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INTRODUCTION: This study aimed to assess the visibility of the indusium griseum (IG) in magnetic resonance (MR) scans of the human fetal brain and to evaluate its reliability as an imaging biomarker of the normality of brain midline development. MATERIAL AND METHODS: The retrospective observational study encompassed T2-w 3T MR images from 90 post-mortem fetal brains and immunohistochemical sections from 41 fetal brains (16-40 gestational weeks) without cerebral pathology. Three raters independently inspected and evaluated the visibility of IG in post-mortem and in vivo MR scans. Weighted kappa statistics and regression analysis were used to determine inter- and intra-rater agreement and the type and strength of the association of IG visibility with gestational age. RESULTS: The visibility of the IG was the highest between the 25 and 30 gestational week period, with a very good inter-rater variability (kappa 0.623-0.709) and excellent intra-rater variability (kappa 0.81-0.93). The immunochemical analysis of the histoarchitecture of IG discloses the expression of highly hydrated extracellular molecules in IG as the substrate of higher signal intensity and best visibility of IG during the mid-fetal period. CONCLUSIONS: The knowledge of developmental brain histology and fetal age allows us to predict the IG-visibility in magnetic resonance imaging (MRI) and use it as a biomarker to evaluate the morphogenesis of the brain midline. As a biomarker, IG is significant for post-mortem pathological examination by MRI. Therefore, in the clinical in vivo imaging examination, IG should be anticipated when an assessment of the brain midline structures is needed in mid-gestation, including corpus callosum thickness measurements.

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Subplate neurons (SpNs) are a heterogeneous neuronal population actively involved in early cortical circuit formation. In rodents, many SpNs survive and form layer 6b. The molecular heterogeneity of SpNs raises the question of whether different subpopulations of SpNs survive through the early postnatal period similarly and whether such diverse SpN populations in the auditory cortex (ACtx) share a common distribution pattern with other sensory systems. To address that, we investigated the expression pattern of multiple specific SpN markers in the ACtx, as well as in the visual (VCtx) and somatosensory (SCtx) cortices as controls, using complexin 3 (Cplx3) antibodies and different SpN-specific Cre-driver mice, such as connective tissue growth factor (CTGF), dopamine receptor D1 (Drd1a), and neurexophilin 4 (Nxph4). We focused on two early time windows in auditory development: (1) during the second postnatal week (PNW) before ear-canal opening and (2) during the third PNW after ear-canal opening. We compared the expression pattern of different SpN markers in ACtx with VCtx and SCtx. At both examined timepoints, Cplx3 and Nxph4 expressing SpNs form the largest and smallest population in the ACtx, respectively. Similar distribution patterns are observable in the VCtx and SCtx during the second PNW but not during the third PNW, for a higher proportion of Drd1a expressing SpNs is detected in the VCtx and CTGF expressing SpNs in the SCtx. This study suggests that different populations of SpNs might contribute differently to the development of individual sensory circuits.

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The deepest layer of the cortex (layer 6b [L6b]) contains relatively few neurons, but it is the only cortical layer responsive to the potent wake-promoting neuropeptide orexin/hypocretin. Can these few neurons significantly influence brain state? Here, we show that L6b-photoactivation causes a surprisingly robust enhancement of attention-associated high-gamma oscillations and population spiking while abolishing slow waves in sleep-deprived mice. To explain this powerful impact on brain state, we investigated L6b's synaptic output using optogenetics, electrophysiology, and monoCaTChR ex vivo. We found powerful output in the higher-order thalamus and apical dendrites of L5 pyramidal neurons, via L1a and L5a, as well as in superior colliculus and L6 interneurons. L6b subpopulations with distinct morphologies and short- and long-term plasticities project to these diverse targets. The L1a-targeting subpopulation triggered powerful NMDA-receptor-dependent spikes that elicited burst firing in L5. We conclude that orexin/hypocretin-activated cortical neurons form a multifaceted, fine-tuned circuit for the sustained control of the higher-order thalamocortical system.

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Association fibers connect different areas of the cerebral cortex over long distances and integrate information to achieve higher brain functions, particularly in humans. Prototyped association fibers are developed to the respective tangential direction throughout the cerebral hemispheres along the deepest border of the subplate during the fetal period. However, how guidance to remote areas is achieved is not known. Because the subplate is located below the cortical surface, the tangential direction of the fibers may be biased by the curved surface geometry due to Sylvian fissure and cortical poles. The fiber length can be minimized if the tracts follow the shortest paths (geodesics) of the curved surface. Here, we propose and examine a theory that geodesics guide the tangential direction of long association fibers by analyzing how geodesics are spatially distributed on the fetal human brains. We found that the geodesics were dense on the saddle-shaped surface of the perisylvian region and sparse on the dome-shaped cortical poles. The geodesics corresponded with the arrangement of five typical association fibers, supporting the theory. Thus, the geodesic theory provides directional guidance information for wiring remote areas and suggests that long association fibers emerge from minimizing their tangential length in fetal brains.

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Anatomists have long expressed interest in neurons of the white matter, which is by definition supposed to be free of neurons. Hypotheses regarding their biochemical signature and physiological function are mainly derived from animal models. Here, we investigated 15 whole-brain human postmortem specimens, including cognitively normal cases and those with pathologic Alzheimer's disease (AD). Quantitative and qualitative methods were used to investigate differences in neuronal size and density, and the relationship between neuronal processes and vasculature. Double staining was used to evaluate colocalization of neurochemicals. Two topographically distinct populations of neurons emerged: one appearing to arise from developmental subplate neurons and the other embedded within deep, subcortical white matter. Both populations appeared to be neurochemically heterogeneous, showing positive reactivity to acetylcholinesterase (AChE) [but not choline acetyltransferase (ChAT)], neuronal nuclei (NeuN), nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d), microtubule-associated protein 2 (MAP-2), somatostatin (SOM), nonphosphorylated neurofilament protein (SMI-32), and calcium-binding proteins calbindin-D28K (CB), calretinin (CRT), and parvalbumin (PV). PV was more richly expressed in superficial as opposed to deep white matter neurons (WMNs); subplate neurons were also significantly larger than their deeper counterparts. NADPH-d, a surrogate for nitric oxide synthase, allowed for the striking morphological visualization of subcortical WMNs. NADPH-d-positive subcortical neurons tended to embrace the outer walls of microvessels, suggesting a functional role in vasodilation. The presence of AChE positivity in these neurons, but not ChAT, suggests that they are cholinoceptive but noncholinergic. WMNs were also significantly smaller in AD compared to control cases. These observations provide a landscape for future systematic investigations.

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Sensory perturbation in one modality results in the adaptive reorganization of neural pathways within the spared modalities, a phenomenon known as "crossmodal plasticity," which has been examined during or after the classic "critical period." Because peripheral perturbations can alter the auditory cortex (ACX) activity and functional connectivity of the ACX subplate neurons (SPNs) even before the critical period, called the precritical period, we investigated if retinal deprivation at birth crossmodally alters the ACX activity and SPN circuits during the precritical period. We deprived newborn mice of visual inputs after birth by performing bilateral enucleation. We performed in vivo widefield imaging in the ACX of awake pups during the first two postnatal weeks to investigate cortical activity. We found that enucleation alters spontaneous and sound-evoked activities in the ACX in an age-dependent manner. Next, we performed whole-cell patch clamp recording combined with laser scanning photostimulation in ACX slices to investigate circuit changes in SPNs. We found that enucleation alters the intracortical inhibitory circuits impinging on SPNs, shifting the excitation-inhibition balance toward excitation and this shift persists after ear opening. Together, our results indicate that crossmodal functional changes exist in the developing sensory cortices at early ages before the onset of the classic critical period.

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Controversy exists as to when conscious pain perception in the fetus may begin. According to the hypothesis of cortical necessity, thalamocortical connections, which do not form until after 24-28 weeks gestation, are necessary for conscious pain perception. However, anesthesiologists and neonatologists treat age-matched neonates as both conscious and pain-capable due to observable and measurable behavioral, hormonal, and physiologic indicators of pain. In preterm infants, these multimodal indicators of pain are uncontroversial, and their presence, despite occurring prior to functional thalamocortical connections, has guided the use of analgesics in neonatology and fetal surgery for decades. However, some medical groups state that below 24 weeks gestation, there is no pain capacity. Thus, a paradox exists in the disparate acknowledgment of pain capability in overlapping patient populations. Brain networks vary by age. During the first and second trimesters, the cortical subplate, a unique structure that is present only during fetal and early neonatal development, forms the first cortical network. In the third trimester, the cortical plate assumes this function. According to the subplate modulation hypothesis, a network of connections to the subplate and subcortical structures is sufficient to facilitate conscious pain perception in the fetus and the preterm neonate prior to 24 weeks gestation. Therefore, similar to other fetal and neonatal systems that have a transitional phase (i.e., circulatory system), there is now strong evidence for transitional developmental phases of fetal and neonatal pain circuitry.

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The development of functionally interconnected networks between primary (S1), secondary somatosensory (S2), and motor (M1) cortical areas requires coherent neuronal activity via corticocortical projections. However, the anatomical substrate of functional connections between S1 and M1 or S2 during early development remains elusive. In the present study, we used ex vivo carbocyanine dye (DiI) tracing in paraformaldehyde-fixed newborn mouse brain to investigate axonal projections of neurons in different layers of S1 barrel field (S1Bf), M1, and S2 toward the subplate (SP), a hub layer for sensory information transfer in the immature cortex. In addition, we performed extracellular recordings in neocortical slices to unravel the functional connectivity between these areas. Our experiments demonstrate that already at P0 neurons from the cortical plate (CP), layer 5/6 (L5/6), and the SP of both M1 and S2 send projections through the SP of S1Bf. Reciprocally, neurons from CP to SP of S1Bf send projections through the SP of M1 and S2. Electrophysiological recordings with multi-electrode arrays in cortical slices revealed weak, but functional synaptic connections between SP and L5/6 within and between S1 and M1. An even lower functional connectivity was observed between S1 and S2. In summary, our findings demonstrate that functional connections between SP and upper cortical layers are not confined to the same cortical area, but corticocortical connection between adjacent cortical areas exist already at the day of birth. Hereby, SP can integrate early cortical activity of M1, S1, and S2 and shape the development of sensorimotor integration at an early stage.

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Subplate neurons are early-born cortical neurons that transiently form neural circuits during perinatal development and guide cortical maturation. Thereafter, most subplate neurons undergo cell death, while some survive and renew their target areas for synaptic connections. However, the functional properties of the surviving subplate neurons remain largely unknown. This study aimed to characterize the visual responses and experience-dependent functional plasticity of layer 6b (L6b) neurons, the remnants of subplate neurons, in the primary visual cortex (V1). Two-photon Ca2+ imaging was performed in V1 of awake juvenile mice. L6b neurons showed broader tunings for orientation, direction, and spatial frequency than did layer 2/3 (L2/3) and L6a neurons. In addition, L6b neurons showed lower matching of preferred orientation between the left and right eyes compared with other layers. Post hoc 3D immunohistochemistry confirmed that the majority of recorded L6b neurons expressed connective tissue growth factor (CTGF), a subplate neuron marker. Moreover, chronic two-photon imaging showed that L6b neurons exhibited ocular dominance (OD) plasticity by monocular deprivation during critical periods. The OD shift to the open eye depended on the response strength to the stimulation of the eye to be deprived before starting monocular deprivation. There were no significant differences in visual response selectivity prior to monocular deprivation between the OD changed and unchanged neuron groups, suggesting that OD plasticity can occur in L6b neurons showing any response features. In conclusion, our results provide strong evidence that surviving subplate neurons exhibit sensory responses and experience-dependent plasticity at a relatively late stage of cortical development.

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Early regional patterning and laminar position of cortical projection neurons is determined by activation and deactivation of transcriptional factors (TFs) and RNA binding proteins (RBPs) that regulate spatiotemporal framework of neurogenetic processes (proliferation, migration, aggregation, postmigratory differentiation, molecular identity acquisition, axonal growth, dendritic development, and synaptogenesis) within transient cellular compartments. Deep-layer projection neurons (DPN), subplate (SPN), and Cajal-Retzius neurons (CRN) are early-born cells involved in the establishment of basic laminar and regional cortical architecture; nonetheless, laminar dynamics of their molecular transcriptional markers remain underexplored. Here we aimed to analyze laminar dynamics of DPN markers, i.e., transcription factors TBR1, CTIP2, TLE4, SOX5, and RBP CELF1 on histological serial sections of the human frontal cortex between 7.5-15 postconceptional weeks (PCW) in reference to transient proliferative, migratory, and postmigratory compartments. The subtle signs of regional patterning were seen during the late preplate phase in the pattern of sublaminar organization of TBR1+/Reelin+ CRN and TBR1+ pioneering SPN. During the cortical plate (CP)-formation phase, TBR1+ neurons became radially aligned, forming continuity from a well-developed subventricular zone to CP showing clear lateral to medial regional gradients. The most prominent regional patterning was seen during the subplate formation phase (around 13 PCW) when a unique feature of the orbitobasal frontal cortex displays a "double plate" pattern. In other portions of the frontal cortex (lateral, dorsal, medial) deep portion of CP becomes loose and composed of TBR1+, CTIP2+, TLE4+, and CELF1+ neurons of layer six and later-born SPN, which later become constituents of the expanded SP (around 15 PCW). Overall, TFs and RBPs mark characteristic regional laminar dynamics of DPN, SPN, and CRN subpopulations during remarkably early fetal phases of the highly ordered association cortex development.

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Human neurodevelopment is characterized by the appearance, development, and disappearance or transformation of various transient structures that underlie the establishment of connectivity within and between future cortical and subcortical areas. Examples of transient structures in the forebrain (among many others) include the subpial granular layer and the subplate zone. We have previously characterized the precise spatiotemporal dynamics of microglia in the human telencephalon. Here, we describe the diversity of microglial morphologies in the subpial granular layer and the subplate zone. Where possible, we couple the predominant morphological phenotype with functional characterizations to infer tentative roles for microglia in a changing neurodevelopmental landscape. We interpret these findings within the context of relevant morphogenetic and neurogenetic events in humans. Due to the unique genetic, molecular, and anatomical features of the human brain and because many human neurological and psychiatric diseases have their origins during development, these structures deserve special attention.

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The cingulate gyrus, as a prominent part of the human limbic lobe, is involved in the integration and regulation of complex emotional, executive, motivational, and cognitive functions, attributed to several functional regions along the anteroposterior axis. In contrast to increasing knowledge of cingulate function in the adult brain, our knowledge of cingulate development is based primarily on classical neuroembryological studies. We aimed to reveal the laminar and cellular development of the various cingulate regions during the critical period from 7.5 to 15 postconceptional weeks (PCW) before the formation of Brodmann type arealization, employing diverse molecular markers on serial histological sections of postmortem human fetal brains. The study was performed by analysis of: (1) deep projection neuron (DPN) markers laminar dynamics, (2) all transient laminar compartments, and (3) characteristic subplate (SP) formation-expansion phase. We found that DPN markers labeling an incipient cortical plate (CP) were the first sign of regional differentiation of the dorsal isocortical and ventral mesocortical belt. Remarkably, increased width of the fibrillar marginal zone (MZ) towards the limbus, in parallel with the narrowing of CP containing DPN, as well as the diminishment of subventricular zone (SVZ) were reliable landmarks of early mesocortical differentiation. Finally, the SP formation pattern was shown to be a crucial event in the isocortical cingulate portion, given that the mesocortical belt is characterized by an incomplete CP delamination and absence of SP expansion. In conclusion, laminar DPN markers dynamics, together with the SVZ size and mode of SP formation indicate regional belt-like cingulate cortex differentiation before the corpus callosum expansion and several months before Brodmann type arealization.

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Mild isolated fetal ventriculomegaly (iFVM) is the most common abnormality of the fetal central nervous system. It is characterized by enlargement of one or both of the lateral ventricles (defined as ventricular width greater than 10 mm, but less than 12 mm). Despite its high prevalence, the pathophysiology of iFVM during fetal brain development and the neurobiological substrate beyond ventricular enlargement remain unexplored. In this work, we aimed to establish the relationships between the structural development of transient fetal brain zones/compartments and increased cerebrospinal fluid volume. For this purpose, we used in vivo structural T2-weighted magnetic resonance imaging of 89 fetuses (48 controls and 41 cases with iFVM). Our results indicate abnormal development of transient zones/compartments belonging to both hemispheres (i.e. on the side with and also on the contralateral side without a dilated ventricle) in fetuses with iFVM. Specifically, compared to controls, we observed enlargement of proliferative zones and overgrowth of the cortical plate in iFVM with associated reduction of volumes of central structures, subplate, and fetal white matter. These results indicate that enlarged lateral ventricles might be linked to the development of transient fetal zones and that global brain development should be taken into consideration when evaluating iFVM.

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Early infantile developmental and epileptic encephalopathies are devastating conditions, generally of genetic origin, but the pathological mechanisms often remain obscure. A major obstacle in this field of research is the difficulty of studying cortical brain development in humans, at the relevant time period in utero. To address this, we established an in vitro assay to study the impact of gene variants on the developing human brain by using living organotypic cultures of the human subplate and neighbouring cortical regions, prepared from ethically sourced, 14-17 post-conception week brain tissue (www.hdbr.org). We were able to maintain cultures for several months, during which time the gross anatomical structures of the cortical plate, subplate and marginal zone persisted, while neurons continued to develop morphologically and form new synaptic networks. This preparation thus permits the study of genetic manipulations and their downstream effects on an intact developing human cortical network. We focused on STXBP1 haploinsufficiency, which is among the most common genetic causes of developmental and epileptic encephalopathy. This was induced using shRNA interference, leading to impaired synaptic function and a reduced density of glutamatergic synapses. We thereby provide a critical proof-of-principle for how to study the impact of any gene of interest on the development of the human cortex.

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The human claustrum is a gray matter structure in the white matter between insula and striatum. Previous analysis found altered claustrum microstructure in very preterm-born adults associated with lower cognitive performance. As the claustrum development is related to hypoxia-ischemia sensitive transient cell populations being at-risk in premature birth, we hypothesized that claustrum structure is already altered in preterm-born neonates. We studied anatomical and diffusion-weighted MRIs of 83 preterm- and 83 term-born neonates at term-equivalent age. Additionally, claustrum development was analyzed both in a spectrum of 377 term-born neonates and longitudinally in 53 preterm-born subjects. Data was provided by the developing Human Connectome Project. Claustrum development showed increasing volume, increasing fractional anisotropy (FA), and decreasing mean diffusivity (MD) around term both across term- and preterm-born neonates. Relative to term-born ones, preterm-born neonates had (i) increased absolute and relative claustrum volumes, both indicating increased cellular and/or extracellular matter and being in contrast to other subcortical gray matter regions of decreased volumes such as thalamus; (ii) lower claustrum FA and higher claustrum MD, pointing at increased extracellular matrix and impaired axonal integrity; and (iii) aberrant covariance between claustrum FA and MD, respectively, and that of distributed gray matter regions, hinting at relatively altered claustrum microstructure. Results together demonstrate specifically aberrant claustrum structure in preterm-born neonates, suggesting altered claustrum development in prematurity, potentially relevant for later cognitive performance.

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This paper discusses possibilities for early detection and early intervention in infants with or at increased risk of neurodevelopmental disorders in low- and middle-income countries (LMICs). The brain's high rate of developmental activity in the early years post-term challenges early detection. It also offers opportunities for early intervention and facilitation of school readiness. The paper proposes that in the first year post-term two early detection options are feasible for LMICs: (a) caregiver screening questionnaires that carry little costs but predict neurodevelopmental disorders only moderately well; (b) the Hammersmith Infant Neurological Examination and Standardized Infant NeuroDevelopmental Assessment (SINDA) which are easy tools that predict neurodisability well but require assessment by health professionals. The young brain's neuroplasticity offers great opportunities for early intervention. Ample evidence indicates that families play a critical role in early intervention of infants at increased risk of neurodevelopmental disorders. Other interventional key elements are responsive parenting and stimulation of infant development. The intervention's composition and delivery mode depend on the infant's risk profile. For instance, in infants with moderately increased risk (e.g., preterm infants) lay community health workers may provide major parts of intervention, whereas in children with neurodisability (e.g., cerebral palsy) health professionals play a larger role.

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Cerebral white matter undergoes a rapid and complex maturation during the early postnatal period. Prior magnetic resonance imaging (MRI) studies of early postnatal development have often been limited by small sample size, single-modality imaging, and univariate analytics. Here, we applied nonnegative matrix factorization, an unsupervised multivariate pattern analysis technique, to T2w/T1w signal ratio maps from the Developing Human Connectome Project (n = 342 newborns) revealing patterns of coordinated white matter maturation. These patterns showed divergent age-related maturational trajectories, which were replicated in another independent cohort (n = 239). Furthermore, we showed that T2w/T1w signal variations in these maturational patterns are explained by differential contributions of white matter microstructural indices derived from diffusion-weighted MRI. Finally, we demonstrated how white matter maturation patterns relate to distinct histological features by comparing our findings with postmortem late fetal/early postnatal brain tissue staining. Together, these results delineate concise and effective representation of early postnatal white matter reorganization.

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Pyramidal neurons with axons that exit from dendrites rather than the cell body itself are relatively common in non-primates, but rare in monkeys and humans.

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During early brain development, the subplate relays thalamocortical afferents to the overlying cortex. Disconnection of thalamic inputs to the prefrontal cortex by lesions of the subplate of the developing prefrontal cortex at early neonatal periods result in adult-onset behavioral abnormalities reminiscent of positive, negative, and cognitive symptoms of schizophrenia. Delayed maturation of γ-amino butyric acid (GABA) function may contribute to certain abnormalities of the prefrontal cortex and clinical manifestations of schizophrenia. Lesions to the subplate have also been implicated in developmental abnormalities of GABA neurotransmission in somatosensory and visual cortices. Therefore, we sought to examine the effects of subplate lesions in the developing prefrontal cortex of rats on the expression of GABA markers [parvalbumin and glutamic acid decarboxylase (GAD67)] and proteins responsible for GABAergic synaptic maturation [potassium-chloride cotransporter (KCC2) and sodium­potassium-chloride cotransporter (NKCC1)]. Lesioned and control rats were sacrificed between postnatal days (P) 5 and 90 and immunolabeled for parvalbumin, GAD67, KCC2, and NKCC1 in the prelimbic area of the prefrontal cortex. We found decreased immunoreactivity of KCC2 on neuronal cell membranes at P11 compared to control rats. However, the overall immunoreactivity of KCC2 and NKCC1 did not differ between lesion and control animals at all time points studied. Lesioned rats also showed decreased expression of parvalbumin, but not GAD67. Our results indicate that mechanisms underlying trafficking and membrane binding of KCC2 may contribute to altered GABA receptor function during development in schizophrenia.

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