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

Cortical wall of human fetal cerebral cortex (early second trimester immunostained for a synaptic marker [red]) revealing the extent of the subplate, which is considerably wider than the cortical plate at this developmental stage.

In 1970, the Boulder Committee described the basic principles of the development of the CNS, derived from observations on the human embryonic cerebrum (The Boulder Committee, 1970).Nearly 30 years later, Bystron et al. (2008) declared a revised nomenclature was needed including a definition of the subplate, which had come to be seen as a distinct and functionally important transient layer.It was defined as a distinct neuronal compartment between the cortical plate and the intermediate zone in the developing cerebral cortex, a relatively larger and more complicated structure in human than in our rodent models (Bystron et al., 2008;Wang et al., 2010).
Along with the marginal zone, the subplate is the earliest site of cortical synapse formation (Bayatti et al., 2008;Kostović & Rakic, 1990;Molliver et al., 1973) and is proposed to play a vital role in integrating spontaneous and sensory-driven activity patterns that sculpt the functional neuronal networks of the mature brain (Molnár et al., 2020).This requires us to find ways of studying its function in human, for instance, in organotypic culture (McLeod et al., 2023), but this also requires us to be sure of where and what the subplate is.The orthodox view is that the human subplate by 15 postconceptional weeks (PCW) is a distinct and relatively large stratum compared to the cortical plate, containing a reduced density of neurons even in the upper subplate and an increased density of axons, synapses, and extracellular matrix (Kostović et al., 2019).It appears that this orthodoxy has been challenged without discussion by the annotation of human brain sections in the BrainSpan Atlas of the Developing Human Brain (ADHB) provided by the Allen Institute (http://www.brainspan.org/) and described by Ding et al. (2022).I would argue that this annotation could misdirect researchers new to this area of research.
In the atlas, at 15 PCW, it appears that the thickness of the histological approaches (acetylcholinesterase [AChE] histochemistry, Alcian Blue staining) for molecular markers, rather than relying on a combination of in situ hybridization and cytoarchitectonics.In situ hybridization may reveal which cells potentially express a protein, but immunohistochemistry tells us exactly where the protein is.Localizing proteins to growing axons, synapses, the extracellular matrix, or radial glial fibers, for instance, can provide important clues as to the boundaries of compartments consisting primarily of axons and other processes.
The principal features of the subplate are immunostaining for the presynaptic protein synaptophysin and the growth cone marker GAP43, which provides a reliable method for revealing the strongly stained subplate and intermediate zone, as well as the unstained cortical plate (Bayatti et al., 2008).By this method, the width of the cortical 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 cortex (Figure 1A; Kostović et al., 2019).Ip et al. (2011) immunostained sections for transcription factors that marked neurons in cortical layers V and VI and the upper subplate alongside GAP43.From these preparations, at 15 PCW, the width of the cortical plate in dorsolateral frontal cortex can be estimated as 800 µm (Figure 1B).More recently,

Junaković et al. (2023) used anti-synaptophysin immunostaining and
Alcian Blue (extracellular matrix [ECM]) staining to define the subplate, showing that the unstained cortical plate has a width of 600-800 µm from 14 PCW onward in dorsolateral cortex.Finally, T1-weighted structural MRI of postmortem fixed brains is able to distinguish the boundary between the cell-dense cortical plate and ECM-dense, and thus hydrophilic, subplate to identify lamina boundaries (Radoš et al., 2006).Using such an approach, Bayatti et al. (2008) demonstrated that 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).

F I G U R E 1
Panel A shows a cross section through the wall of the dorsolateral cortex at 15 PCW immunostained for synaptophysin (SYN).The unstained cortical plate (CP) is sandwiched between the intensely immunoreactive marginal zone (MZ) and subplate (SP).The CP can be estimated as having a radial width of 800 µm, while the SP measures 1100 µm.Panel B shows multiple adjacent sections through the cortical wall immunostained 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 be deduced, 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 section through the fetal parietal cortex at 14.5 PCW from a T1-weighted MRI scan.SP is clearly visible as having a low signal intensity due to its low cellular density and high water content.The CP can be estimated to be 700-µm wide and the SP to be 1000  2022) identify the cortical plate at this stage as a narrow band of very densely packed cells, approximately 300-µm wide, and this is how the reference sections in the AHDB are annotated (Figure 1D,E).I would interpret the layer referred to as cortical plate as containing neurons that have recently migrated to the cortical plate and have the identity of layer V neurons (SOX5, SATB2, ROBO1, and SRGAP1 expressing, according to Ip et al., 2011).This interpretation is backed up by in situ hybridization data provided by Ding et al. (2022), showing this layer expresses LMO4, a layer V marker (Harb et al., 2016).Ding et al. (2022) label the subplate as a slightly less cell-dense layer beneath the cortical plate about 400 µm in width (Figure 1D,E V neurons (Figure 1B; Ip et al., 2011).It does not fit the previously accepted definition of the subplate as a cell-sparse region (Bystron et al., 2008;Kostović et al., 2019).It is widely acknowledged that the subplate seen at 15 PCW is the product of a condensation of the lower levels of layer VI with the presubplate, a layer consisting of older neurons that were part of the preplate prior to splitting of the preplate into marginal zone and subplate by formation of the cortical plate (Duque et al., 2016).Lower layer VI is infiltrated with ingrowing axons separating the neurons out.Possibly this idea has been over applied by Ding et al. (2022) who have incorporated all of layer VI into the subplate.I would contend that the subplate in the reference diagrams of the AHDB at 15 PCW is actually mislabeled as the intermediate zone (Figure 1D), whereas the intermediate zone is designated as the outer fiber zone, which is described as a fiber-rich, cell-sparse layer of the outer subventricular zone (SVZ).This bears resemblance to the multilaminar axonal cellular compartment (MACC; Interestingly, in the set of reference diagrams provided by the ADHB at 21 PCW, the dorsolateral cortical plate is more accurately repre-sented as a layer around 900-to 1000-µm thick (Figure 2A), similar to previous estimations (Kostović & Rakic, 1990;Kostović et al., 2019).
However, the subplate is defined as a lamina thinner than the cortical plate, rather than three to five times wider as previously described (Kostović & Rakic, 1990;Kostović et al., 2019;Radoš et al., 2006).I suggest that the layer described as intermediate zone in the ADHB is actually part of the subplate, and the true intermediate zone has been subsumed by their interpretation of the SVZ.This is supported by examination of the AChE (Figure 2B) and Nissl-stained sections (Figure 2C) supplied by the ADHB alongside the diagrammatic representation.Both stains are homogenous in appearance across the designated SP and IZ, whereas there are clear differences in cytoarchitecture in laminae across the SVZ.A thin band of strongly AChE+ fibers, presumably thalamocortical fibers (Krsnik et al., 2017) In conclusion, the ADHB's interpretation of lamina boundaries in the developing human cortical wall is at odds with the consensus view, yet this is not discussed in the article by Ding et al. (2022) that serves as a guide to the atlas and its accompanying database of in situ hybridization images.With the advent of in utero scanning as a means to detect prenatal lesions and abnormalities and predict neurodevelopmental outcomes, and spatial transcriptomics data that can localize gene expression to regions of the cortical wall, it is important we understand and agree on the location of the subplate, a structure believed to guide the development of the cerebral cortex

CONFLICT OF INTEREST STATEMENT
The author declares no conflicts of interest.
cortical plate has been underestimated, and the boundaries of the subplate and intermediate zone have been misplaced.This contention is based on many articles that have used immunostaining and other This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.© 2024 The Authors.The Journal of Comparative Neurology published by Wiley Periodicals LLC.
-µm wide.Panel D shows an annotated diagram from the BrainSpan Atlas of the Developing Human Brain (AHDB; https://www.brainspan.org/static/atlas) that underestimates the width of both the CP and SP compared to panels A-C, and thus mislocates the SP and intermediate zone (IZ).Panel D shows a corresponding Nissl section 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 fiber layer; MACC, multilaminar axon axonal cellular compartment; PCW, postconceptional weeks; SG, subgranular layer; SVZ, subventricular zone.Panel A is adapted from Kostović et al. (2019) with the permission of the publisher; panels B and C are adapted from Ip et al. (2011) and Bayatti et al. (2008) under a Creative Commons license.Scale bars: (A, B) 200 µm; (C) 5 mm; (D, E) 1 mm.By comparison, Ding et al. ( ).I would interpret this layer as being part of the cortical plate corresponding to established layer VI neurons and maybe some deep layer F I G U R E 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).The annotations 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+ presumptive thalamocortical 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 C mark an alignment of cells along fiber tracks emanating from (and to) the internal capsule (IC) located deeper than the AChE+ fibers and forming the IZ.The SP, on the other hand, is easily recognized as a largely homogenous cell sparse region of the section.Panel D shows an example of an Alcian Blue-stained section from anterior cerebral cortex.This staining clearly distinguishes subplate from the IZ as well as the CP.The SP is approximately three times the width of the CP in this location at this developmental stage.Panel D is adapted from Duque 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) 2 mm; (D) 500 µm.
Figure 1A) described by Žunić Išasegi et al. (2018), which is essentially the lower part of the intermediate zone where axons mingle with neuroprogenitors of the outer SVZ.My interpretation of the reference Nissl section provided by the ADHB is depicted in Figure 1E and envisages a larger cortical plate and subplate and smaller intermediate zone.
, forms the outer margin of the intermediate zone (Figure 2B, arrowheads), with Nissl staining revealing alignment of cells along fiber tracks emanating from and to the internal capsule.These structures fall within the boundary of the SVZ in the diagram supplied by the ADHB (Figure 2A), but I would interpret these structures as forming the intermediate zone (Figure 2B,C).Alcian Blue staining provides an excellent method for distinguishing subplate from intermediate zone (Figure 2D, adapted from Duque et al., 2016), showing the subplate to be a more prominent structure than either the intermediate zone or cortical plate.