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 , 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 , alterations in specific GABAergic receptors [68, 69], changes in markers of the cholinergic system [70, 71], focal increases in interneurons , 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  that are consistent with alterations in serotonergic receptor systems in human autistic brain .
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 . Probst bundles have been previously described in BTBR mice  and in other mouse models of callosal agenesis (for example, netrin1 , NF1a , ddN , RI-I , Emx-1 ) and in human patients with partial agenesis of the corpus callosum . 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 (), modulate axon growth , and prevent axon regeneration . 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 . NG2 cells form synaptic junctions with axons, and participate in glutamatergic [100, 101] and GABAergic  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 . 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 . Many different mouse models exhibit callosal abnormalities , yet only very few reportedly exhibit behavioral deficits resembling autism , 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.  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 , mutant Disc1 transgenic mice  and Reelin knockout mice . Moreover, reduced hippocampal neurogenesis has been reported in the Emx1 knockout mouse, a model that also exhibits callosal agenesis . Intriguingly, Disc1 truncation produces callosal abnormalities in mice , and Disc1 single nucleotide polymorphisms are associated with autism, with the strongest association occurring in males . Reduced hippocampal neurogenesis is associated with stress, depression and defects in cognitive function ; the observation that stress abnormalities have been reported in BTBR mice  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 , 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  and 5-hydroxytryptamine 5-HT6 receptor selective antagonists  and AMPA receptor modulators  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  suggests the potential translational relevance of at least one neuropathology finding in the present study.