Histological and MRI Study of the Development of the Human Indusium Griseum.

To uncover the ontogenesis of the human indusium griseum (IG), 28 post-mortem fetal human brains, 12-40 postconceptional weeks (PCW) of age, and 4 adult brains were analyzed immunohistochemically and compared with post-mortem magnetic resonance imaging (MRI) of 28 fetal brains (14-41 PCW). The morphogenesis of the IG occurred between 12 and 15 PCW, transforming the bilateral IG primordia into a ribbon-like cortical lamina. The histogenetic transition of sub-laminated zones into the three-layered cortical organization occurred between 15 and 35 PCW, concomitantly with rapid cell differentiation that occurred from 18 to 28 PCW and the elaboration of neuronal connectivity during the entire second half of gestation. The increasing number of total cells and neurons in the IG at 25 and 35 PCW confirmed its continued differentiation throughout this period. High-field 3.0 T post-mortem MRI enabled visualization of the IG at the mid-fetal stage using T2-weighted sequences. In conclusion, the IG had a distinct histogenetic differentiation pattern than that of the neighboring intralimbic areas of the same ontogenetic origin, and did not show any signs of regression during the fetal period or postnatally, implying a functional role of the IG in the adult brain, which is yet to be disclosed.

Coevolution in the timing of GABAergic and pyramidal neuron maturation in primates.

The cortex of primates is relatively expanded compared with many other mammals, yet little is known about what developmental processes account for the expansion of cortical subtype numbers in primates, including humans. We asked whether GABAergic and pyramidal neuron production occurs for longer than expected in primates than in mice in a sample of 86 developing primate and rodent brains. We use high-resolution structural, diffusion MR scans and histological material to compare the timing of the ganglionic eminences (GE) and cortical proliferative pool (CPP) maturation between humans, macaques, rats, and mice. We also compare the timing of post-neurogenetic maturation of GABAergic and pyramidal neurons in primates (i.e. humans, macaques) relative to rats and mice to identify whether delays in neurogenesis are concomitant with delayed post-neurogenetic maturation. We found that the growth of the GE and CPP are both selectively delayed compared with other events in primates. By contrast, the timing of post-neurogenetic GABAergic and pyramidal events (e.g. synaptogenesis) are predictable from the timing of other events in primates and in studied rodents. The extended duration of GABAergic and pyramidal neuron production is associated with the amplification of GABAerigc and pyramidal neuron numbers in the human and non-human primate cortex.

Maintenance of Lognormal-Like Skewed Dendritic Spine Size Distributions in Dentate Granule Cells of TNF, TNF-R1, TNF-R2, and TNF-R1/2-Deficient Mice.

Dendritic spines are sites of synaptic plasticity and their head size correlates with the strength of the corresponding synapse. We recently showed that the distribution of spine head sizes follows a lognormal-like distribution even after blockage of activity or plasticity induction. As the cytokine tumor necrosis factor (TNF) influences synaptic transmission and constitutive TNF and receptor (TNF-R)-deficiencies cause changes in spine head size distributions, we tested whether these genetic alterations disrupt the lognormality of spine head sizes. Furthermore, we distinguished between spines containing the actin-modulating protein synaptopodin (SP-positive), which is present in large, strong and stable spines and those lacking it (SP-negative). Our analysis revealed that neither TNF-deficiency nor the absence of TNF-R1, TNF-R2 or TNF-R 1 and 2 (TNF-R1/R2) degrades the general lognormal-like, skewed distribution of spine head sizes (all spines, SP-positive spines, SP-negative spines). However, TNF, TNF-R1 and TNF-R2-deficiency affected the width of the lognormal distribution, and TNF-R1/2-deficiency shifted the distribution to the left. Our findings demonstrate the robustness of the lognormal-like, skewed distribution, which is maintained even in the face of genetic manipulations that alter the distribution of spine head sizes. Our observations are in line with homeostatic adaptation mechanisms of neurons regulating the distribution of spines and their head sizes.

Fetal development of the human amygdala.

The intricate development of the human amygdala involves a complex interplay of diverse processes, varying in speed and duration. In humans, transient cytoarchitectural structures deliquesce, leading to the formation of functionally distinct nuclei as a result of multiple interdependent developmental events. This study compares the amygdala's cytoarchitectural development in conjunction with specific antibody reactivity for neuronal, glial, neuropil, and radial glial fibers, synaptic, extracellular matrix, and myelin components in 39 fetal human brains. We recognized that the early fetal period, as a continuation of the embryonic period, is still dominated by relatively uniform histogenetic processes. The typical appearance of ovoid cell clusters in the lateral nucleus during midfetal period is most likely associated with the cell migration and axonal growth processes in the developing human brain. Notably, synaptic markers are firstly detected in the corticomedial group of nuclei, while immunoreactivity for the panaxonal neurofilament marker SMI 312 is found dorsally. The late fetal period is characterized by a protracted migration process evidenced by the presence of doublecortin and SOX-2 immunoreactivity ventrally, in the prospective paralaminar nucleus, reinforced by vimentin immunoreactivity in the last remaining radial glial fibers. Nearing the term period, SMI 99 immunoreactivity indicates that perinatal myelination becomes prominent primarily along major axonal pathways, laying the foundation for more pronounced functional maturation. This study comprehensively elucidates the rate and sequence of maturational events in the amygdala, highlighting the key role of prenatal development in its behavioral, autonomic, and endocrine regulation, with subsequent implications for both normal functioning and psychiatric disorders.

Loss of tumor necrosis factor (TNF)-receptor 1 and TNF-receptor 2 partially replicate effects of TNF deficiency on dendritic spines of granule cells in mouse dentate gyrus.

The cytokine tumor necrosis factor (TNF) is involved in the regulation of physiological and pathophysiological processes in the central nervous system. In previous work, we showed that mice lacking constitutive levels of TNF exhibit a reduction in spine density and changes in spine head size distribution of dentate granule cells. Here, we investigated which TNF-receptor pathway is responsible for this phenotype and analyzed granule cell spine morphology in TNF-R1-, TNF-R2-, and TNF-R1/R2-deficient mice. Single granule cells were filled with Alexa568 in fixed hippocampal brain slices and immunostained for the actin-modulating protein synaptopodin (SP), a marker for strong and stable spines. An investigator blind to genotype investigated dendritic spines using deconvolved confocal image stacks. Similar to TNF-deficient mice, TNF-R1 and TNF-R2 mutants showed a decrease in the size of small spines (SP-negative) with TNF-R1/R2-KO mice exhibiting an additive effect. TNF-R1 mutants also showed an increase in the size of large spines (SP-positive), mirroring the situation in TNF-deficient mice. Unlike the TNF-deficient mouse, none of the TNF-R mutants exhibited a reduction in their granule cell spine densities. Since TNF tunes the excitability of networks, lack of constitutive TNF reduces network excitation. This may explain why we observed alterations in spine head size distributions in TNF- and TNF-R-deficient granule cells. The changes in spine density observed in the TNF-deficient mouse could not be linked to canonical TNF-R-signaling. Instead, noncanonical pathways or unknown developmental functions of TNF may cause this phenomenon.

Layer-specific changes of KCC2 and NKCC1 in the mouse dentate gyrus after entorhinal denervation.

The cation-chloride cotransporters KCC2 and NKCC1 regulate the intracellular Cl- concentration and cell volume of neurons and/or glia. The Cl- extruder KCC2 is expressed at higher levels than the Cl- transporter NKCC1 in mature compared to immature neurons, accounting for the developmental shift from high to low Cl- concentration and from depolarizing to hyperpolarizing currents through GABA-A receptors. Previous studies have shown that KCC2 expression is downregulated following central nervous system injury, returning neurons to a more excitable state, which can be pathological or adaptive. Here, we show that deafferentation of the dendritic segments of granule cells in the outer (oml) and middle (mml) molecular layer of the dentate gyrus via entorhinal denervation in vivo leads to cell-type- and layer-specific changes in the expression of KCC2 and NKCC1. Microarray analysis validated by reverse transcription-quantitative polymerase chain reaction revealed a significant decrease in Kcc2 mRNA in the granule cell layer 7 days post-lesion. In contrast, Nkcc1 mRNA was upregulated in the oml/mml at this time point. Immunostaining revealed a selective reduction in KCC2 protein expression in the denervated dendrites of granule cells and an increase in NKCC1 expression in reactive astrocytes in the oml/mml. The NKCC1 upregulation is likely related to the increased activity of astrocytes and/or microglia in the deafferented region, while the transient KCC2 downregulation in granule cells may be associated with denervation-induced spine loss, potentially also serving a homeostatic role via boosting GABAergic depolarization. Furthermore, the delayed KCC2 recovery might be involved in the subsequent compensatory spinogenesis.

Constitutive tumor necrosis factor (TNF)-deficiency causes a reduction in spine density in mouse dentate granule cells accompanied by homeostatic adaptations of spine head size.

The majority of excitatory synapses terminating on cortical neurons are found on dendritic spines. The geometry of spines, in particular the size of the spine head, tightly correlates with the strength of the excitatory synapse formed with the spine. Under conditions of synaptic plasticity, spine geometry may change, reflecting functional adaptations. Since the cytokine tumor necrosis factor (TNF) has been shown to influence synaptic transmission as well as Hebbian and homeostatic forms of synaptic plasticity, we speculated that TNF-deficiency may cause concomitant structural changes at the level of dendritic spines. To address this question, we analyzed spine density and spine head area of Alexa568-filled granule cells in the dentate gyrus of adult C57BL/6J and TNF-deficient (TNF-KO) mice. Tissue sections were double-stained for the actin-modulating and plasticity-related protein synaptopodin (SP), a molecular marker for strong and stable spines. Dendritic segments of TNF-deficient granule cells exhibited ∼20% fewer spines in the outer molecular layer of the dentate gyrus compared to controls, indicating a reduced afferent innervation. Of note, these segments also had larger spines containing larger SP-clusters. This pattern of changes is strikingly similar to the one seen after denervation-associated spine loss following experimental entorhinal denervation of granule cells: Denervated granule cells increase the SP-content and strength of their remaining spines to homeostatically compensate for those that were lost. Our data suggest a similar compensatory mechanism in TNF-deficient granule cells in response to a reduction in their afferent innervation.

Prenatal development of the human entorhinal cortex.

Little is known about the development of the human entorhinal cortex (EC), a major hub in a widespread network for learning and memory, spatial navigation, high-order processing of object information, multimodal integration, attention and awareness, emotion, motivation, and perception of time. We analyzed a series of 20 fetal and two adult human brains using Nissl stain, acetylcholinesterase (AChE) histochemistry, and immunocytochemistry for myelin basic protein (MBP), neuronal nuclei antigen (NeuN), a pan-axonal neurofilament marker, and synaptophysin, as well as postmortem 3T MRI. In comparison with other parts of the cerebral cortex, the cytoarchitectural differentiation of the EC begins remarkably early, in the 10th week of gestation (w.g.). The differentiation occurs in a superficial magnocellular layer in the deep part of the marginal zone, accompanied by cortical plate (CP) condensation and multilayering of the deep part of CP. These processes last until the 13-14th w.g. At 14 w.g., the superficial lamina dissecans (LD) is visible, which divides the CP into the lamina principalis externa (LPE) and interna (LPI). Simultaneously, the rostral LPE separates into vertical cell-dense islands, whereas in the LPI, the deep LD emerges as a clear acellular layer. In the 16th w.g., the LPE remodels into vertical cell-dense and cell-sparse zones with a caudorostral gradient. At 20 w.g., NeuN immunoreactivity is most pronounced in the islands of layer II cells, whereas migration and differentiation inside-out gradients are seen simultaneously in both the upper (LPE) and the lower (LPI) pyramidal layers. At this stage, the EC adopts for the first time an adult-like cytoarchitectural organization, the superficial LD becomes discernible by 3T MRI, MBP-expressing oligodendrocytes first appear in the fimbria and the perforant path (PP) penetrates the subiculum to reach its molecular layer and travels along through the Cornu Ammonis fields to reach the suprapyramidal blade of the dentate gyrus, whereas the entorhinal-dentate branch perforates the hippocampal sulcus about 2-3 weeks later. The first AChE reactivity appears as longitudinal stripes at 23 w.g. in layers I and II of the rostrolateral EC and then also as AChE-positive in-growing fibers in islands of superficial layer III and layer II neurons. At 40 w.g., myelination of the PP starts as patchy MBP-immunoreactive oligodendrocytes and their processes. Our results refute the possibility of an inside-out pattern of the EC development and support the key role of layer II prospective stellate cells in the EC lamination. As the early cytoarchitectural differentiation of the EC is paralleled by the neurochemical development, these developmental milestones in EC structure and connectivity have implications for understanding its normal function, including its puzzling modular organization and potential contribution to consciousness content (awareness), as well as for its insufficiently explored deficits in developmental, psychiatric, and degenerative brain disorders.

Understanding Emotions: Origins and Roles of the Amygdala.

Emotions arise from activations of specialized neuronal populations in several parts of the cerebral cortex, notably the anterior cingulate, insula, ventromedial prefrontal, and subcortical structures, such as the amygdala, ventral striatum, putamen, caudate nucleus, and ventral tegmental area. Feelings are conscious, emotional experiences of these activations that contribute to neuronal networks mediating thoughts, language, and behavior, thus enhancing the ability to predict, learn, and reappraise stimuli and situations in the environment based on previous experiences. Contemporary theories of emotion converge around the key role of the amygdala as the central subcortical emotional brain structure that constantly evaluates and integrates a variety of sensory information from the surroundings and assigns them appropriate values of emotional dimensions, such as valence, intensity, and approachability. The amygdala participates in the regulation of autonomic and endocrine functions, decision-making and adaptations of instinctive and motivational behaviors to changes in the environment through implicit associative learning, changes in short- and long-term synaptic plasticity, and activation of the fight-or-flight response via efferent projections from its central nucleus to cortical and subcortical structures.

Structural Changes in the Cortico-Ponto-Cerebellar Axis at Birth are Associated with Abnormal Neurological Outcomes in Childhood.

White matter lesions in hypoxic-ischemic encephalopathy (HIE) are considered to be the important substrate of frequent neurological consequences in preterm infants. The aim of the study was to analyze volumes and tractographic parameters of the cortico-ponto-cerebellar axis to assess alterations in the periventricular fiber system and crossroads, corticopontine and corticospinal pathways and prospective transsynaptic changes of the cerebellum.Term infants (control), premature infants without (normotypic) and with perinatal HIE (HIE) underwent brain magnetic resonance imaging at term-equivalent age (TEA) and at 2 years. Cerebrum, cerebellum, brainstem divisions and ventrodorsal compartments volumetric analysis were performed, as well as fractional anisotropy (FA) and apparent diffusion coefficient (ADC) of corticopontine, corticospinal pathways and middle cerebellar peduncles. Amiel-Tison scale at TEA and the Hempel test at 2 years were assessed.Cerebellum, brainstem and its compartments volumes were decreased in normotypic and HIE groups at TEA, while at 2 years volumes were significantly reduced in the HIE group, accompanied by decreased volume and FA and increased ADC of corticopontine and corticospinal pathways. Negative association of the brainstem, cerebellum, mesencephalon, pons, corticopontine volumes and corticospinal pathway FA at TEA with the neurological score at 2 years. Cerebellum and pons volumes presented as potential prognostic indicators of neurological outcomes.Our findings agree that these pathways, as a part of the periventricular fiber system and crossroads, exhibit lesion-induced reaction and vulnerability in HIE. Structural differences between normotypic and HIE group at the 2 years suggest a different developmental structural plasticity.

The actin-modulating protein synaptopodin mediates long-term survival of dendritic spines.

Large spines are stable and important for memory trace formation. The majority of large spines also contains synaptopodin (SP), an actin-modulating and plasticity-related protein. Since SP stabilizes F-actin, we speculated that the presence of SP within large spines could explain their long lifetime. Indeed, using 2-photon time-lapse imaging of SP-transgenic granule cells in mouse organotypic tissue cultures we found that spines containing SP survived considerably longer than spines of equal size without SP. Of note, SP-positive (SP+) spines that underwent pruning first lost SP before disappearing. Whereas the survival time courses of SP+ spines followed conditional two-stage decay functions, SP-negative (SP-) spines and all spines of SP-deficient animals showed single-phase exponential decays. This was also the case following afferent denervation. These results implicate SP as a major regulator of long-term spine stability: SP clusters stabilize spines, and the presence of SP indicates spines of high stability.

3D-reconstruction and functional properties of GFP-positive and GFP-negative granule cells in the fascia dentata of the Thy1-GFP mouse.

Granule cells of the mouse fascia dentata are widely used in studies on neuronal development and plasticity. In contrast to the rat, however, high-resolution morphometric data on these cells are scarce. Thus, we have analyzed granule cells in the fascia dentata of the adult Thy1-GFP mouse (C57BL/6 background). In this mouse line, single neurons in the granule cell layer are GFP-labeled, making them amenable to high-resolution 3D-reconstruction. First, calbindin or parvalbumin-immunofluorescence was used to identify GFP-positive cells as granule cells. Second, the dorsal-ventral distribution of GFP-positive granule cells was studied: In the dorsal part of the fascia dentata 11% and in the ventral part 15% of all granule cells were GFP-positive. Third, GFP-positive and GFP-negative granule cells were compared using intracellular dye-filling (fixed slice technique) and patch-clamp recordings; no differences were observed between the cells. Finally, GFP-positive granule cells (dorsal and ventral fascia dentata) were imaged at high resolution with a confocal microscope, 3D-reconstructed in their entirety and analyzed for soma size, total dendritic length, number of segments, total number of spines and spine density. Sholl analysis revealed that dendritic complexity of granule cells is maximal 150-200 mum from the soma. Granule cells located in the ventral part of the hippocampus revealed a greater degree of dendritic complexity compared to cells in the dorsal part. Taken together, this study provides morphometric data on granule cells of mice bred on a C57BL/6 background and establishes the Thy1-GFP mouse as a tool to study granule cell neurobiology.

Structural basis of developmental plasticity in the corticostriatal system.

Previous studies have shown that in developing monkey corticostriatal fibres terminate around striatal cytoarchitectonic compartments--cell islands, showing transfiguration around 105th embryonic day (E105) of gestation. In the present study we have analyzed these striatal cytoarchitectonic islands and acetylcholinesterase (AChE) rich patches in the developing human brain considering them as structural indicators of the development of the corticostriatal pathways. Postmortal brain tissue of 27 fetuses and prematurely born infants, ranging from 11-34 postovulatory weeks (POW) whose deaths were attributed to non neurological causes, were processed by Nissl method, AChE histochemistry and imunocytochemical technique (synaptophysin). All specimens are part of the Zagreb Neuroembryological Collection. Initial AChE patches, presumably corresponding to the dopaminergic islands, were seen as early as 10 POW whereas cytoarchitectonical cell islands were not observed until 14 POW The main developmental change occurs between 20-24 POW when AChE negative cell poor zones develop around cell islands. This transient AChE pattern of striatal organization reaches its peak around 28 POW being most prominent along lateral border of putamen. In one case of periventricular hemorrhagic lesion with premortem survival period we have found reorganization of AChE patches in the putamen which indicates structural plasticity of corticostriatal pathways. In conclusion we propose that cell poor zones serve as waiting compartments for growing corticostriatal fibers which approach striatum through subcallosal bundle and external capsule. The period of the existence of striatal compartments (14-30 POW) is a sensitive period for structural plasticity and vulnerability after periventricular lesions.

Relationship of apolipoprotein E and age at onset to Parkinson disease neuropathology.

Previous studies investigating the association between apolipoprotein E (APOE) genotypes and Parkinson disease (PD) have yielded conflicting results, and only a few have addressed APOE as a possible determinant of PD pathology. Therefore, we aimed to evaluate the relationship between APOE and PD as well as APOE and PD pathology. We studied 108 pathologically verified patients with PD and 108 controls pair-matched for age and gender. Allele frequencies of APOE differed between patients with PD and controls (p = 0.02). The frequency of epsilon4 allele increased (p = 0.01), whereas that of epsilon3 allele decreased with advancing PD pathology (p = 0.002). Only age of PD onset was an independent predictor for the rate of progression of PD pathology in which late-onset patients appeared to reach end point PD pathology more rapidly than early-onset patients (p = 0.001). In conclusion, our findings suggest that APOE may express its effect on the risk of PD by modifying the occurrence of PD pathology, but age of PD onset seems to be the principal determinant of the progression rate of PD pathology.

Excitotoxic hippocampal neuron loss following sustained electrical stimulation of the perforant pathway in the mouse.

Prolonged electrical stimulation of the perforant pathway in the rat evokes epileptiform discharges in dentate granule cells and irreversibly damages hilar neurons. In this in vivo study, we demonstrate that similar perforant path stimulation in C57Bl/6 mice causes the same pattern of hippocampal neuron loss. Therefore, this mouse model of seizure-induced hippocampal injury can be used for a wide variety of studies in genetically altered conditions not available in rats.

A general homeostatic principle following lesion induced dendritic remodeling.

INTRODUCTION: Neuronal death and subsequent denervation of target areas are hallmarks of many neurological disorders. Denervated neurons lose part of their dendritic tree, and are considered "atrophic", i.e. pathologically altered and damaged. The functional consequences of this phenomenon are poorly understood. RESULTS: Using computational modelling of 3D-reconstructed granule cells we show that denervation-induced dendritic atrophy also subserves homeostatic functions: By shortening their dendritic tree, granule cells compensate for the loss of inputs by a precise adjustment of excitability. As a consequence, surviving afferents are able to activate the cells, thereby allowing information to flow again through the denervated area. In addition, action potentials backpropagating from the soma to the synapses are enhanced specifically in reorganized portions of the dendritic arbor, resulting in their increased synaptic plasticity. These two observations generalize to any given dendritic tree undergoing structural changes. CONCLUSIONS: Structural homeostatic plasticity, i.e. homeostatic dendritic remodeling, is operating in long-term denervated neurons to achieve functional homeostasis.

The relevance of human fetal subplate zone for developmental neuropathology of neuronal migration disorders and cortical dysplasia.

The human fetal cerebral cortex develops through a series of partially overlapping histogenetic events which occur in transient cellular compartments, such as the subplate zone. The subplate serves as waiting compartment for cortical afferent fibers, the major site of early synaptogenesis and neuronal differentiation and the hub of the transient fetal cortical circuitry. Thus, the subplate has an important but hitherto neglected role in the human fetal cortical connectome. The subplate is also an important compartment for radial and tangential migration of future cortical neurons. We review the diversity of subplate neuronal phenotypes and their involvement in cortical circuitry and discuss the complexity of late neuronal migration through the subplate as well as its potential relevance for pathogenesis of migration disorders and cortical dysplasia. While migratory neurons may become misplaced within the subplate, they can easily survive by being involved in early subplate circuitry; this can enhance their subsequent survival even if they have immature or abnormal physiological activity and misrouted connections and thus survive into adulthood. Thus, better understanding of subplate developmental history and various subsets of its neurons may help to elucidate certain types of neuronal disorders, including those accompanied by epilepsy.

Perinatal growth of prefrontal layer III pyramids in Down syndrome.

We analyzed the dendritic differentiation of layer IIIc pyramidal neurons of prefrontal cortex (prospective area 9) in the brains of a premature infant and a 2.5-month-old infant with Down syndrome and two age-matched control subjects during the peak period of dendritic growth and differentiation. Our quantitative analysis supports qualitative observation and revealed no significant differences in the tempo and mode of dendritic differentiation between normal and Down syndrome cases. Thus we have concluded that the children with Down syndrome from our study begin their lives with morphologically normal layer III pyramidal neurons. Our findings suggest that pathologic changes of key prefrontal input-output neuronal elements begin to develop in Down syndrome after 2.5 months of postnatal age.