Supernumerary neurons within the cerebral cortical subplate in autism spectrum disorders

Highlights

• The cortical subplate is present early in development prior to the cortical plate.

• Neuron densities within the subplate of adult subjects are markedly increased autism.

• The ratio of cell types also indicates the retention of an immature state in ASD.

• Subplate pathology could be an early factor in connectivity alterations in autism.

Abstract

Autism spectrum disorders (ASDs) involve alterations to cortical connectivity that manifest as reduced coordinated activity between cortical regions. The neurons of the cortical subplate are a major contributor to establishing thalamocortical, corticothalamic and corticocortical long-range connections and only a subset of this cell population survives into adulthood. Previous reports of an indistinct gray-white matter boundary in subjects with ASD suggest that the adjacent subplate may also show organizational abnormalities. Frozen human postmortem tissue samples from the parietal lobe (BA7) were used to evaluate white-matter neuron densities adjacent to layer VI with an antibody to NeuN. In addition, fixed postmortem tissue samples from frontal (BA9), parietal (BA7) and temporal lobe (BA21) locations, were stained with a Golgi-Kopsch procedure, and used to examine the morphology of these neuronal profiles. Relative to control cases, ASD subjects showed a large average density increase of NeuN-positive profiles of 44.7 percent. The morphologies of these neurons were consistent with subplate cells of the fusiform, polymorphic and pyramidal cell types. Lower ratios of fusiform to other cell types are found early in development and although adult ASD subjects showed consistently lower ratios, these differences were not significant. The increased number of retained subplate profiles, along with cell type ratios redolent of earlier developmental stages, suggests either an abnormal initial population or a partial failure of the apoptosis seen in neurotypical development. These results indicate abnormalities within a neuron population that plays multiple roles in the developing and mature cerebral cortex, including the establishment of long-range cortical connections.

Introduction

Numerous studies have suggested that autism spectrum disorders (ASDs) involve a disruption of cortical connectivity that results in a reduction of synchronized and coordinated brain activity between regions (Dinstein et al., 2011). This fundamental network change may underlie the behavioral symptomology seen in the disorder (Cherkassky et al., 2006, D’Mello and Stoodley, 2015, Just, 2004, Just et al., 2012, Olivito et al., 2018). Functional magnetic resonance imaging (fMRI) and electroencephalography (EEG) studies support this claim in a number of specific cognitive domains that include language comprehension (Gao et al., 2019, Just, 2004), visuospatial processing (Damarla et al., 2010), theory of mind abilities (Kana et al., 2009), working memory (Koshino et al., 2008, Koshino et al., 2005), and the recognition of emotional faces (Mennella et al., 2017). The cellular substrates that underlie this abnormality are not well documented, although anatomical work in postmortem tissue demonstrates increased synaptic spine densities on certain cortical projection neurons (Hutsler and Zhang, 2010), as well as abnormalities in axon characteristics in the frontal lobe white matter in autism (Zikopoulos and Barbas, 2010). Finally, diffusion tensor imaging methods that are sensitive to the organization of white matter tracts continue to demonstrate reduced anisotropy in ASD subjects (Fitzgerald et al., 2019).

One potential microanatomical substrate of altered connectivity in ASD comes from the examination of the cortical subplate, a neurodevelopmental zone arising from the cortical preplate that in later development lies directly subjacent to cortical layer VI (Bystron et al., 2008, Hutsler and Casanova, 2016). Animal studies have shown that the subplate is critical to the development of both thalamocortical and corticothalamic connections (Kast and Levitt, 2019, McConnell et al., 1994, Shatz and Luskin, 1986, Allendoerfer, 1994). Subplate neurons also play a role in the establishment of cortico-cortical connections, send projections to the contralateral hemisphere, and play an important role in guiding axons into the overlying cortex (deAzevedo et al., 1997, Ghosh et al., 1990, Ghosh and Shatz, 1992). Ablation of the subplate in animals demonstrates a number of resulting irregularities including the failure of ocular dominance column formation, and altered innervation of both the cortex and thalamus (for review see Kanold and Luhmann, 2010). Following their neurodevelopmental role, many neurons of the cortical subplate undergo apoptosis, however a number of neurons persist (Chun and Shatz, 1989; Allendoerfer, 1994) and become embedded into the mature cortical circuitry where they may act as “modulators” or “gatekeepers” of cortical afferents (Friedlander and Torres-Reveron, 2009, Kostovic et al., 2011, Luhmann, 2009). Animal models show that these surviving subplate neurons maintain their axonal and dendritic connections with the overlying cortex (Friauf and Shatz, 1991) and their abnormal presence has been observed in human postmortem samples in a number of neurological disorders.

In individuals with schizophrenia, researchers have demonstrated an increased density of subplate neurons in the dorsolateral prefrontal cortex (Eastwood and Harrison, 2005, Yang et al., 2011), parahippocampal gyrus (Eastwood and Harrison, 2005), and superior temporal gyrus (Eastwood and Harrison, 2003). Kostovic et al. (Kostovic et al., 2011) postulate that an increased presence of inhibitory (GABAergic) subplate neurons in schizophrenia results in excess inhibition and a functional disconnection between the frontal lobe and the limbic system. An increase in the density of superficial white matter neurons has also been demonstrated in the temporal lobe of individuals with seizure disorders (Andres, 2004, Rossini et al., 2011, Thom et al., 2001). The high comorbidity of seizure disorders, along with the proposed theory of an excitatory-inhibitory imbalance in the cerebral cortex (Bozzi et al., 2018, Nelson and Valakh, 2015, Robertson et al., 2016), suggests that ASD might be an additional syndrome involving subplate alterations.

Neuroanatomical evidence for a potential disruption to the subplate in ASD has been suggested by the presence of a diffuse boundary between layer VI and the subcortical white matter which is at least partially driven by excessive cell profiles in the subplate zone (Avino and Hutsler, 2010). Simms et al. (2009) qualitatively noted supernumerary cells in the subplate region of the anterior cingulate cortex in autism and older qualitative descriptions exist from case studies showing a poorly defined transition from cortical gray to white matter (Bailey et al., 1998). Genetic evidence for subplate involvement in the etiology of autism comes from subplate-specific genes in mouse models that are associated with autism (Hoerder-Suabedissen et al., 2013). Despite these varied and suggestive findings, a systematic analysis of this neuronal compartment has yet to be performed in autism (see Serati et al., 2019). Given the roles of the cortical subplate to first construct and then modulate cortical connectivity, the fact that ASD involves disrupted cortical connectivity, and the converging lines of evidence indicating a subplate disruption in autism, we sought to document the presence of neuronal profiles in ASD relative to neurotypical subjects. First, the density of subplate neurons was quantified using a neuron-specific antibody (anti-Neun). Second, we examined the morphological characteristics of these cells to see how they compared to previously described cell types that are found within the subplate. Because the ratio of subplate cell types systematically changes as development proceeds, we also examined the proportions of these morphologically identified cell types as an indicator of the developmental processes that might contribute to alterations in the number of persistent subplate neurons in ASD.