Background
Autism is a behaviorally defined neurodevelopmental disorder consisting of impairment in reciprocal social interactions, deficits in communication, and restrictive and repetitive patterns of behaviors and interests. Preclinical and clinical advancement of novel therapies for the treatment of autism spectrum disorders (ASDs) would be greatly augmented by the existence of translational animal models for preclinical testing. Because the diagnostic and clinical outcomes of autism are based solely on behavioral outcome measures, neurobehavioral assessments have been a key focus of preclinical animal models. Several recent mouse models, generated using both forward and reverse genetic approaches, exhibit one or more behavioral deficits characteristic of autism [
1‐
7].
It is widely acknowledged that the use of rodents to model complex neuropsychiatric disorders has significant limitations. Indeed, some neuroanatomic substrates that have been implicated in autism (for example, regions of the brain such as the fusiform gyrus [
8] and cell types such as von Economo neurons [
9]) are not present in rodents. However, in other complex central microtubule associated disorders, novel drug candidates can be tested in genetically defined mouse models to advance disease-modifying treatments. For example, genetically modified mice overexpressing amyloid plaques can be used to test novel drug candidates, despite the fact that these mice do not exhibit other cardinal features of human Alzheimer disease such as neurofibrillary tangles and neurodegeneration. With regard to autism, current human molecular genetics suggest that no single gene defines susceptibility to idiopathic autism [
10]. Although several single-gene mouse models of monogenic forms of ASD exhibit behavioral deficits of autism [
6,
11], it is likely that no single-gene mouse model will be reliable for evaluating novel therapeutic candidates. Indeed, even in more homogenous single gene disorders such as fragile × syndrome, recent studies report heterogeneity in molecular signatures, which may translate to differential treatment responses [
12].
An alternative and equally attractive approach is to employ both forward and reverse genetic mouse models in drug development for ASD. Mouse models of specific chromosomal aberrations represent a viable and appealing strategy for phenotypic characterizations in psychiatric diseases [
4,
13]. Inbred mouse strains can be powerful tools to decipher genes that confer susceptibility to distinct behavioral phenotypes. For example, forward genetics combined with quantitative trait loci mapping has defined specific loci that underlie a phenotype of behavioral despair [
14,
15]. Such approaches enhance confidence in the predictive validity of animal models for complex human diseases.
The BTBR inbred mouse recapitulates the three core behavioral features that define ASD, including deficits in social interactions as juveniles and adults, unusual vocalizations as infants, and repetitive stereotyped behaviors [
3,
6,
16,
17]. This strain is unique in that few mouse strains effectively model all three behavioral characteristics of autism [
18]. More recent data in this model suggest that pharmacotherapy and behavioral intervention can reverse specific subdomains of the deficits [
6,
19‐
22]. One striking feature of the BTBR mouse is the complete absence of a corpus callosum. Agenesis of the corpus callosum in the BTBR model represents the most robust and fully penetrant callosal defect in any strain identified to date [
23]. Other mouse strains display either callosal dysgenesis (partial reduction in callosal anatomy (for example,
Disc1 truncation mice [
24]) or less penetrant agenesis phenotypes (for example, in BALB/c mice, 10 to 18% of mice show callosal agenesis, and the incidence is gender-specific [
25]). Because the callosal agenesis phenotype is fully penetrant in BTBR mice, this model represents a unique opportunity to explore the consequences of callosal abnormalities on brain structure and function. There is strong evidence that callosal abnormalities may be a crucial component associated with the core behavioral deficits of ASD. Human callosal abnormalities are rather common, with a reported frequency of 0.7 to 5.3% in the USA. Although many cases are asymptomatic, many patients born with callosal agenesis reportedly have cognitive and social impairments [
26,
27]. Despite the fact that most idiopathic cases of ASD do not show complete callosal agenesis, reductions in the volume of the corpus callosum is one of the most consistent neuroimaging finding in the brains of patients with autism [
28‐
31]. A recent study showing that the corpus callosum undergoes changes in response to experience-dependent plasticity in the adult brain [
32] suggests that alterations in white-matter microstructure can be modified by experience, and may be amenable to treatment. Such findings support mechanistic studies aimed at understanding the relationship between callosal abnormalities and ASD.
One approach to investigating the consequences of callosal defects is to characterize cellular and molecular changes in preclinical models, in which confounding factors such as postmortem delay and antemortem medications can be controlled. There is a paucity of reports describing the precise neuroanatomic, neurophysiological and neurochemical profile of the BTBR mouse. Neuroanatomic changes described to date in the BTBR strain are probably associated with absence of corpus callosum, and include the presence of Probst bundles [
20], reduced hippocampal commissure [
23] and an increased number of unmyelinated axons in the anterior commissure [
33]. Functional changes reported in the BTBR model include augmented stress [
34] and abnormal hippocampal neurophysiology [
35,
36].
We hypothesized that more complete histopathological characterization of the BTBR brain would reveal abnormal cellular and anatomic features that may correlate with the behavioral deficits and callosal abnormalities. In this study, we profiled a panel of neuronal, glial, synaptic and plasticity markers in the brains of BTBR and age-matched control mice. The markers were chosen to investigate neuropathologies that have been implicated in autism. The control inbred strain chosen for investigation, the B6 mouse, exhibits high levels of sociability, a low level of repetitive behaviors, and an intact corpus callosum [
18,
37]. Particular emphasis was placed on the forebrain limbic system, given the intimate relationship between the development of the corpus callosum and the limbic system, and the involvement of limbic system structures in ASD [
38]. The relationship between behavioral deficits and neuropathologic findings in the BTBR model is discussed in terms of relevance to the translatability of the model to ASD.
Discussion
We report extensive histopathological characterization of the brain of the BTBR inbred mouse strain, and describe novel changes in specific markers within key forebrain regions. Specifically, a significant increase in the expression of the oligodendrocyte precursor NG2 in the ACC and marked reductions in the number of neural precursors positive for DCX, PSA-NCAM and NeuroD in the hippocampus were seen. Despite the presence of complete callosal agenesis, surprisingly few changes in the majority of markers were found in most brain regions (Table
2). Given the number of neuronal-, glial-, synaptic- and neurotransmitter-related markers we examined, the modest extent of global changes in neurostructural proteins in response to such a marked perturbation of normal brain development is striking. No evidence of structural or antigenic changes was seen in most brain regions using markers such as MAP2 for neuronal dendrites, Timm staining for mossy fibers, and AchE histochemistry for cholinergic pathways, and no specific changes in the expression of excitatory (VGluT1) or inhibitory (GAD67, GAD65, PVA) markers were seen. Such results do not exclude the possibility that subtle changes might be found using higher-resolution approaches such as electron microscopy. Moreover, non-histologic assays such as neurochemistry and electrophysiology represent complimentary mechanistic approaches for addressing the contribution of functional deficits to the autism-like behaviors seen in the BTBR mouse.
Our intent was to profile a panel of diverse cellular and molecular markers in the BTBR mouse as opposed to addressing one or a few specific mechanistic hypotheses. Given that the BTBR model is proposed to mimic the human behavioral impairments of autism, the relevance of our findings to human neuropathology of autism is worth consideration. The current understanding of the neuropathology of human idiopathic autism is based on relatively limited numbers of cases. The most consistent findings reported to date include defective cortical minicolumns [
61], reduction in neuron number and size in key brain areas, and increased dendritic spine density [
38,
62‐
66]. Additional findings include loss of Purkinje cells [
67], alterations in specific GABAergic receptors [
68,
69], changes in markers of the cholinergic system [
70,
71], focal increases in interneurons [
72], and increased glial activation [
73‐
75]. Based on this information, some comparisons with our present findings include lack of changes in brain weight, and absence of changes in cholinergic fiber density and PVA-expressing interneurons in BTBR brain, at least as analyzed qualitatively. These results suggest that differences exist between the pathology of BTBR and human autism. Another difference is that we found no evidence of glial activation in the BTBR brain. The gliosis seen in postmortem examination of human autism cases might have arisen from environmental, inflammatory (both of which have been implicated in autism) or other epigenetic mechanisms that are not present in the mouse model. Additionally, seizures, which occur in up to 30% of patients with ASD [
76,
77], or concomitant drug therapy can alter glial morphology or activation [
78,
79]. These confounding factors cannot be controlled for in human postmortem studies, and pose particular challenges in ASD, for which a paucity of human postmortem brain tissue is available. Such factors re-emphasize the need for relevant translational animal models for ASD. Future studies using more comprehensive evaluations such as spine morphology, minicolumn assessments and receptor autoradiography represent suitable techniques to compare the neuropathology of the BTBR mouse more directly with postmortem human autistic brain. Indeed, receptor autoradiography in BTBR mice has revealed neurochemical changes in the serotonergic system [
22] that are consistent with alterations in serotonergic receptor systems in human autistic brain [
80].
In +the present study, we defined specific cellular and anatomic changes in glia, a population of cells that would be expected to change as a consequence of white matter defects. There was reduced expression of the myelin markers MBP and CNPase in midline forebrain structures, findings that would be predicted from reduced white matter content and callosal agenesis. Likewise, misorientation of glial fibers in the alveus and cingulum are probably a consequence of callosal agenesis, given that it is known that existing fibers can be rerouted in different orientations. Alterations in CNPase and MBP in focal regions of the cortex may be related to the presence of Probst bundles, which have been defined as aberrant, longitudinally oriented, white matter bundles near the midline that are believed to be composed of rerouted callosal fibers [
27]. Probst bundles have been previously described in BTBR mice [
20] and in other mouse models of callosal agenesis (for example, netrin1 [
81], NF1a [
82], ddN [
83], RI-I [
84], Emx-1 [
52]) and in human patients with partial agenesis of the corpus callosum [
85]. The relationship of the small ectopic white-matter bundles in the ACC to Probst bundles is unclear, and additional experiments, such as fiber tract tracing experiments, which were historically used to define Probst bundles [
86,
87], may be illuminating. Fiber tracing experiments may also help characterize the misoriented glial fibers in the alveus and cingulum. CNPase and MBP immunoreactive areas in regions of ectopic white matter bundles were devoid of the synaptic antigens synaptophysin, PSD95, VGluT1, synapsin 2 and drebrin. This is expected, given that the ectopic structures are composed of white matter. However, it is noteworthy that disruption of synaptic cytoarchitecture and integrity in the ACC may lead to significant functional consequences for higher-order functions such as attention, executive function, and integration of emotion and cognitive processes. In autism, abnormalities in the ACC have been identified using neuroimaging methods [
88‐
91]. Notably, the ACC is reported to display anatomic and functional alterations in several human neuropsychiatric conditions, including schizophrenia and bipolar disease, in addition to autism [
92‐
95]. Translational research of higher order functional circuits using endpoints such as neuroimaging and electroencephalography in both mouse and human callosal agenesis/dysgenesis and in patients with ASD may address key hypotheses about the relationship between ACC alterations and neuropsychiatric conditions.
When comparing the relative changes of all the markers evaluated in our study, the most robust changes were seen for DCX, PSA-NCAM, BDNF and NG2 expression. It is of particular interest that these markers are known to play a role in neuronal development and plasticity. Selective changes in such a panel of neurodevelopmental proteins are consistent with a congenital defect in neurodevelopment. The chondroitin sulfate proteoglycan NG2 is known to regulate cell proliferation and motility ([
96]), modulate axon growth [
97], and prevent axon regeneration [
98]. NG2 cells in both white and gray matter engage not only in the genesis of oligodendrocytes during development, but also in remyelination of demyelinated axons in the adult nervous system [
99]. NG2 cells form synaptic junctions with axons, and participate in glutamatergic [
100,
101] and GABAergic [
102] signaling with neurons. Very little is known about the changes in oligodendrocyte precursors in disease states, with the exception of multiple sclerosis, in which NG2 has been suggested to prevent remyelination [
103]. Whether the increased expression of NG2-positive cells represents an attempt to remyelinate the absent corpus callosum, or may function to participate in defective neuron-glia communication is unknown. Future experiments aimed at quantifying the number of NG2 positive cells and the changes in NG2 positive processes may further elucidate the mechanisms of specific alterations in polydendrocytes in this and other models of callosal anomalies. Expression of these specific neuronal and glial progenitors in human postmortem brain from cases of autism or callosal agenesis has not been reported to date. This finding represents an important area for future investigation in human postmortem brain, and is an example of how the BTBR mouse model has the potential to uncover novel neuroanatomical features of human disease.
The relationship between callosal anomalies and behavioral impairment in mice is complex. Several studies have examined the behavioral phenotypes of mouse models of callosal abnormalities; interestingly, very mild phenotypes have been reported across a wide range of basic behavioral assays [
23]. Many different mouse models exhibit callosal abnormalities [
104], yet only very few reportedly exhibit behavioral deficits resembling autism [
18], indicating that the relationship, as in humans, is not a simple one. The BALB/c mouse is one example other than the BTBR mouse that exhibits social deficits and callosal abnormalities [
1,
23,
105], yet other models of callosal dysgenesis (such as the J1 strain, which is phylogenetically similar to BTBR) do not exhibit impaired social interactions. To address a putative relationship more directly, Yang
et al. [
20] reported that surgical transection of the corpus callosum in B6 mice at postnatal day 7 did not result in the same autism-like behavioral deficits seen in the BTBR model, leading the authors to suggest that callosal abnormalities are not responsible for the behavioral deficits in autism. Similarly, commissurotomy in humans, also termed split-brain or disconnection syndrome, does not result in the same behavioral abnormalities as in cases of callosal agenesis [
27,
106]. Clearly, further studies in animals and in humans are required to directly address the hypothesis that callosal abnormalities contribute to the behavioral deficits of autism.
The most profound changes in the present study occurred in the hippocampus. The hippocampus plays an important role in memory functions, emotional behavior, processing of novel information and integrating social information, all domains affected in ASD. Several relevant genetic mouse models of neuropsychiatric disorders emphasize the potential role of reduced neurogenesis on autism-related behaviors, including chromosome 22q11 deletion [
13], mutant
Disc1 transgenic mice [
24] and
Reelin knockout mice [
107]. Moreover, reduced hippocampal neurogenesis has been reported in the
Emx1 knockout mouse, a model that also exhibits callosal agenesis [
53]. Intriguingly,
Disc1 truncation produces callosal abnormalities in mice [
24], and
Disc1 single nucleotide polymorphisms are associated with autism, with the strongest association occurring in males [
108]. Reduced hippocampal neurogenesis is associated with stress, depression and defects in cognitive function [
109]; the observation that stress abnormalities have been reported in BTBR mice [
34] suggests a putative link.
Reduction in BDNF mRNA in the hippocampus is consistent with the reduction in neurogenesis. Hippocampal BDNF mRNA levels were most dramatically reduced in the dentate gyrus, with less significant reductions noted in the CA1 region. Recently, Silverman
et al. reported reduced levels of BDNF protein in BTBR compared with B6 hippocampus, using biochemical methods [
21], consistent with the present study. These results are also consistent with findings in other models of stress in which reduced neurogenesis is accompanied by changes in BDNF; however, the magnitude of reductions in the BTBR model in our studies is more profound than has been reported under conditions of stress [
110‐
112]. Future experiments evaluating the effects of therapies that regulate neurogenesis and/or BDNF levels represent opportunities to decipher the role of these changes in reversing behavioral abnormalities. Therapeutic targets such as histamine H3 [
113] and 5-hydroxytryptamine 5-HT6 receptor selective antagonists [
114] and AMPA receptor modulators [
21] represent good examples. With respect to relevance to human autism, patients with ASD have volumetric and structural changes in the hippocampus as revealed by neuroimaging approaches [
115,
116]. The recent postmortem study illustrating that impairments in neurogenesis may occur in some cases of human autism [
117] suggests the potential translational relevance of at least one neuropathology finding in the present study.
Competing interests
Eight (DS, SO, EA, HS, RR, BC, MP, DM) of the twelve authors are employees of Pfizer Global Research and Development, which funded this study at the time of submission of the manuscript.
Authors' contributions
DS designed and directed the studies, interpreted data and wrote the manuscript. SO performed and coordinated all histopathology studies, procured samples and tissues, prepared figures and tables, and contributed to data interpretation and preparation of the manuscript. DM evaluated the pathology results of most of the markers used in the study, interpreted results and assisted in preparation of the paper. HS and SO performed the Aperio image analysis, and cut and stained the mossy-fiber sections for specific markers. FD carried out all the free-floating experiments and special stains. MP, BC and RR contributed intellectually to the design and interpretation of the results, and the editing of the manuscript. SS and AT performed the staining and stereology experiments for the neurogenesis measures. VV contributed intellectually to the design and interpretation of the neurogenesis experiments and contributed to the manuscript. The BDNF experiments and data analysis were performed by EA. All authors reviewed the manuscript before submission.