Developmental Disorders of the Nervous System

Embryological and Fetal Development of the Nervous System

Neuroembryology integrated with molecular genetics provides the key to understanding congenital malformations of the nervous system. Modern neuroembryology or ontogenesis encompasses not only classical descriptive morphogenesis but also the molecular genetic programming of development and the immunocytochemical demonstration of maturation of neuronal and glial proteins in individual cells and sequences of neurotransmitter biosynthesis, synapse formation, and myelination. Neuroimaging and electrocerebral maturation, as determined by electroencephalogram (EEG) in preterm infants, contribute other aspects of ontogenesis of normal and abnormal brain formation that are particularly relevant to clinical neurology. Maturation refers to both growth, a measure of physical characteristics over time, and development, the acquisition of metabolic functions, reflexes, sensory awareness, motor skills, language, and intellect. Molecular development , by contrast with molecular biology, refers to the maturation of cellular function by changes in molecular structures such as the phosphorylation of neurofilaments. In neurons, it also includes the development of an energy production system that actively maintains a resting membrane potential, the synthesis of secretory molecules as neurotransmitters, and the formation of membrane receptors. Membrane receptors respond to various transmitters at synapses, to a variety of trophic and adhesion molecules, and during development to substances that attract or repel growing axons in their intermediate and final trajectories. Molecular biology is the basis of linking a DNA sequence to a specific gene and a particular locus on a specific chromosome, and ultimately making a correlation with normal function and a particular disease.

Table 89.1 shows known genetic loci and mutations in human central nervous system (CNS) malformations. In most cases, mutations affect the genetic programming of the spatial and temporal sequences of developmental processes. Molecular genetic data are rapidly becoming available because of intense interest in this key to understanding neuroembryology in general and neural induction in particular. Other aspects of current investigative interest include the roles of neurotropic factors, hormones, ion channels, and neurotransmitter systems in fetal brain development. Genetic manipulation in animals has created many genetic models of human cerebral malformations. These contribute greatly to our understanding of human dysgeneses and provide insights into the pathogenesis of epilepsy and other functional results of dysgeneses.

Maturation progresses in a predictable sequence with precise timing. Insults that adversely affect maturation influence events occurring at a particular time. Some are brief (e.g., a single exposure to a toxin), whereas others act over many weeks or throughout gestation (e.g., congenital infections, maternal diabetes mellitus, and genetic or chromosomal defects). Even brief insults may have profound influences on later development by interfering with processes essential to initiate the next stage of development. Often this makes the timing of an adverse event difficult. Timing of onset of mutated genetic expression or of embryonic or fetal exposure to a teratogenic exogenous toxin is one of the most important determinants of the nature and extent of cerebral malformations ( ).

The anatomical and physiological correlates of neurological maturation reflect the growth and development of the individual neuron and its synaptic relations with other neurons. The mature neuron is a secretory cell with an electrically polarized membrane. Though endocrine and exocrine cells are secretory and muscle cells possess excitable membranes, only neurons embrace both functions. Some epithelial cells are adherent to neighboring cells forming a sheet of epithelium or glandular villi, and have weakly polarized membranes, but they are not excitable. The precursors of neurons are neither secretory nor excitable. The cytological maturation of neurons is an aspect of ontogenesis that is as important as is their spatial relations with other cells, both for future function and for the pathogenesis of some functional neurological disorders of infancy such as neonatal seizures ( ).

Neuroblasts are postmitotic neuroepithelial cells committed to neuronal lineage. These cells have not yet achieved all functions of mature neurons such as membrane polarity, secretion, and synaptic relations with other neurons, and often they are still migratory. Use of the term blast is different for neural development than for hematopoieses, in which blast cells are still in the mitotic cycle or may even be neoplastic. The events of neural maturation after initial induction and formation of the neural tube are each predictive of specific types of malformation of the brain and of later abnormal neurological function. These are (1) neurulation or formation of the neural tube, (2) mitotic proliferation of neuroblasts, (3) programmed death of excess neuroblasts, (4) neuroblast migration, (5) growth of axons and dendrites, (6) electrical polarity of the cell membrane and the energy pump to maintain a resting membrane potential, (7) synaptogenesis, (8) biosynthesis of neurotransmitters, and (9) myelination of axons.

Malformations of the nervous system are unique. No two individual cases are identical, even when categorized as the same anatomical malformation, such as alobar holoprosencephaly (HPE), syndromic or isolated agenesis of the corpus callosum, and types 1 and 2 lissencephaly. Functional expression of anatomically similar cases also may vary widely. For example, two cases of HPE with nearly identical imaging findings and similar histological patterns of cortical architecture and subcortical heterotopia at autopsy may differ in that one infant may have epilepsy refractory to pharmacological control, whereas the other may have no clinical seizures at all. The difference may be at the level of synaptic organization and the relative maturation of afferent input and neuronal maturation ( ). A discussion of the critical sequence of events in neural maturation follows.

Neurulation

Neurulation refers to the formation and closure of the neural tube. The formation of the neural tube from the neural plate starts with the establishment of the axis in the neural plate. The three early axes—longitudinal, horizontal, and vertical—persist during life and correspond to the basic body plan of all vertebrates ( ). Gastrulation occurs at 16 days’ gestation in the human; the Henson node and primitive streak establish bilateral symmetry as the basic body plan and the three axes of the body, as well as of the future neural tube. A flat neural plate is formed around the primitive streak and is the earliest differentiation of a neuroepithelium. The lateral margins of this neuroepithelial neural plate contain the precursors of neural crest cells. Shortly thereafter, grooving and bending of the neural plate occurs in the rostrocaudal axis. Subsequent closure of the lateral margins of the folding neural placode ensues in the dorsal midline to form the neural tube. To accomplish closure, intercellular filaments interdigit cells of the two sides to form a veil at midline closure points and the neuropores. At this time, the neural crest separates bilaterally at the two fusing lips of the closing neural tube, and its cells migrate along predetermined pathways to form the peripheral nervous system including autonomic ganglion cells and their axons and Schwann cells, chromaffin tissue, melanocytes, adipocytes, blood vessels, and various other cells derived from all three of the traditional germ layers: ectoderm, mesoderm, and endoderm. Because of the pervasiveness of neural crest derivatives and the expression of the same genes in all germ layers, Hall has proposed that the neural crest be regarded as a fourth germ layer with status equal to the other three ( ). Neural crest cells terminally differentiate only after reaching their final destination. The inhibitory function of versican, a chondroitin sulfate proteoglycan, is an important factor of the extracellular matrix for neural crest cell migration ( ).

The process just described is primary neurulation . Another process, secondary neurulation , occurs in the most caudal regions of the spinal cord and is limited to the lower sacral region, the part of the incipient spinal cord that formed caudal to the posterior neuropore, which is not at the extreme posterior end of the neural placode. During secondary neurulation, rather than the ependyma forming from the dorsal surface of the placode, which then becomes folded, a central canal grows rostrally from the posterior end of the solid cylinder of neural tissue within its core. It may or may not reach the central canal of primary neurulation more rostrally, and often in the midgestational or earlier fetus in particular, a transverse section through the lower sacral spinal cord reveals two ependymal-lined central canals, both in the vertical axis and one above the other. This is a normal condition, by contrast with two central canals side-by-side in the horizontal axis, at any level of the spinal cord, which represents duplication from the overexpression of a dorsalizing gene in the vertical axis of the neural tube and is found in some malformations.

Following neurulation, an associated process begins: segmentation of the neural tube and its compartmental division into neuromeres (called rhombomeres in the hindbrain). Segmentation of the neural tube is one of three independent segmentation processes in the vertebrate body, the others being the branchial arches and the somites ( ).

Mitotic Proliferation of Neuroblasts (Neuronogenesis)

After formation of the neural tube, proliferation of neuroepithelial cells in the ventricular zone associated with mitoses at the ventricular surface generates neurons and glial cells. The rate of division is greatest during the early first trimester in the spinal cord and brainstem and during the late first and early second trimester in the forebrain. Within the ventricular zone of the human fetal telencephalon, only 33 mitotic cycles provide the total number of neurons required for the mature human cerebral cortex (10 cycles in rodents), because of an exponential increase ( ). Most mitotic activity in the neuroepithelium occurs at the ventricular surface, and the orientation of the mitotic spindle determines the subsequent immediate fate of the daughter cells. If the cleavage plane is perpendicular to the ventricular surface, the two daughter cells become equal neuroepithelial cells preparing for further mitosis. If, however, the cleavage is parallel to the ventricular surface, the two daughter cells are unequal (asymmetrical cleavage). In that case, the one at the ventricular surface becomes another neuroepithelial cell, whereas the one away from the ventricular surface separates from its ventricular attachment and becomes a postmitotic neuroblast ready to migrate to the cortical plate. Furthermore, the products of two genes that determine cell fate, called numb and notch , are on different sides of the neuroepithelial cell. Therefore, with symmetrical cleavages, both daughter cells receive the same amount of each, but. With asymmetrical cleavage, the cells receive unequal ratios of each, which also influences their subsequent development ( ). The orientation of the mitotic spindle requires centractin . The mitotic spindle, the strands of which are microtubules, is linked to the plasma membrane during the splitting of the cytoplasm (cytokinesis) by a protein complex called centralspindlin ( ).

Active mitoses cease well before the time of birth in most parts of the human nervous system, but a few sites retain a potential for postnatal mitoses of neuroblasts. One recognized site is the periventricular region of the cerebral hemispheres ( ). Another is the external granular layer of the cerebellar cortex, where occasional mitoses persist until 1 year of age. Postnatal regeneration of these neurons after destruction of most by irradiation or cytotoxic drugs occurs in animals and may occur in humans as well. Primary olfactory receptor neurons also retain a potential for regeneration. In fact, if a constant turnover of these neurons in the olfactory epithelium did not occur throughout life, the individual would become anosmic after a few upper respiratory infections, which transiently denude the intranasal epithelium.

Neuronogenesis also involves the biosynthesis of cell-specific proteins. Many of these are detectable in the germinal matrix as evidence of early commitment of cells not only to a neuronal lineage but also to a fate as a specific type of neuron. The previously held concept that germinal matrix cells were uniformly undifferentiated postmitotic neuroepithelial cells was incorrect. But a population of “stem cells” with mitotic potential also is present in the subventricular zone and just beneath the hippocampal dentate gyrus ( ). These have generated considerable interest because of a potential for regeneration of the damaged adult brain and because they may be induced to mature as neurons ( ). Transplanted stem cells have an increased risk of neoplastic transformation, however ( ). Cultures of stem cells not only can generate neurons but also may even generate a poorly formed miniature cortex or whole brain ( ).

Disorders of Neuronogenesis

Destructive processes may destroy so many neuroblasts that regeneration of the full complement of cells is impossible. This happens when the insult persists for a long time or is repetitive, destroying each subsequent generation of dividing cells. Inadequate mitotic proliferation of neuroblasts results in hypoplasia of the brain ( Fig. 89.1 ). Such brains are small and grossly malformed, either because of a direct effect on neuroblast migration or by destruction of the glial cells with radial processes that guide migrating nerve cells. The entire brain may be affected, or portions may be selectively involved. Cerebellar hypoplasia often is a selective interference with proliferation of the external granular layer. In some cases, cerebral hypoplasia and microcephaly are the result of precocious development of the ependyma before all mitotic cycles of the neuroepithelium are complete, because ependymal differentiation arrests mitotic activity at the ventricular surface. The mutation of a gene that programs neuronogenesis may be another explanation for generating insufficient neuroepithelial cells. In somatic mutations that give rise to hamartomatous malformations of the brain, such as hemimegalencephaly and tuberous sclerosis, the genetic program for neuronal lineage, differentiation, and cellular growth is altered such that proliferation may be deficient and those neuroblasts that do form are dysmorphic, often megalocytic, and do not function normally, including becoming epileptogenic.

Programmed Cell Death (Apoptosis)

Normal mitotic proliferation produces excessive neuroblasts in every part of the nervous system. Reduction of this abundance by 30%–50% is by a programmed process of cell death, or apoptosis, until achieving the definitive number of immature neurons. The factors that arrest the process of apoptosis in the fetus are multiple and are in part genetically determined. Cells that do not match with targets are more vulnerable to degeneration than those that achieve synaptic contact with other cells. Endocrine hormones and neuropeptides modulate apoptosis. Some homeotic genes such as c-fos are important in the regulation of apoptosis in the nervous system, and other suppressor genes stop the expression of apoptotic genes. Caspase-3 is a key mediator of apoptosis, a protease activated as early as neural tube formation; it also is active in many neurodegenerative diseases ( ). During apoptosis, cells break up into membrane-bound fragments, a process regulated by the protein pannexin-1, which has its own membrane channels; it can be deregulated by quinolone antibiotics ( ).

Two phases of apoptosis are distinguished. One involves as-yet undifferentiated neuroepithelial cells or neuroblasts with incomplete differentiation; the other phase involves fully differentiated neurons of the fetal brain. The first phase begins during embryonic life and may extend to midgestation in some parts of the brain (e.g., periventricular telencephalic neuroepithelium) until ependyma differentiates at the ventricular surface. The second phase may be ongoing throughout life, as occurs in primary olfactory neurons of the nasal mucosa, and in the olfactory bulb and hippocampus, closely associated with a reservoir of stem cell progenitors.

In addition to cellular apoptosis, mitochondria within cells also undergo a similar autophagy (mitophagy), largely mediated by the genes Parkin and PINK1, mutations of which explain some hereditary neurodegenerative diseases ( ).

Neuroblast Migration

No neurons of the mature human brain occupy their site of generation from the neuroepithelium. They migrate to their mature site to establish the proper synaptic connections with appropriate neighboring neurons and send their axons in short or long trajectories to targets. The subependymal germinal matrix ( Fig. 89.2 ) is the subventricular zone of the embryonic concentric layers and consists of postmitotic premigratory neuroblasts and glioblasts. In general, the movement of maturing nerve cells is centrifugal, radiating toward the surface of the brain. The cerebellar cortex is exceptional in that external granule cells first spread over the surface of the cerebellum and then migrate into the folia. Migration of neuroblasts begins at about 6 weeks’ gestation in the human cerebrum and is not completed until at least 34 weeks of fetal life, although the majority of germinal matrix cells after midgestation are glioblasts. Glioblasts continue to migrate until early in the postnatal period. Within the brainstem, neuroblast migration is complete by 2 months’ gestation. Cerebellar external granule cells continue migrating throughout the first year of life.

Neuroblast migration permits a three-dimensional spatial relationship to develop between neurons, which facilitates the formation of complex synaptic circuits. The timing and sequence of successive waves of migrating neuroblasts are precise. In the cerebral cortex, immature nerve cells reach the pial surface and then form deeper layers as more recent arrivals replace their position at the surface. Neurons forming the most superficial layers of neocortex are thus the last to have migrated, although in the three-layered hippocampus, the most superficial neurons represent the earliest migratory wave. Three major groups of molecules control neuroblast migration ( ): (1) molecules of the cytoskeleton that determine the initiation (filamin-A and ADP-ribosylation factor GEF2) and ongoing progression (doublecortin and LIS1) of neuroblast movement; (2) signaling molecules involved in lamination, including reelin and other proteins not yet associated with human diseases; and (3) molecules modulating glycosylation that provide stop signals to migrating neuroblasts (e.g., POMT1 [protein O -mannosyl-transferase], involved in Walker-Warburg syndrome; POMGnT1 [protein O -mannose β-1,2- N -acetylglucosaminyltransferase], involved in muscle-eye-brain disease; and fukutin, involved in Fukuyama muscular dystrophy).

The laminated arrangement of the mammalian cerebral cortex requires a large cortical surface area to accommodate increasing numbers of migrating neuroblasts and glioblasts. Initially the cortical plate shows no histological layering, a process beginning at about midgestation, but rather has an immature columnar architecture. The lamination is superimposed upon this columnar pattern, but columnar architecture is still seen postnatally, particularly at the crowns of gyri and the depths of sulci. Even before histological lamination is evident, ribonucleic acid (RNA) probes for specific neuronal identities can already detect future organization of the cortical plate ( ). Convolutions provide this large surface area without incurring a concomitant increase in cerebral volume. The formation of gyri and sulci is thus a direct result of migration ( Fig. 89.3 ). Most gyri form in the second half of gestation, which is a period of predominant gliogenesis and glial cell migration. Therefore, the proliferation of glia in the cortex and subcortical white matter may be more important than neuroblast migrations in the formation of convolutions, but the growth of dendrites and synaptogenesis also may influence gyration by contributing mass to the neuropil.

Major Mechanisms of Neuroblast Migration: Radial Glial Fiber Guides and Tangential Migration along Axons

The majority of neuroblasts arriving at the cortical plate do so by means of radial glial guides from the subventricular zone. A second route, tangential migration, uses axons as the guides for the migratory neuroblasts. The genetically determined programming of neuroblast migration begins when cells are still undifferentiated neuroepithelial cells and even before all their mitotic cycles are complete. Neuroepithelial cells express the gene products of the lissencephaly gene ( LIS1 ), as do ependymal cells and Cajal-Retzius cells of the molecular layer of cerebral cortex. The expression of this gene is defective in type 1 lissencephaly (Miller-Dieker syndrome), a severe disorder of neuroblast migration ( ). An understanding of its function in migration is incomplete. The guidance of most neurons of the forebrain to their predetermined site from the germinal matrix (embryonic subventricular zone) is by long radiating fibers of specialized fetal astrocytes ( Fig. 89.4 ). The elongated processes of these glial cells span the entire wall of the fetal cerebral hemisphere; their cell bodies are in the periventricular region, and their terminal end-feet are on the limiting pial membrane at the surface of the brain (see Fig. 89.4 ). Radial glial cells are the first astroglial cells of the human nervous system converted into a mature fibrillary astrocyte of the subcortical white matter; some are still present at birth. Mature astrocytes are present throughout the CNS by 15 weeks’ gestation, and gliogenesis continues throughout fetal and postnatal life. Several types of glial cells are recognizable between 20 and 36 weeks’ gestation.

Facilitating the mechanical process of neuroblasts gliding along a radial glial fiber are several specialized proteins at the radial glial fiber surface membrane or extracellular space. An example is astrotactin, secreted by the neuroblast ( ). Glial cells and neural cell adhesion molecules also facilitate gliding ( ). These adhesion molecules must be deactivated when the migratory neuroblast reaches the neural plate so that the next arriving neuroblast on the same radial glial fiber can bypass the first to establish the inside-out arrangement of the cortical plate, with the earliest migratory waves forming the deep layers and the last arrivals forming the superficial layers. Fetal ependymal cells have radiating processes that resemble those of the radial glial cell but do not extend beyond the germinal matrix and secrete molecules in the extracellular matrix. Some adhesion molecules are present in the extracellular matrix ( ). These molecules serve as lubricants, as adhesion molecules between the membranes of the neuroblast and the radial glial fiber, and as nutritive and growth factors. They stimulate cell movement. Deficient molecules lead to defective migration. For example, the abnormality of the L1 adhesion molecule is the defective genetic program in X-linked hydrocephalus accompanied by polymicrogyria and pachygyria. Other inhibitory cell adhesion molecules also are essential for detachment of neuroblasts from radial glia ( ).

The process of transformation of radial glial cells into astrocytes and ependymal cells begins during the first half of gestation and completes postnatally. During midgestation when neuronal migration is at a peak, many radial glial cells remain attached to the ventricular and pial surfaces, increasing in length and curving with the expansion and convolution of the cerebral wall. From 28 weeks’ gestation to 6 years of age, astrocytes of the frontal lobe shift from the periventricular to the subcortical region. The centrifugal movement of this band of normal gliosis marks the end of neuronal migration in the cerebral mantle. Ependyma does not completely line the lateral ventricles until 22 weeks’ gestation. Studies of messenger RNA (mRNA) in individual glioblasts indicate that these immature glial precursors already exhibit differences related to their final differentiation ( ).

Radial glial cells also act as resident stem cells in the fetal brain. In the presence of injury, such as a cortical microinfarct, radial glia are capable of differentiating as neurons to replace those that were lost. Radial glia express nestin and other primitive proteins found only in cells of multipotential lineage or that participate in early developmental processes, such as floor-plate ependymal cells.

In addition to the radial migration to the cerebral cortex, tangential migration also occurs, but the number of neuroblasts is far smaller ( ). These migrations perpendicular to the radial fibers probably use axons rather than glial processes as guides for migratory neuroblasts. This explains why not all cells in a given region of cortex are from the same clone or vertical column. Most of the tangentially migrating neuroblasts in the cerebral cortical plate are generated in the fetal ganglionic eminence, a deep telencephalic structure of the germinal matrix that gives origin to basal ganglionic neurons and to the γ-aminobutyric acid (GABA)-ergic inhibitory interneurons of the cerebral cortex. These neurons in the cortex from tangential migration have some unique metabolic features and distinctive immunoreactivities in tissue section for antibodies against soluble calcium-binding molecules, such as calretinin and parvalbumin ( ). Calretinin-reactive inhibitory interneurons in the cerebral cortex comprise about 12% of total neurons and are a subset of total neurons arriving at the cortical plate by tangential migration, which represent about 20% of total cortical neurons. These also include a population of disinhibitory interneurons that suppress the activity of inhibitory interneurons ( ).

Tangential migrations occur in the brainstem and olfactory bulb as well as in the cerebrum. The subpial region is another site of neuroblast migration that does not use radial glial cells. Calretinin-reactive neurons are in the cerebellum as well as the cerebral cortex ( ), particularly Purkinje cells, basket cells, and neurons of the dentate and inferior olivary nuclei of the cerebellar system, but not those of the pontine nuclei, which similarly originated in the rhombic lip of His.

Disorders of Neuroblast Migration

Nearly all malformations of the brain are a direct result of faulty neuroblast migration, or at least involve a secondary impairment of migration. Imperfect cortical lamination, abnormal gyral development, subcortical heterotopia, and other focal dysplasias relate to some factor that interferes with neuronal migration, whether vascular, traumatic, metabolic, or infectious. The most severe migratory defects occur in early gestation (8–15 weeks), often associated with even earlier events in the gross formation of the neural tube and cerebral vesicles. Heterotopia of brainstem nuclei also occurs. Later defects of migration are expressed as disorders of cortical lamination or gyration such as lissencephaly, pachygyria, and cerebellar dysplasias. Insults during the third trimester cause subtle or focal abnormalities of cerebral architecture that may express in infancy or childhood as epilepsy.

Most disturbances of neuroblast migration involve arrested migration before the journey is complete. These disorders are divisible into three anatomical phases, depending on where the migratory arrest occurred. An example of neuroblasts never having begun migration from the periventricular region is periventricular nodular heterotopia, an X-linked genetic disorder due to defective expression of the gene, filamin-A ( FLNA ). Subcortical laminar heterotopia results when neuroblasts begin migration but arrest in the subcortical white matter before reaching the cortical plate. This is another X-linked recessive trait but is due to a different gene called doublecortin ( DCX ). The term double cortex is sometimes used, but this name is incorrect because unlike a true cortex, the subcortical heterotopia lacks lamination. If the neuroblasts reach the cortical plate but lack correct lamination, accompanying this abnormal architecture of the cortical plate are abnormalities of gyration such as lissencephaly or pachygyria. Several different genes, including LIS1 and reelin ( RLN ), are important in cortical plate organization ( ) and mutated in malformations of the terminal phase of neuroblast migration.

Lissencephaly is a condition of a smooth cerebral cortex without convolutions. Normally at midgestation, the brain is essentially smooth; the interhemispheric, sylvian, and calcarine fissures are the only ones formed. Gyri and sulci develop between 20 and 36 weeks’ gestation, and the mature pattern of gyration is evident at term, although some parts of the cerebral cortex (e.g., frontal lobes) are still relatively small. In lissencephaly type 1 (Miller-Dieker syndrome), the cerebral cortex remains smooth. Lesser degrees of this gross morphological defect exist, with a few excessively wide gyri (pachygyria) or multiple excessively small gyri (polymicrogyria). The histopathological pattern is that of a four-layer cortex in which the outermost layer (1) is the molecular layer, as in normal six-layered neocortex. Layer 2 corresponds to layers 2 through 6 of normal neocortex, layer 3 is cell-sparse as a persistent fetal subplate zone, and layer 4 consists of incompletely migrated neurons in the subcortical intermediate zone. In lissencephaly type 2 (Walker-Warburg syndrome), poorly laminated cortex with disorganized and disoriented neurons is seen histologically, and the gross appearance of the cerebrum is one of a smooth brain or a few poorly formed sulci ( Fig. 89.5 ). The term cobblestone refers to the aspect of the surface, with multiple shallow furrows not corresponding to normal sulci. The cerebral mantle may be thin, suggesting a disturbance of cell proliferation as well as of neuroblast migration. Malformations of the brainstem and cerebellum often are present as well (see Fig. 89.5 ). Lissencephaly type 1 and type 2 (Walker-Warburg syndrome, Fukuyama muscular dystrophy, muscle-eye-brain disease of Santavuori) are genetic diseases. LIS1 was the first gene discovered in the lissencephalies, but many more have now been identified ( ). Lissencephaly also results from nongenetic disturbances of neuroepithelial proliferation or neuroblast migration, including destructive encephaloclastic processes such as congenital infections during fetal life. More recently it has been recognized that the lissencephalies, including those resulting from mutations in LIS1, DCX, and ARX genes, are disturbances not only in radial migration but also involve tangentially migrating neuroblasts ( ).

Other abnormal patterns of gross gyration of the cerebral cortex occur secondary to neuroblast migratory disorders. Pachygyria signifies abnormally large, poorly formed gyri and may be present in some regions of cerebral cortex, with lissencephaly in other regions. Polymicrogyria refers to excessively numerous and abnormally small gyri that similarly may coexist with pachygyria. The small gyri often show fusion of adjacent molecular zones and other gaps in the pial membrane and leptomeninges that also result in overmigration ( ). However, polymicrogyria does not necessarily always denote a primary migratory disorder of genetic origin. Small, poorly formed gyri may occur in zones of fetal ischemia, and they regularly surround porencephalic cysts due to middle cerebral artery occlusion in fetal life.

In the cerebral hemisphere, most germinal matrix cells become neurons during the first half of gestation, and most form glia during the second half of gestation. Nonetheless, a small number of germinal matrix cells are neuronal precursors, migrating into the cerebral cortex in late gestation. Because the migration of the external granular layer in the cerebellar cortex is incomplete until 1 year of age, a potential for acquired insults to interfere with late migrations persists throughout the perinatal period. Anatomical lesions such as periventricular leukomalacia, intracerebral hemorrhages and abscesses, hydrocephalus, and traumatic injuries may disrupt the delicate radial glial guide fibers and prevent normal migration even though the migrating cell itself may escape the focal destructive lesion. Damaged radial glial cells tend to retract their processes from the pial surface. The migrating neuron travels only as far as its retracted glial fibers carry it. If this fiber retracts into the subcortical white matter, the neuroblast stops there and matures, becoming an isolated heterotopic nodule composed of several nerve cells that were migrating at the same time in the same place. In these nodules, neurons of various cortical types differentiate without laminar organization and with haphazard orientations of their processes, but a few extrinsic axons may prevent total synaptic isolation of the nodule. Interference with the glial guide fibers in the cerebral cortex itself results in neurons either not reaching the pial surface or not being able to reverse direction and then descending to a deeper layer. The consequence is imperfect cortical lamination, which interferes with the development of synaptic circuits. These disturbances of late neuroblast migration do not produce the gross malformations of early gestation and may be undetectable by imaging techniques. They may account for many neurological sequelae after the perinatal period, including seizures, perceptual disorders, impairment of gross or fine motor function, learning disabilities, and intellectual disability.

In sum, either defective genetic programming or acquired lesions in the fetal brain that destroy or interrupt radial glial fibers may cause disorders of neuroblast migration. Cells may not migrate at all and become mature neurons in the periventricular region, as occurs in X-linked periventricular nodular heterotopia ( ) and in some cases of congenital cytomegalovirus infection. Cells may become arrested along their course as heterotopic neurons in deep subcortical white matter, as occurs in many genetic syndromes of lissencephaly-pachygyria and in many metabolic diseases including cerebrohepatorenal (Zellweger) syndrome and many aminoacidurias and organic acidurias. The same aberration may occur in acquired insults to the radial glial cell during ontogenesis. Cells may overmigrate beyond the limits of the pial membrane into the meninges as ectopic neurons, either singly or in clusters known as marginal glioneuronal heterotopia , or brain warts. Rarely, herniation of the germinal matrix into the lateral ventricle may occur through gaps in the ependyma; those cells mature as neurons, forming a non-neoplastic intraventricular mass that may or may not obstruct cerebrospinal fluid (CSF) flow. Whether disoriented radial glial fibers actually guide neuroblasts to an intraventricular site or neuroblasts are physically pushed in a direction of less resistance is uncertain.

Fissures and Sulci of Cortical Structures

Fissures and sulci are grooves that form in laminated cortices, principally cerebral and cerebellar. Such folding accomplishes a need for an enlarging surface area without a concomitant increase in tissue volume as development proceeds. Without gyration of the cerebral cortex and foliation of the cerebellar cortex, the brain would be so large and voluminous at birth that neither the neonate nor the mother would survive delivery. Fissures and sulci both result from mechanical forces during fetal growth, but they differ in that fissures form from external forces and sulci form from internal forces imposed by the increased volume of neuronal cytoplasm and the formation of neuropil, the processes of neurons and glial cells ( ). The ventricular system acts as another external force, surrounded by but outside of the brain parenchyma. Whereas fissures generally form earlier and often are deeper than sulci, these are not the most important differences. Box 89.1 lists the various fissures of the brain, and Fig. 89.6 is a drawing of the development of the human telencephalic flexure, which becomes, after closure of the operculum, the sylvian fissure. It should be noted that the ventral bending of the primitive oval-shaped telencephalic hemisphere results in the original posterior pole becoming the temporal—not the occipital—lobe, and that the lateral ventricle bends with the brain. The occipital horn of the lateral ventricle is a more recent diverticulum of the original simple ventricle and, as such, remains the most variable part of the ventricular system, symmetrical in only 25% of normal individuals. Cerebellar folia are the equivalent of cerebral cortical gyri. A temporally and spatially precise sequence of the development of fissures, sulci, and cerebellar folia is genetically programmed and enables the neuroradiologist and neuropathologist to also assess maturational delay of this aspect of ontogenesis. The gestational age of a premature infant may be determined to within a 2-week period or less from the convolutional pattern of the brain.

Growth of Axons and Dendrites

During the course of neuroblast migration, neurons remain largely undifferentiated cells, and the embryonic cerebral cortex at midgestation consists of vertical columns of tightly packed cells between radial blood vessels and extensive extracellular spaces. Cytodifferentiation begins with a proliferation of organelles, mainly endoplasmic reticulum and mitochondria in the cytoplasm, and clumping of condensed nuclear chromatin at the inner margin of the nuclear membrane. Rough endoplasmic reticulum becomes swollen, and ribosomes proliferate.

The outgrowth of the axon always precedes the development of dendrites, and the axon forms connections before the differentiation of dendrites begins. Ramón y Cajal first noted the projection of the axon toward its destination and named this growing process the cone d’accroissement (growth cone). The tropic factors that guide the growth cone to its specific terminal synapse, whether chemical, endocrine, or electrotaxic, have been a focus of controversy for many years. However, we now know that diffusible molecules secreted along their pathway by the processes of fetal ependymal cells and perhaps some glial cells guide growth cones during their long trajectories. Some molecules (e.g., brain-derived neurotropic growth factor, netrin, S-100β protein) attract growing axons, whereas others (e.g., the glycosaminoglycan keratan sulfate—not to be confused with another very different protein, keratin) strongly repel them and thus prevent aberrant decussations and other deviations.

The proteoglycan keratan sulfate has been known since 1990 to be an important molecule in the dorsal median septum of the spinal cord that prevents rostrally growing dorsal column axons from crossing the midline before their intended destinations in the nuclei gracilis and cuneatus at the caudal medulla oblongata; aberrant decussation would confuse the brain about laterality of sensory stimuli ( ). Keratan sulfate is selective, however, repelling excitatory glutamatergic axons while facilitating inhibitory GABAergic axons. The great majority of dorsal root ganglion neurons that project axons into the dorsal columns are glutamatergic, by contrast with spinothalamic fibers that mainly are GABAergic; ascending axons of the nuclei gracilis and cuneatus to the thalamus also are GABAergic. Another repulsive factor for guidance of olfactory axons away from septal receptors is a secreted protein called Slit , which is the ligand for the Slit receptor Robo ( ). Commissural axons also are enabled to cross the ventral median septum of the spinal cord that repulses longitudinal axons growing rostrally or caudally in the longitudinal axis of the neural tube and early fetal spinal cord ( )

Keratan sulfate also occurs in the forebrain and is strongly expressed in early fetal life in the thalamus and globus pallidus, later appearing in the molecular zone and later diffusely in the cortical plate, finally becoming more localized in the deep cortical laminae and the U-fiber layer, where it impedes the penetration of axons from deep white-matter heterotopia so that they cannot integrate into cortical synaptic circuitry and epileptic networks ( ). Granulofilamentous keratan sulfate also binds to neuronal somatic membranes, but not to dendritic spines, explaining why axosomatic synapses are inhibitory and axodendritic synapses are excitatory ( ). An additional function of keratan sulfate in the brain, where is it secreted by astrocytes into the intercellular matrix, is to surround axonal fascicles so that axons can neither enter nor exit the fascicles except at programmed places. Both large and long fascicles, such as the corticospinal tract, and short fascicles, such as the coarse local axonal bundles within the globus pallidus and similar but smaller “pencil fibers of Wilson” within the corpus striatum, are insulated ( ). Keratan sulfate also has a wider distribution in the body in organs other than the CNS. It is strongly expressed in cornea, cartilage, bone, synovium, connective tissues, and other sites ( ). It may explain why cartilage is not penetrated by nerves except at designated foramina.

Matrix proteins such as laminin and fibronectin also provide a substrate for axonal guidance. Cell-to-cell attractions operate as the axon approaches its final target. Despite the long delay between the migration of an immature nerve cell and the beginning of dendritic growth, the branching of dendrites eventually accounts for more than 90% of the synaptic surface of the mature neuron. The pattern of dendritic ramification is specific for each type of neuron. Spines form on the dendrites as short protrusions with expanded tips, providing sites of synaptic membrane differentiation. The Golgi method of impregnation of neurons and their processes with heavy metals such as silver or mercury, used for more than a century, continues to be one of the most useful methods for demonstrating dendritic arborizations. Among the many contributions of this technique to the study of the nervous system, beginning with the elegant pioneering work of Ramón y Cajal, none has surpassed its demonstration of the sequence of normal dendritic branching in the human fetus. Newer immunocytochemical techniques for demonstrating dendrites also are now available, such as microtubule-associated protein 2. These techniques are applicable to human tissue resected surgically, as in the surgical treatment of epilepsy, and to the tissue secured at autopsy.

Electrical Polarity of the Cell Membrane

The development of membrane excitability is one of the important markers of neuronal maturation, but knowledge is incomplete about the exact timing and duration of this development. Membrane polarity establishes before synaptogenesis and before the synthesis of neurotransmitters begins. Because the maintenance of a resting membrane potential requires considerable energy expenditure to fuel the sodium-potassium pump, the undifferentiated neuroblast would be incapable of maintaining such a dynamic condition as a resting membrane potential. The development of ion channels within the neural membrane is another important factor in the maturation of excitable membranes and the maintenance of resting membrane potentials.

Synaptogenesis

Synapse formation follows the development of dendritic spines and polarization of the cell membrane. The relation of synaptogenesis to neuroblast migration differs in different parts of the nervous system. In the cerebral cortex, synaptogenesis always follows neuroblast migration. In the cerebellar cortex, however, the external granule cells develop axonal processes that become the long parallel fibers of the molecular layer and make synaptic contact with Purkinje cell dendrites before migrating through the molecular and Purkinje cell layer to their mature internal position within the folium. Synaptophysin immunoreactivity is a useful marker for studying normal and abnormal synaptogenesis in the fetus and newborn. Throughout the brain, the precisely programmed sequence of synaptogenesis can be identified in sections of fetal brain of various gestational ages ( ).

Afferent nerve fibers reach the neocortex early, before lamination occurs in the cortical plate. The first synapses are axodendritic and occur both external to and beneath the cortical plate in the future layers I and VI, which contain the first neurons that have migrated.

An excessive number of synapses form on each neuron, with subsequent elimination of those that are not required. Outside the CNS, muscle fibers also begin their relation with the nervous system by receiving multiple sources of innervation from multiple motor neurons, later retaining only one. Transitory synapses also form at sites on neurons where they no longer exist in the mature condition. The SMNs of newborn kittens display prominent synapses on their initial axonal segment, where they never occur in adult cats. Somatic spines are an important synaptic site on the embryonic Purkinje cell, but these spines and their synapses disappear as the dendritic tree develops. A structure/function correlation is possible in the developing visual cortex. In preterm infants of 24 to 25 weeks’ gestation, the visual evoked potentials (VEPs) recorded at the occiput exhibit initial long-latency negativity, but by 28 weeks’ gestation, a small positive wave precedes this negativity. The change in this initial component of the VEP corresponds to dendritic arborization and the formation of dendritic spines that occurs at that time.

The EEG of the premature infant follows a predictable and time-linked progression in maturation. The EEG reflects synaptogenesis more closely than any other feature of cerebral maturation and thereby provides a noninvasive and clinically useful measure of neurological maturation in the preterm infant. Fetal EEG may even detect neurological disease and seizures in utero.