Clowry GJ

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Received: 25 September 2023 Revised: 16November 2023 Accepted: 27 February 2024DOI: 10.1002/cne.25605COMMEN TAR YIs there a consensus on the location and composition of thehuman subplate?Gavin J. ClowryBiosciences Institute and Centre for Transformative Neuroscience, Newcastle University, Newcastle upon Tyne, UKCorrespondenceGavin J. Clowry, Biosciences Institute, Newcastle University, Framlington Place, Newcastle upon TyneNE2 4HH, UK. Email: gavin.clowry@ncl.ac.ukIn 1970, the Boulder Committee described the basic principles of thedevelopment of the CNS, derived from observations on the humanembryonic cerebrum (The Boulder Committee, 1970). Nearly 30 yearslater, Bystron et al. (2008) declared a revised nomenclature wasneeded including a definition of the subplate, which had come to beseen as a distinct and functionally important transient layer. It wasdefined as a distinct neuronal compartment between the cortical plateand the intermediate zone in the developing cerebral cortex, a rel-atively larger and more complicated structure in human than in ourrodent models (Bystron et al., 2008;Wang et al., 2010).Alongwith themarginal zone, the subplate is the earliest site of cor-tical synapse formation (Bayatti et al., 2008; Kostović & Rakic, 1990;Molliver et al., 1973) and is proposed to play a vital role in integratingspontaneous and sensory-driven activity patterns that sculpt the func-tional neuronal networks of themature brain (Molnár et al., 2020). Thisrequires us to find ways of studying its function in human, for instance,in organotypic culture (McLeod et al., 2023), but this also requires usto be sure of where and what the subplate is. The orthodox view isthat the human subplate by 15 postconceptional weeks (PCW) is adistinct and relatively large stratum compared to the cortical plate,containing a reduced density of neurons even in the upper subplateand an increased density of axons, synapses, and extracellular matrix(Kostović et al., 2019). It appears that this orthodoxy has been chal-lenged without discussion by the annotation of human brain sectionsin the BrainSpan Atlas of the Developing Human Brain (ADHB) pro-vided by the Allen Institute (http://www.brainspan.org/) and describedbyDing et al. (2022). I would argue that this annotation couldmisdirectresearchers new to this area of research.In the atlas, at 15 PCW, it appears that the thickness of thecortical plate has been underestimated, and the boundaries of thesubplate and intermediate zone have been misplaced. This contentionis based on many articles that have used immunostaining and otherThis is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, providedthe original work is properly cited.© 2024 The Authors. The Journal of Comparative Neurology published byWiley Periodicals LLC.histological approaches (acetylcholinesterase [AChE] histochemistry,Alcian Blue staining) for molecular markers, rather than relying ona combination of in situ hybridization and cytoarchitectonics. In situhybridization may reveal which cells potentially express a protein, butimmunohistochemistry tells us exactly where the protein is. Localiz-ing proteins to growing axons, synapses, the extracellular matrix, orradial glial fibers, for instance, can provide important clues as to theboundaries of compartments consisting primarily of axons and otherprocesses.The principal features of the subplate are immunostaining forthe presynaptic protein synaptophysin and the growth cone markerGAP43, which provides a reliable method for revealing the stronglystained subplate and intermediate zone, as well as the unstained cor-tical plate (Bayatti et al., 2008). By this method, the width of thecortical plate in cross section can be estimated to be around 800 µm,and the subplate to be 1000 µm, at 15 PCW in the dorsolateral cor-tex (Figure 1A; Kostović et al., 2019). Ip et al. (2011) immunostainedsections for transcription factors that marked neurons in cortical lay-ers V and VI and the upper subplate alongside GAP43. From thesepreparations, at 15 PCW, the width of the cortical plate in dorsolateralfrontal cortex can be estimated as 800 µm (Figure 1B). More recently,Junaković et al. (2023) used anti-synaptophysin immunostaining andAlcianBlue (extracellularmatrix [ECM]) staining to define the subplate,showing that the unstained cortical plate has a width of 600–800 µmfrom 14 PCW onward in dorsolateral cortex. Finally, T1-weightedstructural MRI of postmortem fixed brains is able to distinguish theboundary between the cell-dense cortical plate and ECM-dense, andthus hydrophilic, subplate to identify lamina boundaries (Radoš et al.,2006). Using such an approach, Bayatti et al. (2008) demonstratedthat at 14.5 PCW, the cortical plate is approximately 700-µm wide,and the subplate is 1000-µm wide in the dorsolateral parietal cortex(Figure 1C).J Comp Neurol. 2024;532:e25605. wileyonlinelibrary.com/journal/cne 1 of 4https://doi.org/10.1002/cne.256052 of 4 CLOWRYF IGURE 1 Panel A shows a cross section through the wall of the dorsolateral cortex at 15 PCW immunostained for synaptophysin (SYN). Theunstained cortical plate (CP) is sandwiched between the intensely immunoreactivemarginal zone (MZ) and subplate (SP). The CP can be estimatedas having a radial width of 800 µm, while the SPmeasures 1100 µm. Panel B showsmultiple adjacent sections through the cortical wallimmunostained for a variety of markers for cortical layers and axons to the same scale as panel A. The boundary between layers V and VI can bededuced, along with the boundary with the SP. Again, the CP can be estimated as having a width of 800 µm. Panel C shows a coronal sectionthrough the fetal parietal cortex at 14.5 PCW from a T1-weightedMRI scan. SP is clearly visible as having a low signal intensity due to its lowcellular density and high water content. The CP can be estimated to be 700-µmwide and the SP to be 1000-µmwide. Panel D shows an annotateddiagram from the BrainSpan Atlas of the Developing Human Brain (AHDB; https://www.brainspan.org/static/atlas) that underestimates the widthof both the CP and SP compared to panels A–C, and thusmislocates the SP and intermediate zone (IZ). Panel D shows a corresponding Nisslsection from the ADHB to the same scale but annotated by the author to show the current consensus for the location of CP and SP. IFL, inner fiberlayer; MACC, multilaminar axon axonal cellular compartment; PCW, postconceptional weeks; SG, subgranular layer; SVZ, subventricular zone.Panel A is adapted fromKostović et al. (2019) with the permission of the publisher; panels B and C are adapted from Ip et al. (2011) and Bayattiet al. (2008) under a Creative Commons license. Scale bars: (A, B) 200 µm; (C) 5mm; (D, E) 1mm.By comparison, Ding et al. (2022) identify the cortical plate at thisstage as a narrow band of very densely packed cells, approximately300-µm wide, and this is how the reference sections in the AHDB areannotated (Figure 1D,E). I would interpret the layer referred to as cor-tical plate as containing neurons that have recently migrated to thecortical plate and have the identity of layer V neurons (SOX5, SATB2,ROBO1, and SRGAP1 expressing, according to Ip et al., 2011). Thisinterpretation is backed up by in situ hybridization data provided byDing et al. (2022), showing this layer expresses LMO4, a layer Vmarker(Harb et al., 2016).Ding et al. (2022) label the subplate as a slightly less cell-denselayer beneath the cortical plate about 400 µm in width (Figure 1D,E).I would interpret this layer as being part of the cortical plate corre-sponding to established layer VI neurons and maybe some deep layer 10969861, 2024, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/cne.25605 by Newcastle University, Wiley Online Library on [19/03/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons LicenseCLOWRY 3 of 4F IGURE 2 Panels A–C are adapted from the BrainSpan Atlas of the Developing Human Brain (ADHB) (https://www.brainspan.org/static/atlas)and compare an annotated diagram of the dorsolateral cortex at 21 PCW (A) with adjacent AChE (B) and Nissl-stained sections (C). Theannotations in panels B and C have been provided by the author. It can be seen in panel B that there is a bundle of AChE+ presumptivethalamocortical fibers (arrowheads) more commonly interpreted as forming the boundary between the intermediate zone (IZ) and subplate (SP),but according to panel A, they would form the boundary between the subventricular zone (SVZ) and IZ. Arrows in in panel Cmark an alignment ofcells along fiber tracks emanating from (and to) the internal capsule (IC) located deeper than the AChE+ fibers and forming the IZ. The SP, on theother hand, is easily recognized as a largely homogenous cell sparse region of the section. Panel D shows an example of an Alcian Blue-stainedsection from anterior cerebral cortex. This staining clearly distinguishes subplate from the IZ as well as the CP. The SP is approximately three timesthe width of the CP in this location at this developmental stage. Panel D is adapted fromDuque et al. (2016) with the permission of the publisher.AChE, acetylcholinesterase; EC, external capsule; PVf, periventricular fiber-rich zone; SG, subgranular layer; VZ, ventricular zone; PCW,postconceptional weeks. Scale bars: (A–C) 2mm; (D) 500 µm.V neurons (Figure 1B; Ip et al., 2011). It does not fit the previouslyaccepted definition of the subplate as a cell-sparse region (Bystronet al., 2008; Kostović et al., 2019). It is widely acknowledged that thesubplate seen at 15 PCW is the product of a condensation of the lowerlevels of layer VI with the presubplate, a layer consisting of older neu-rons thatwere part of the preplate prior to splitting of the preplate intomarginal zone and subplate by formation of the cortical plate (Duqueet al., 2016). Lower layer VI is infiltratedwith ingrowing axons separat-ing the neurons out. Possibly this idea has been over applied by Dinget al. (2022) who have incorporated all of layer VI into the subplate.I would contend that the subplate in the reference diagrams ofthe AHDB at 15 PCW is actually mislabeled as the intermediate zone(Figure 1D), whereas the intermediate zone is designated as the outerfiber zone, which is described as a fiber-rich, cell-sparse layer of theouter subventricular zone (SVZ). This bears resemblance to the multil-aminar axonal cellular compartment (MACC; Figure 1A) described byŽunić Išasegi et al. (2018), which is essentially the lower part of theintermediate zone where axons mingle with neuroprogenitors of theouter SVZ. My interpretation of the reference Nissl section providedby the ADHB is depicted in Figure 1E and envisages a larger corticalplate and subplate and smaller intermediate zone.Interestingly, in the set of referencediagramsprovidedby theADHBat 21 PCW, the dorsolateral cortical plate is more accurately repre-sented as a layer around 900- to 1000-µm thick (Figure 2A), similar toprevious estimations (Kostović & Rakic, 1990; Kostović et al., 2019).However, the subplate is defined as a lamina thinner than the corti-cal plate, rather than three to five times wider as previously described(Kostović & Rakic, 1990; Kostović et al., 2019; Radoš et al., 2006). Isuggest that the layer described as intermediate zone in the ADHBis actually part of the subplate, and the true intermediate zone hasbeen subsumed by their interpretation of the SVZ. This is supportedby examination of the AChE (Figure 2B) and Nissl-stained sections(Figure 2C) supplied by the ADHB alongside the diagrammatic rep-resentation. Both stains are homogenous in appearance across thedesignated SP and IZ, whereas there are clear differences in cytoar-chitecture in laminae across the SVZ. A thin band of strongly AChE+fibers, presumably thalamocortical fibers (Krsnik et al., 2017), formsthe outer margin of the intermediate zone (Figure 2B, arrowheads),with Nissl staining revealing alignment of cells along fiber tracks ema-nating from and to the internal capsule. These structures fall within theboundary of the SVZ in the diagram supplied by the ADHB (Figure 2A),but I would interpret these structures as forming the intermediatezone (Figure 2B,C). Alcian Blue staining provides an excellent methodfordistinguishing subplate from intermediate zone (Figure2D, adaptedfromDuque et al., 2016), showing the subplate to be amore prominentstructure than either the intermediate zone or cortical plate. 10969861, 2024, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/cne.25605 by Newcastle University, Wiley Online Library on [19/03/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License4 of 4 CLOWRYIn conclusion, the ADHB’s interpretation of lamina boundaries inthe developing human cortical wall is at odds with the consensusview, yet this is not discussed in the article by Ding et al. (2022) thatserves as a guide to the atlas and its accompanying database of in situhybridization images. With the advent of in utero scanning as a meansto detect prenatal lesions and abnormalities and predict neurodevel-opmental outcomes, and spatial transcriptomics data that can localizegeneexpression to regions of the corticalwall, it is importantweunder-stand and agree on the location of the subplate, a structure believed toguide the development of the cerebral cortexCONFLICT OF INTEREST STATEMENTThe author declares no conflicts of interest.DATA AVAILABILITY STATEMENTData sharing is not applicable to this article as no new data werecreated or analyzed in this studyORCIDGavin J. Clowry https://orcid.org/0000-0002-6489-8329PEER REVIEWThe peer review history for this article is available at https://publons.com/publon/10.1002/cne.25605REFERENCESBayatti, N., Moss, J. A., Sun, L., Ambrose, P., Ward, J. F., Lindsay, S., &Clowry, G. J. (2008). 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Journal of Comparative Neurology, 532, e25605.https://doi.org/10.1002/cne.25605 10969861, 2024, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/cne.25605 by Newcastle University, Wiley Online Library on [19/03/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

Is there a consensus on the location and composition of the human subplate?

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Is there a consensus on the location and composition of the human subplate?

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