as gabrb3^+/+ mice in locating a buried piece of cookie (data not shown). Although these observations do not eliminate the possibility of an olfaction confound, they do shed doubt on olfactory irregularities as being the primary explanation for the observed social impairment exhibited by gabrb3^-/- mice. Taken together, the deficits that gabrb3^-/- mice exhibit in regards to sociability, social novelty, nest building and previous observations that gabrb3^-/- mouse mothers fail to care for their young [39], add up to strongly indicate that normal social behavior is significantly impaired in gabrb3^-/- mice.

Gabrb3^-/- mice were also found to exhibit significant deficits in a variety of exploratory parameters. The diagrams depicted in Figure 7 represent a typical path that either a gabrb3^+/+ or gabrb3^-/- might pursue. Differences between gabrb3^+/+ and gabrb3^-/- mice can be gleaned from such diagrams, including the hyperactivity typically exhibited by gabrb3^-/- mice (high path density in outer region), low exploration of the novel object (low path density in the inner ring) as well as the stereotypical circling pattern typical of these mice (see arrow Fig. 7). Whereas, gabrb3^+/+ mice exhibit the opposite pattern, lower path density in the outer region and a higher path density in the inner ring containing the novel object, reflecting the higher amount of time spent investigating the object. Compared to control mice, gabrb3^-/- mice took longer to initiate the investigation of a novel object placed in the center of the testing chamber and spent less time devoted to exploring the novel object. Whereas, the total number of contacts a mouse made with the novel object did not appear to be as informative as the total amount of time the mouse actually spent investigating the object (Table 3).

Another component of exploratory behavior is rearing, which is associated with a rodent's motivational state and arousal level when exposed to a novel environment [67]. Consequently, novel stimuli, in all sensory modalities, are highly effective in attracting and focusing attention. Significantly less rearing occurred in gabrb3^-/- compared to gabrb3^+/+ and C57BL/6J, but not 129/SvJ mice (Table 3). However, the duration of an average rearing event in 129/SvJ mice was observed to be much longer than in the other genotypes (Figure 8B), therefore, potentially accounting for the lower rearing number. On the whole, 129/SvJ mice appeared to be more inquisitive, in regards to the exploratory parameters assessed, then the other mouse genotypes. Gabrb3^-/- mice also exhibited a lower rearing frequency and lower mean rearing duration compared to control mice (Fig. 8 A and B). Studies conducted by Aspide et al., [5, 6] have demonstrated that the frequency and duration of rearing episodes exhibited by a rodent placed in a novel environment also indexes the scanning and orienting phase of attention (non-selective attention). The significant reduction in both the frequency and duration of rearing episodes exhibited by the gabrb3^-/- mice, relative to controls (Fig. 8A & B) suggests gabrb3^-/- mice exhibit a deficit in non-selective attention. This is of interest as individuals with ASD often display deficits across many attentional domains, including selective and sustained attention with the inability to shift and orient attention rapidly and accurately among spatial targets and between sensory modalities [2, 35, 75]. This attentional deficit likely contributes to an autistic individuals reduced tendency and motivation to thoroughly explore novel environments [64] and has been suggested to be a contributing factor in the limited social interactions that autistic individuals display toward others [31].

In addition to the above behavioral deficits, gabrb3^-/- mice have been previously found to be poor swimmers, exhibit difficulty walking on grid floors, often run in circles and do not perform as well as controls on the accelerating rotarod [27, 39]. Therefore, one possible explanation for the previously observed deficits and reduction in rearing, may involve changes to the vestibular system of the inner ear, which controls balance and orientation in space. Subsequently, two studies have reported changes in components of the vestibular system of the gabrb3^-/- mouse [42, 49]. While mindful of these observations, the current assessments made were not dependent on high levels of motor coordination or postural control. Furthermore, upon close observation of gabrb3^-/- mice, they were found to be quite capable of rearing and able to groom themselves for extended periods while sitting back on their hindlimbs. Therefore, the moderate motor coordination and potential postural control issues associated with gabrb3^-/- mice may contribute, but are unlikely to be the primary cause of reduced rearing behavior. Another potential confound to the behavioral assessments performed on gabrb3^-/- mice is that these mice are prone to seizures [27, 39]. Therefore, an accumulated seizure load, especially in the older gabrb3^-/- mice could potentially contribute to the observed behavioral differences as well as differences in brain morphology. We are not able to comment on whether there would be differences in the cerebellar hypoplasia between younger and older gabrb3^-/- mice as the cerebellar assessments were all done on mice between 51-52 weeks of age. However, younger gabrb3^-/- mice were found to perform in a similar fashion to older gabrb3^-/- mice in regards to the behaviors assessed in this study (data not shown). Furthermore, as demonstrated in the gabrb3^-/- mouse, GABAergic deficits leads to numerous phenotypic traits including epilepsy, therefore, one would anticipate that ASD cases that exhibit GABAergic deficits may likewise present with epilepsy. Subsequently, about 25% of the ASD population exhibit epilepsy [34] and would also be subject to an accumulated seizure load as they age. This increasing seizure load could also potentially influence the manifestation of the ASD phenotype over the lifetime of this population.

A variety of neuroimaging studies suggest that the cerebellum is involved in more than just motor coordination, but also in a diverse set of higher cognitive functions [45], many of which have been observed to be affected in ASD. These include the processing of spatial information [44, 63], spatial orientation [41], exploratory behavior [62] and shifting attention [1]. The cerebellar vermis, especially lobules VI-VII, have been reported as being abnormal in ASD [64]. Semi-quantitative assessments of the cerebellar vermal regions of gabrb3^-/- mice relative to gabrb3^+/+ mice found significant reductions in the surface areas of lobules II-VII (Fig. 10) with non-significant reductions occurring in lobules VIII-X. As large sections of lobules IX and X mature earlier than lobules VI-VII, the current observation suggests that the developmental aberrations leading to reductions in the surface areas of lobules VI-VII in gabrb3^-/- mice may have, in part, occurred later in vermal development [4, 40]. Similarly, the L1 cell adhesion molecule knockout mouse, which also exhibit hypoplasia of the cerebellar vermis, especially in lobule VI, also displays a reduced tendency to explore unfamiliar environments, novel objects placed within their environments, are in continual motion and display stereotypical circling [30]. It is not clear what the circling pattern in these mice or gabrb3^-/- mice represents, though it is reminiscent of the peripheral circling exhibited by rodents with induced cerebellar lesions. Moreover, it has been noted that there is a proclivity towards spinning (circling) in the ASD population [15]. The observed hypoplasia of the cerebellar vermis of the gabrb3^-/- mice could conceivably contribute to a variety of behavioral deficits observed in gabrb3^-/- mice including poor exploratory performance, reduced rearing, impaired contextual memory, poor motor coordination, hyperactivity, reduced digging, stereotypical circling and hypotonia [27, 37, 39]. It is noteworthy that the reduced exploratory and presence of stereotypical patterns observed in ASD have both been significantly correlated with the magnitude of cerebellar hypoplasia in vermal lobules VI-VII [64]. Furthermore, lobules VI and VII are part of the oculomotor vermis, which controls saccadic eye and head movements [32] and are therefore essential for adequate exploration of the external world. Although, it is compelling to consider that the behavioral deficits exhibited by gabrb3^-/- mice are linked with its cerebellar hypoplasia, disrupting other brain regions can also lead to similar behavioral deficits. For example lesioning of the hippocampus in normal mice likewise results in poor nest building and reduced rearing and exploration [25]. Subsequently, gabrb3^-/- mice also exhibit abnormal GABA_A receptor binding and function in the hippocampus [39, 43]. In addition, we recently discovered that the locus coeruleus (LC) in gabrb3^-/- mice have an expanded plexus of LC dendrites relative to gabrb3^+/+, C57Bl/6J and 129/SvJ mice [37]. This is of potential interest as the LC has also been implicated in processes involved in focusing, orienting, scanning and shifting attention [7, 28, 77]. However, while the cerebellum [3] and hippocampus [33] have both been implicated in ASD, to date, the LC has not been highly investigated in regards to ASD.