Supernumerary neurons within the cerebral cortical subplate in autism spectrum disorders
Graphical abstract
Subplate neurons reside in the white matter (WM) adjacent to the deepest layers of the cerebral cortex (Layer VI). During development these early generated cells play an important role in organizing the cerebral cortex, and establishing connections between cortical regions. Relative to neurotypical subjects (A), an increase in the number of neuronal profiles is found in individuals with autism spectrum disorder (B). The morphological features of these profiles suggest that they belong to the initial population of subplate neurons.
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.
Section snippets
Results
A neuron-specific marker was used to examine the overall subplate neuron density in autistic subjects relative to an age-matched neurotypical group utilizing frozen tissue blocks from parietal lobe region BA9. To further identify these neurons, we also documented their morphological types and proportions in Golgi-stained tissues, and compared these findings to the adult cell complement in neurotypical subjects. This latter approached utilized long-term fixed tissue blocks taken from the
Discussion
There is a large increase in the density of NeuN-positive cell profiles within the subplate zone of the cerebral cortex in individuals diagnosed with autism spectrum disorders. Golgi-Kopsch staining revealed that cells within this zone are predominantly either fusiform or polymorphic and comport with previous descriptions of subplate-type neurons. The lower ratios found in ASD are consistent with ratios found at earlier stages in NT development, but the magnitude of this difference was not
Tissue samples
Both formalin-fixed and frozen cortical tissue blocks were acquired through the Autism Tissue Program, the Harvard Brain Tissue Resource Center, University of Maryland’s Brain and Tissue Bank; the Autism Brain Network, and the University of Miami Brain Endowment Bank. Formalin-fixed tissue blocks included eulaminate isocortex from the parietal (BA 7), frontal (BA 9), and temporal (BA 21) lobes (von Economo and Koskinas, 1925) that were used in a previous golgi study (Hutsler and Zhang, 2010).
Funding
This work was supported by a Bilinski Foundation Fellowship to Dr. Avino.
CRediT authorship contribution statement
Thomas Avino: Conceptualization, Methodology, Software, Writing - original draft, Funding acquisition. Jeffrey J. Hutsler: Conceptualization, Formal analysis, Resources, Supervision, Writing - original draft, Writing - review & editing, Visualization.
Acknowledgments
The authors thank the Autism Tissue Program, the Harvard Brain Tissue Resource Center, University of Maryland Brain and Tissue Bank; Autism Brain Network, and the University of Miami Brain Endowment Bank for their invaluable assistance in collecting the tissues utilized in the present study. We would also like to thank the families of these subjects for generously donating the tissue used in this research. This study would not have been possible without their invaluable contributions. Finally,
References (90)
- et al.
Abnormal cell patterning at the cortical gray-white matter boundary in autism spectrum disorders
Brain Res.
(2010) Autism as a sequence: From heterochronic germinal cell divisions to abnormalities of cell migration and cortical dysplasias
Med. Hypotheses
(2014)- et al.
Cingulate white matter neurons in schizophrenia and bipolar disorder
Biol. Psychiatry
(2009) - et al.
Disrupted neural synchronization in toddlers with autism
Neuron
(2011) - et al.
Interstitial white matter neuron density in the dorsolateral prefrontal cortex and parahippocampal gyrus in schizophrenia
Schizophr. Res.
(2005) - et al.
The changing roles of neurons in the cortical subplate
Front Neuroanat
(2009) - et al.
Analysis of thick brain sections by obverse—Reverse computer microscopy: Application of a new, high clarity Golgi—Nissl stain
J. Neurosci. Methods
(1981) Early human brain development: Starring the subplate
Neurosci. Biobehav. Rev.
(2018)- et al.
Increased dendritic spine densities on cortical projection neurons in autism spectrum disorders
Brain Res.
(2010) - et al.
Sigmoid fits to locate and characterize cortical boundaries in human cerebral cortex
J. Neurosci. Methods
(2013)
Autism as a neural systems disorder: a theory of frontal-posterior underconnectivity
Neurosci. Biobehav. Rev.
Precision in the development of neocortical architecture: From progenitors to cortical networks
Prog. Neurobiol.
Functional connectivity in an fMRI working memory task in high-functioning autism
Neuroimage
Excitation/inhibition imbalance in animal models of autism spectrum disorders
Biol. Psychiatry
Cajal-Retzius cells and the development of the neocortex
Trends Neurosci.
Excitation-inhibition dysbalance as predictor of autistic phenotypes
J. Psychiatr. Res.
Toward a better understanding of neuronal migration deficits in autism spectrum disorders
Front. Cell Dev. Biol.
A reliable Golgi-Kopsch modification
Brain Res. Bull.
Reduced GABAergic action in the autistic brain
Curr. Biol.
The role of the subplate in schizophrenia and autism: A systematic review
Neuroscience
Increased interstitial white matter neuron density in the dorsolateral prefrontal cortex of people with schizophrenia
Biol. Psychiatry
The subplate, a transient neocortical structure: Its role in the development of connections between thalamus and cortex
Annu. Rev. Neurosci.
Human cortical dysplasia and epilepsy: an ontogenetic hypothesis based on volumetric MRI and NeuN neuronal density and size measurements
Cereb. Cortex
The Isocortex of Man
Neurobiological bases of autism-epilepsy comorbidity: a focus on excitation/inhibition imbalance
Eur. J. Neurosci.
Development of the human cerebral cortex: Boulder Committee revisited
Nat. Rev. Neurosci.
Focal cortical dysplasias in autism spectrum disorders
Acta Neuropathol. Commun.
Proliferation and apoptosis in the developing human neocortex
Anat. Rec.
Functional connectivity in a baseline resting-state network in autism
NeuroReport
Interstitial cells of the adult neocortical white matter are the remnant of the early generated subplate neuron population
J. Comp. Neurol.
3D global and regional patterns of human fetal subplate growth determined in utero
Brain Struct. Funct.
Cerebro-cerebellar circuits in autism spectrum disorder
Front. Neurosci.
Cortical underconnectivity coupled with preserved visuospatial cognition in autism: Evidence from an fMRI study of an embedded figures task
Autism Res.
Callosal neurons in the cingulate cortical plate and subplate of human fetuses
J. Comp. Neurol.
Interstitial white matter neurons express less reelin and are abnormally distributed in schizophrenia: towards an integration of molecular and morphologic aspects of the neurodevelopmental hypothesis
Mol. Psychiatry
Widespread disrupted white matter microstructure in autism spectrum disorders
J. Autism Dev. Disord.
Functional synaptic circuits in the subplate during fetal and early postnatal development of the cat visual cortex
J. Neurosci.
Changing patterns of synaptic input to subplate and cortical plate during development of visual cortex
J. Neurophysiol.
The language network in autism: Atypical functional connectivity with default mode and visual regions
Autism Res.
Requirement for subplate neurons in the formation of thalamocortical connections
Nature
Involvement of subplate neurons in the formation of ocular dominance columns
Science
Brain hyper-reactivity to auditory novel targets in children with high-functioning autism
Brain
Neuropathological spectrum of cortical dysplasia in children with severe focal epilepsies
Acta Neuropathol.
Development, evolution and pathology of neocortical subplate neurons
Nat. Rev. Neurosci.
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