During the early fetal period (8-11PCW), the first fibers from the thalamus and basal forebrain ran sagittally through the expanding intermediate zone and initially dispersed the subventricular zone
Analysis of serial sections through the telencephalon in the youngest group examined (8.5–11 PCW) revealed a well-developed intermediate zone (IZ), situated between the subventricular zone (SVZ), and the presubplate and cortical plate (CP) compartments (Fig. 1). On Nissl-stained sections, the IZ appeared pale; this was due to the richness of fibers and paucity of cell bodies. Fibers in the occipital and frontal portions of the cerebral hemisphere ran in an antero-posterior sagittal direction. Large bundles of cross-sectioned fibers (Fig. 1b, asterix) were separated by strands of migratory neurons which were oriented in a radial direction, respectively, from ventricle to pia (Fig. 1b, arrowheads).
In contrast to the sagittal orientation of fibers in the frontal and occipital cortex, the midlateral regions of the telencephalon contained radially oriented fiber bundles (Fig. 1c, d) which originated from the thalamus, and were directed in a fan-like radiation to the midlateral telencephalon (not shown, but described in our previous publications; Kostović and Goldman-Rakic 1983; Kostovic and Rakic 1984; Krsnik et al. 2017).
At this early fetal age, the SVZ is not “delaminated” from the ventricular zone (VZ), as seen on Nissl-stained-thick celloidin or paraffin sections (Fig. 1a, b), and the distinction of these two proliferative zones was transparent only on very thin sections or on sections stained immunohistochemically for axons. Specifically, fibers entering the IZ showed transient SNAP25 immunoreactivity (Fig. 1c, d). On sections which were immunostained for SNAP25, the external boundary of the IZ (Fig. 1c, IZ) was sharply delineated from the presubplate (Fig. 1d, asterisk) and the VZ stood out as a non-fibrillar layer. The border between sagittal fibers of the IZ and SVZ was not too evident due to the fact that sagittal fibers already ran through the external superficial portion of the SVZ causing the initial stratification of the SVZ (Fig. 1d). Consequently, SVZ was not homogenous on sections immunostained for the neuroepithelial progenitor cell marker SOX2 (Fig. 1e, f) or the general proliferative marker Ki67 (Fig. 1g, h); however, the VZ was well delineated and homogenous (Fig. 1).
In older specimens from this period, there was an increase in the thickness of the IZ (Fig. 2a, b). For the first time, during this phase, it was possible to identify fibers entering in longitudinal and sagittal directions of the IZ and visualize the first division of sagittally running fibers into the deep internal sagittal stratum (ISS) and the superficial external sagittal stratum (ESS) which corresponds to the primordial external capsule (EC) (Fig. 2c). AChE staining of growing thalamic and basal forebrain fibers (Kostović and Rakić 1984; Kostović 1986) revealed that fibers of the ISS ran into the IC and further into the thalamus. Fibers from the ESS were followed into the AChE-reactive basal forebrain (Fig. 2c, arrow). Due to the growth of the callosal fiber system after 10.5 PCW (see Rakic and Yakovlev 1968), we were able to visualize the initial “delamination” of the VZ from the SVZ, into the medial neocortical areas, which were invaded by the initial growth of the corpus callosum (CC).
In vitro MRI scanning (Fig. 2d) of corresponding developmental phases showed basic trilaminar organization (Radoš et al. 2006; see also the three-layer organization described by; Huang et al. 2013), in which the internal lamina (towards the ventricle) was cell dense proliferative VZ/SVZ, the external lamina (towards the pia) was cell dense CP, and the intermediate lamina corresponds to the IZ with sagittal and tangential SS. It is important to note that, on the conventional in vitro MRI, one cannot visualize the border between the SVZ and IZ. Consequently, the exact position of the initial ISS and ESS was not discernible within the IZ. However, by following the exit of the IC (internally) and the external border of the striatum (externally), it was possible to estimate the approximate subpial depth of the initial ISS (Fig. 2d, one arrow) and ESS (Fig. 2d, two arrows), even using in vitro MRI images.
The beginning of the mid-fetal period (12.5–15 PCW): fibers from the axonal SS and CC invaded the proliferative zones
During this restricted period, massive sagittal thalamocortical and cortico-subcortical fibers, emanating from the IC, penetrated through ganglionic eminence and the SVZ, intermingling with proliferative cells (Fig. 3f). At the points where fibers showed radial orientation, we observed that proliferative neurons formed radially oriented strands (Fig. 3f, arrowheads). On more frontal and occipital regions, where fibers showed sagittal orientation, proliferative neurons were dispersed in sublaminas (Fig. 3b). During this period, sagittally running callosal fibers (Fig. 3e, CC) began to penetrate through the deepest portion of the SVZ, adjacent to the VZ, splitting the SVZ into inner and regularly serrated SVZ, juxtaposed to the VZ, and a larger, proper outer SVZ (OSVZ). The periventricular fibers, running between inner SVZ and OSVZ, were described previously by our group (Kostović et al. 2002a), and correspond to the inner fibrillar layer by Smart et al. 2002, and GAP43 and SRGP1 reactive periventricular system described by Molnár and Clowry (2012) in a 15 PCW old brain. We named the splitting of proliferative zones into a deep ventricular contingent (VZ and inner SVZ) and a more superficial contingent, as “delamination”. In current neuroembryological literature, this term is used to describe the process in which neural precursors leave the VZ and become intermediate progenitors (Arai and Taverna 2017).
The expansion of sagittally and tangentially directed fibers (axonal SS) from the IZ also occured on its own superficial (pial) side, where these fibers penetrated into the presubplate zone, invaded the deep portion of the CP, and participated in SP formation. Fibers of both the ESS, originating from the basal forebrain, and prospective thalamic fibers from the ISS participated in an invasion of the deep portion of the CP and SP formation. After this morphogenetic event, the expanded synaptic SP becomes the closest cortical compartment to the ESS, which was clearly visible on sections stained with AChE (Fig. 3d, arrow). Expansion of the SP from the deep loose portion of the CP (Kostović and Rakić 1990; Duque et al. 2016) appeared to ‘‘create’’ more space for the ingrowth of SS fibers, as discernable on in vivo MRI scans showing SP formation (Fig. 3g).
Midgestation (15–22 PCW): spread of fibers from the ESS and ISS to all neocortical regions and the establishment of a new multilaminar axonal-cellular compartment (MACC)
During midgestation, thalamic fibers situated in the ISS, and basal forebrain fibers situated in the ESS, grew tangentially and longitudinally in a sagittal direction towards the frontal and occipital pole (Fig. 4). Separation of the ISS and ESS was clearly visible at the exit of the posterior limb of the IC (PLIC; Fig. 4c, triangle). SS fibers, when approaching the SP, showed an oblique course (Fig. 4d, arrows). The ISS ran in close proximity to the SVZ (Fig. 4d), where AChE-reactive fibers showed loose arrangement. ESS fibers showed two types of fiber arrangement: more superficial fibers were strongly stained with AChE and correspond to the EC, while deeper running fibers were interrupted by radially oriented striations which were not reactive to AChE staining (Fig. 4b, arrowheads). A very interesting band was visible on the outer border of the EC which was not reactive to AChE staining (Fig. 4b, asterisk). During midgestation, the spatial relationship between SS fibers, migratory cell strands, and the glial elements became more complex, particularly in the occipital lobe, and this clearly defined and organized cellular–fibrillar architecture remains present in the later period, around 22 PCW (Fig. 5b, e).
During this period, the ISS intermingled with cells of the OSVZ (terminology introduced by Smart 2002), while the ESS intermingled with outer/external transient cell bands. In general, compartments containing SS fibers were very cellular. The multiple alternation of fibrillar strata and cellular layers leads to profound transformation of the OSVZ and deep IZ, which then jointly formed a new compartment in the cerebral wall. This compartment showed a very characteristic multilaminar organization on Nissl-stained sections (Fig. 5b) and was composed of fibrillar (axonal) and cell layers. We believe that the most appropriate term for this compartment is the “multilaminar axonal-cell compartment” (MACC). The cell density in this new compartment was higher than in the early IZ and the abundance of fibers was more prominent than in early SVZ. The cytoarchitectonics and fibrillar arrangement (Fig. 5b) of the MACC in the occipital lobe was geometrically well defined. Sharp delineation of the occipital ESS (Fig. 5b, marked with 7) was very characteristic and precisely spatially ordered: fiber bundles within this stratum were separated by strands of cells which merged at the external and internal sides of the stratum, with a tangentially oriented outer proliferative cell layer (Fig. 5b, marked with 6) and an external proliferative transient cell band (Fig. 5b, e, marked with 8). Radially oriented strands and sagittally running fibers give this stratum a palisade-like appearance (Smart et al. 2002; Zecevic et al. 2005), corresponding to the outer fibrillar layer, described by Smart et al. (2002) (OFL, discussed later). Thus, the layers of the deep periventricular compartment were defined as follows: (1) ventricular zone; (2) inner SVZ (ISVZ); (3) PVFZ; (4–6) complex cellular/fibrillar stratum composed of OSVZ mixed with fibers of the ISS containing an inner (proliferative) cell layer (4) together with bulk of sagittal fibers (5) which dispersed cells of the OSVZ, and outer (proliferative) cell layer (6); ESS (7) with thalamocortical projection fibers and an outermost fiber stratum (EC) containing fibers from basal forebrain and (8) external (proliferative) transient cell band (Fig. 5b, e). Prospective associative fibers develop along the deep border with the SP (Fig. 5b, asterisk). On in vivo MRI scans, not all layers were visible and well delineated. On Fig. 5c, f, one can see a single T2 hypointense ventricular zone, the PVFZ, and a single T2 hypointense band which probably corresponds to the MACC of the histological section.
Comparison between cytoarchitectonically defined layers and sublayers of the MACC, and the distribution of various glial markers, showed partial compatibility, where the closest compatibility was shown by the prospective radial glia marker PAX6 (Fig. 6e), which showed accumulation in the outer proliferative cell layer (Fig. 6e; arrow; also marked as number 6 on Fig. 5b). Distinct monolayer corresponding to the external transient cell band in medial and distal portions of the occipital lobe (Fig. 6f–i, arrow) shows precise alignment along the growth “corridor” of the ESS and led us to refer to these cells as “corridor cells”. GFAP staining showed stronger immunoreactivity in the ESS, where radial fibers intersect with sagittal-running fibers, leaving fiber-rich zones unstained (Fig. 6b, arrowhead). The microglial marker, CD45, showed alignment of microglia along the roots of the SS (Fig. 6d, arrow) where the SS emanated from the crossroad area (Fig. 6d, asterisk). In contrast to these findings, the oligodendroglial marker, OLIG2 (Fig. 6c), was distributed throughout the axonal SS without laminar preference.
In the developing frontal lobe (Fig. 5d, e), the arrangement of SS fibers and proliferative cellular zones was different and geometrically less strictly defined than in the occipital lobe. The first difference was in the periventricular fiber-rich (callosal) zone (PVFZ). Specifically, massive callosal fibers in the developing genu of corpori callosi surrounded the developing anterior horn of lateral ventricles from the medial, dorsal, and lateral sides. The PVFZ corresponding to the inner fibrillar layer (IFL) described by Smart et al. (2002), was intersected with closely spaced striations composed of cellular elements, previously described as the callosal septa (Jovanov-Milosevic et al. 2009). In the occipital lobe, periventricular callosal fibers formed a sagittally oriented thinner sheet along the lateral wall of the ventricles (fore-runner of the tapetum) but avoided the calcarine area. Furthermore, thalamocortical and cortico-subcortical fibers of the ISS were less compact and almost completely dispersed in the OSVZ (Fig. 5e).
Period corresponding to extremely early preterms (22–28 PCW)
During this period, sagittally running fibers, originating from the thalamus and basal forebrain, accumulated in the superficial SP, below the CP, and gradually penetrated this cell dense layer. The AChE-reactive fibers of the thalamus and basal forebrain, which ‘‘exit’’ from the axonal SS, ran in oblique short trajectories before entering the SP. In the basal portion of the occipital lobe, SS fibers were more compact, and the ESS was close to the ISS (Fig. 7c). In the dorsal portion of the parietooccipital part of brain, the SS is wider as a consequence of widely spaced ISS and ESS (Fig. 7b). The exact arrangement of different classes of projection, commissural, and associative fibers within the SS was difficult to determine on regular histological sections stained with Nissl, but this arrangement was more obvious on preparations stained with AChE. The position of AChE-reactive fibers from the pulvinar (Kostović and Rakić 1984) and basal forebrain (Kostović 1986) was visualized with histochemical AChE staining (Fig. 7). AChE-unreactive commissural CC bordered the medial side of the ISS and OSVZ. It was situated along the roof of the ventricle, was of a large size, formed a ‘‘callosal plate’’, and was easily identified (Fig. 7a, CC). Fibers from the pulvinar occupied a medial position within the occipital SS, while fibers from the basal forebrain formed the EC, within the most external part of the SS (Kostović and Rakić 1984). Research has shown that primary visual projection is well developed by that time (Hevner 2000; Vasung et al. 2017), but this is not reactive to AChE staining during the early preterm period, had already formed a three-dimensional loop (Kostović and Rakić 1984; Krsnik et al. 2017), and represents a prominent component of the SS. The position of efferent, corticofugal fibers within the axonal SS is difficult to determine without tracing methods. In the brains of experimental animals, it has been shown that efferent fibers occupy a deeper position in ‘‘fetal’’ white matter (Bicknese et al. 1994; Molnár et al. 1998). This corresponds to the classical rules (Sachs 1892), which state that, during development, long, earlier-developing, fiber systems are closer to the ventricles, while later-developing short pathways are closer to cortex. Corticofugal fibers were not reactive to AChE staining and obviously correspond to AChE-unreactive zones within the SS. They also develop relatively early; corticospinal fibers were found to reach decussation level in the brainstem by 15 PCW and the lower spinal cord by 24 PCW (Eyre et al. 2000; Eyre 2003; Staudt 2007), while corticopontine fibers are an early component of the periventricular fiber system (Vasung et al. 2011). In the superior part of the occipital lobe, there is a narrow zone of AChE unreactivity (Fig. 7b, asterisk), separating the EC and deeper parts of the SP. This zone marks the prospective development of associative fiber bundles.
In general, the multilaminar organization of cell layers and fiber strata (Fig. 8) resembles the organization seen in the midgestational brain in that the VZ is reduced in size, but the OSVZ and ISS remain very cellular. During this period, the distribution of glial and proliferative markers (Fig. 8e–j), and the distribution of migratory neurons, also resemble midgestational organization, where fiber-rich strata alternate with cell-rich zones. Cell strands in the ESS are less pronounced, but this part of the SS still shows a palisade-like appearance. Pallisadic arrangement (radial striations) was visible both on Nissl (Fig. 8b) and AChE staining (Fig. 8d). Proliferative cells were readily found in both the ISS and ESS (Fig. 8i, j), but were rarely seen in the SP. A cell-rich band, situated in the ESS, is more pronounced in the basal, ventral portion of the occipital lobe, and together with a parallel external proliferative layer, appears as two tracks (Fig. 8b, between arrows). The position of this band is an important landmark for visualization of the SS on MRI (Fig. 9a, d, number 3). The stratified appearance of the SS on MRI was enhanced by the differential laminar distribution of glia (Fig. 9b, c, e, f). Microglial distribution (Fig. 9b, c) and GFAP-immunoreactive radial glia (Fig. 9e, f) delineated the crossroad areas (Fig. 9c, f, asterisk; see also Judaš et al. 2005), and these periventricular crossing fibers continue into the SS (Fig. 9c, f double arrow). The spatial distribution of these glial elements enhanced the visualization of layers on MR images (Fig. 9a, d).
The period corresponding to very preterm and moderate-to-late preterm birth (28–36 PCW): white matter was divided into four segments (I–IV) and all major fiber systems were already present in axonal SS
The rapid development of distal white matter segments III and IV (centrum semiovale and gyral white matter; Fig. 10) is primarily related to the development of the associative fiber system (Vasung et al. 2010, 2017; Mitter et al. 2015). In parallel with this process, there is a reduction of SP thickness (Kostović and Rakić 1990) and a reduction in SP volume (Vasung et al. 2016). The lateral delineation of SS towards SP is now significantly changed, in particular in frontal lobe (Fig. 10a, b, CS), due to the development of centrum semiovale (Fig. 10b, III). Centrum semiovale is composed of massive associative cortico-cortical fibers, which intermingle with other projection pathways. Associative fibers are AChE-unreactive, and, therefore, centrum semiovale, which is in the core of cerebral wall is less AChE-stained and stands out between AChE-stained SP and CP on superficial, and EC on the deep side. An important landmark for medial delineation of axonal SS is the sagittal extensions of the CC. A large cross-sectional area of the CC (Fig. 10a, b) stood out on sections stained with AChE, because callosal fibers are AChE-unreactive, which is in contrast to AChE-reactive SS fibers. AChE-reactive fibers originating from the thalamus and the basal forebrain within the occipital and frontal SS form three discrete “tracks”: the internal “track” which borders with the CC and corresponds to the ISS, and two external “tracks” which correspond to the ESS. A comparison between sections stained with Nissl (Fig. 11a, b) and AChE (Fig. 11c, d) showed that transient cell layers, seen on Nissl staining, were partially in congruence with fibers which were reactive to AChE staining. However, no complete compatibility was evident, since AChE-reactive fibers are just one component of the SS. The most reliable identification of all fiber systems is for the PVFZ: these fibers are not AChE-reactive and continue into the massive callosal system at genu and splenial levels. The voluminous PVFZ of the preterm brain may also be delineated on preparations stained with Nissl by virtue of a pale, cell-poor appearance. Transient cell bands were still present along the outer and inner borders of the ESS; the external transient cell band on the outer border (number 8 on Fig. 11b) and a parallel-running cell layer on the inner surface of the ESS (number 6 on Fig. 11b), together with the inner cell layer of the SVZ (number 4 on Fig. 11b), contain proliferative cells, which are important constituents of transient layers and participate in stratification of the occipital SS. However, the overall number of cells which were immunoreactive for SOX2 was reduced compared to the extremely preterm period (not shown). Despite a reduction in the number of proliferative cells, transient cell bands remain as an important landmark of stratification in the occipital SS of the preterm brain and represent the substrate of the “two-track appearance” on occipital lobe sections stained with Nissl (marked as numbers 6 and 8 on Fig. 11b), and resembled the “two-track appearance” of AChE staining. As stated earlier, these two cell bands actually delineate very conspicuous fibrillar ESS (marked as number 7 on Fig. 11b), which shows fewer radial striations compared to the previous period of development, probably caused by the diminishment of migratory cells, which migrate radially through sagittally oriented fibers of the ESS. The laminar organization of the occipital SS leads to their visibility on in vivo MRI scans (Fig. 11e). The occipital SS appear as alternating layers of differing signal intensity on T2-weighted images. From ventricle to SP, and following differing signal intensity, the following strata were visible:  T2 hypointense lamina corresponding to proliferative periventricular zones (Fig. 11e, number 1);  T2 hyperintense deep lamina (Fig. 11e, number 2);  a T2 hypointense band (Fig. 11e, number 3, arrow);  T2 hyperintense lamina which merges with an SP T2 hyperintense compartment (Fig. 11e, number 4). Two T2 hypointense bands surrounded the T2 hyperintense lamina to form a characteristic “triplet” structure (two bands and a central lamina), which could also be identified in all infants born prematurely (aged 26–32 PCW; 13 of 13 examined cases). The signal intensity of the two outer hypointense bands corresponded to the signal intensity of the CP, indicating high packing cellular density, while the central lamina showed signal intensity similar to the white matter. It was difficult to define which microstructural elements visible on Nissl and AChE preparations corresponded to this “triplet” MRI scans, because laminas shown on MRI, due to the limitations of MRI resolution, may include several sublayers, which can only be separated by histological preparations. To be specific, we identified eight sublaminas on Nissl-stained sections during this (Fig. 11b) and earlier developmental stages (Fig. 5), which led us to presume that the MRI “triplet” structure represented the fusion of several cellular and fiber strata. In addition, MRI scans depict the callosal periventricular system (future tapetum) in a variable and inconsistent manner.
Term age: The MACC was replaced by predominantly fibrillar, deep white matter segments. Delineation of the axonal SS was enhanced by the initial myelination, and had a different appearance in normal newborns born at term when compared to normotypic infants born prematurely and scanned at TEA
At term, cellular bands disappeared from the axonal SS and fibrillar component within the MACC, which became more compact rather than its earlier laminated appearance (Fig. 12c). This process was more obvious in the occipital/parietal regions than in the frontal lobe. In the occipital/parietal region, sagittal commissural, projection, and associative fibers ran in parallel, for a long distance along the posterior horn of the lateral ventricles, to form several fibrillar strata. In addition, in the occipital lobe, axonal strata were compressed in dense fiber bundles, and became more compact. The closest stratum to the ventricle is the tapetum; the following stratum streaming from the PLIC, containing thalamocortical radiation, is mixed with efferent pathways. The visual radiation runs within the ventral portion of the ESS. The most superficial are the associative sagittal pathways. The SVZ was no longer visible in sections taken from the frontal lobe due to the massive callosal system, aligned along the thalamocortical and associative fibers, which radiated more superficially and entered the centrum semiovale. Analysis of proliferative markers in the VZ, ISVZ, OSVZ, and SS (Fig. 12e, l) showed resolution of the MACC due to the disappearance of some transient layers, an overall reduction of proliferative activity in the remaining cell layers, and more compact axonal bundles. Only scattered proliferative cells were present in the fiber-rich strata. The VZ and ISVZ were reduced in their extent, and curved along the lateral angle of the anterior horn of the lateral ventricles, forming a “pocket” surrounded by callosal fibers. The VZ, and eventually the SVZ, formed a band along the roof of lateral ventricles, below the transversal portion of the CC and merged with, the so-called, subcallosal zone (Kostović et al. 2002b). In newborns, the appearance of the SS was marked with a new event, the initial myelination of projection fibers, presumably from thalamic nuclei (Fig. 12f, g). At the coronal section of the occipital lobe (Fig. 12f, g), the myelination pattern was very typical; namely, the fibers continuing from the SS, became more compact, forming a more sharply delineated irregular C-shaped trajectory. When approaching the border between areas 17/18, fibers formed a fine plexus in the gyral white matter of area 18 and only a small number of fibers continued into the lower lip of the calcarine fissure. Myelinated fibers in the C-shape sagittal trajectory (Fig. 12f) were very close to the bottom of the calcarine fissure, but did not exit from the stratum and did not enter into the calcarine cortex.
On MRI scans, there were slight differences in appearance of the occipital SS in normal infants born at term and in normotypic infants born prematurely and scanned at TEA. The “triplet” structure was found in all normal term born controls (Fig. 12a) (five out of five cases). On these scans of normal term infants, there was a well-delineated stratum of low T2 signal intensity (Fig. 12a, arrow), which ran parallel to the two strata of high T2 signal intensity. The position and shape of two external tracks within the “triplet” structure corresponded to myelinated stratum, and is marked as an arrow on Fig. 12g. In premature infants, scanned at TEA, SS stratification (the “triplet” structure) was less pronounced on MRI and appeared relatively immature (Fig. 12h). The T2 hypointense band was thin and discontinued and the lower portion of the band was not well delineated (Fig. 12h, arrow). This immature appearance of the “triplet” was found in 8 out of 13 cases (62%). The “triplet” image was missing in two cases (15%) and was poorly delineated in 3 out of 13 cases (23%). Developmental changes on MR scans and in histological structures were best revealed using illustration of landmark phases (Fig. 12). The differences in “triplet” appearance between the occipital SS in preterm infants (Fig. 12a) scanned at TEA and at term (Fig. 12h) were probably caused by three events: an increase in myelination of the visual pathways (compare Fig. 12g, n), the disappearance of transient proliferative bands (compare Fig. 12e, l), and the pronounced compactness of fibers (compare Fig. 12c, j).