Subplate Neurons

The importance of the subplate neurons in cerebral organizational events was defined by an elegant series of studies both in experimental animals and human brain. Cells destined to be subplate neurons are generated in the germinative zones and migrate both radially and tangentially to the primitive marginal zone at approximately 7 weeks of gestation before generation and migration of neurons of the cortical plate ( Fig. 7.1 and Table 7.2 ). Initially, these cells are part of the preplate that is split by approximately 10 weeks of gestation by the developing cortical plate into the subplate neurons below and the Cajal-Retzius neurons of the marginal zone above. (The cortical plate neurons give rise to layers II through VI of the cerebral cortex.) Thus the subplate zone contains some of the earliest born neurons of the cerebral cortex, which during this developmental period are more mature than the overlying cortical plate. The subplate neurons rapidly exhibit morphological differentiation and express a variety of receptors for neurotransmitters (gamma-aminobutyric acid [GABA], excitatory amino acids), neuropeptides, and growth factors (nerve growth factor, neuropeptide gamma, somatostatin, calbindin).

Schematic summary of development of human prefrontal cortex.

At the earliest age studied (10.5 weeks), the preplate zone has been split by the early-arriving neurons of the cortical plate into neurons of the marginal zone (MZ) above and of the subplate zone below. Note the exuberant neuronal development of the subplate zone into the third trimester of gestation. CP , Cortical plate; F , frontal; IZ , intermediate zone; SP _L , subplate zone (lower); SP _P , subplate-preplate zone; SP _U , subplate zone (upper); SV, subventricular zone; V, ventricular zone; WM, white matter.

(From Mrzljak L, Uylings HBM, Kostovic I, et al. Prenatal development of neurons in the human prefrontal cortex: I. A qualitative Golgi study. J Comp Neurol. 1988;271:355.)

The subplate neurons elaborate a dendritic arbor with spines, receive synaptic inputs from ascending afferents from thalamus and distant cortical sites, and extend axonal collaterals to overlying cerebral cortex and to other cortical and subcortical sites (thalamus, other cortical regions, corpus callosum). In addition to the subplate neurons , the subplate zone contains other tissue components, such as radial glial processes, radially and tangentially migrating neurons, early developing astrocytes, microglia, and oligodendrocyte precursors, that are also found in other fetal compartments. Yet, this zone is distinguished by an extensive extracellular space that is filled with hydrophilic extracellular matrix (ECM) and heterogeneous contingents of waiting cortical afferents and transient synapses. This hydrophilic feature underlies the visibility of the zone in T2-weighted magnetic resonance imaging (MRI) scans of the human fetus from approximately 18 to 26 weeks of gestation ( Fig. 7.2 ).

Reconstructed T2-weighted MRI image obtained in vivo from a 25-week fetus.

Note the high signal intensity in the region of the subplate zone, consistent with its hydrophilic nature.

(Courtesy Dr. Caitlin Rollins and Computational Radiology Laboratory, Boston Children’s Hospital.)

The subplate zone is also molecularly distinct , as determined by the identification of subplate-enriched genes by transcriptome profiling of different fetal layers. Gene expression profiling of the subplate zone in midgestation fetal human brains indicates that the human subplate is functionally enriched for synaptic plasticity and generally shows signs of more advanced maturity compared with the overlying cortical plate. Thus some of the molecular hallmarks of the subplate zone during early development primarily relate to cell maturity, and as subplate cells form, they extend axons and receive synaptic inputs earlier than the cortical plate. Importantly, the subplate is specifically rich in chondroitin sulfate proteoglycans (CSPGs), and the subplate transcriptome is enriched for genes involved in the production of ECM and proteoglycans. CSPGs are known to interact with laminin, fibronectin, tenascin, and collagen, and their differential distribution supports a role in axonal pathfinding and cell migration.

The functions of the subplate neurons now appear to be particularly far-reaching (see Table 7.2 ). ^a

a References .

Concomitant studies of subplate neurons of developing human cerebral cortex provide a crucial link with the experimental studies (see Fig. 7.3 ). The subplate neuron layer in human cortex reaches a peak between approximately 24 and 32 weeks of gestation. At this peak time, the width of the subplate zone is approximately four times that of the cortical plate. Programmed cell death (apoptosis) of this layer appears to begin generally late in the third trimester, and approximately 90% of subplate neurons have disappeared after approximately the sixth month of postnatal life. Slightly different time courses for peak development and regression of the subplate neurons exist for somatosensory and visual cortices. In the subplate dissolution stage, which occurs in humans at greater than 35 postconceptional weeks, subplate neurons decline in number and the volume of the subplate zone decreases. The reduction in volume reflects primarily a decrease in extracellular space and fewer axon bundles within the subplate zone. A distinct subplate zone is no longer identifiable by about 6 months post term in humans, but large neurons embedded in white matter are thought to be the remaining subplate cells, which are referred to as interstitial white matter neurons.

Cytoarchitectonics of the subplate zone in the visual area.

Shown are A, an 18-week old human and, B, an E78 monkey fetus displayed in plastic 1-µm-thick sections. The subplate zone (SP) is characterized by low cell density and presence of mature neurons. The border between the subplate zone and the white matter (WM) is relatively sharp because of the presence of well-delineated fiber bundles (arrows) in the white matter. External limit of fibers is marked by arrowhead . Note remarkable similarities between the lamination pattern and the cortical plate (CP)-subplate thickness ratio in man and monkey. Bar = 100 µm applies to both illustrations. MZ , Marginal zone.

(From Kostovic I, Rakic P. Developmental history of the transient subplate zone in the visual and somatosensory cortex of the macaque monkey and human brain. J Comp Neurol. 1990;297:441.)

Lamination

Attainment of the proper alignment, orientation, and layering of cortical neurons, defined as lamination, occurs during and following neuronal migration (see Table 7.1 ). These events are among the earliest in cortical organization. The microcircuitry of the cerebral cortex underlying cognitive processing is dependent on precise interrelationships between variable numbers of excitatory pyramidal neurons and inhibitory nonpyramidal (granular) neurons in cortical modules. The cortical layers are specialized compartments that contain neurons with unique properties that underlie specific roles in neural circuitry. The normal cerebral cortex is composed of two main classes of neurons: (1) pyramidal neurons, which comprise 75% to 85% of total cortical neurons, are glutamatergic, and project outside the cortex; and (2) nonpyramidal neurons (interneurons), which comprise 15% to 25% of cortical neurons, are GABAergic and project within the cortical layers.

The neocortex begins to transform from an undifferentiated cortical plate to a highly specialized structure at around 30 gestational weeks ( Fig. 7.4 ). At this age, the cortical plate becomes composed of six layers in which each layer is characterized by a specific composite of pyramidal and nonpyramidal neurons. Dramatic changes in lamination, laminar thickness, and pyramidal and nonpyramidal cell differentiation and density in the last half of gestation are consistent with neuroimaging findings of marked increases in cortical thickness and surface area over this same time period (see later). Pyramidal neurons are known to originate from radial progenitors (radial glial cells) in the subventricular zone (SVZ); these early precursors produce neurons destined particularly for the deeper cortical layers and reach the cortex by radial migration before the second half of gestation (see Chapter 6 ). The early differentiation of pyramidal neurons in layer V is consistent with their early origin and migration. Over the second half of gestation and into infancy, there is a striking increase in the overall thickness of the cortex and in layers I–III, V, and VI. The increase in thickness in layers I–III in particular over the last half of gestation likely reflects their expansion by late migrating (GABAergic) interneurons, given that the SVZ continues to actively generate mainly GABAergic neurons beyond midgestation (see Chapters 5 and 6 ).

Development of human cortical plate in the frontal association cortex.

See text for details. The scale bar is one millimeter.

(From Andiman SE, et al. The cerebral cortex overlying periventricular leukomalacia: analysis of pyramidal neurons. Brain Pathol . 2010;20:803–814, with permission.)

Gyral Development

Gyrification is the process whereby folding patterns of sulci and gyri develop on the surface of the brain. Several of these patterns are asymmetrical between the right and left side of the cerebral hemispheres, and several distinguish the human brain from that of other species. The gyrus is a ridge of cerebral cortex, whereas the sulcus is a depression or furrow on either side of the ridge. Gyrification results in a dramatic increase in the cortical surface area within the limited and rigid confines of the skull, and thus, in the volume of cortical gray matter . As discussed later, a striking increase in cerebral cortical volume in the human infant from approximately 28 to 40 weeks after conception has been shown by quantitative MRI measurements of cortical gray matter volumes. Thus a fourfold increase in cerebral cortical gray matter volume could be documented ( Fig. 7.5 ). The changes in cortical gyral development and cortical surface area that accompany and presumably are caused by the increase in cortical volume can be seen by MRI ( Fig. 7.6 ). The formation of gyri and sulci in the brain also allows for compact wiring that promotes and enhances efficient neural processing.

Cerebral cortical gray matter volume as a function of postconceptional age.

Quantitative volumetric MR determinations of absolute cerebral cortical gray matter volume as a function of postconceptional age in a series ( n = 35) of preterm and full-term infants.

(From Huppi PS, Warfield S, Kikinis R, et al. Quantitative magnetic resonance imaging of brain development in premature and mature newborns. Ann Neurol. 1998;43:224–235.)

Measurements of cortical surface area and volume by advanced magnetic resonance imaging (MRI) (upper) and conventional MR images of gyral development (lower) during the last 14 weeks of gestation. Data derived from study of 113 preterm infants.

(From Kapellou O, Counsell SJ, Kennea NL, Dvet L, et al. Abnormal cortical development after premature birth shown by altered allometric scaling of brain growth. PLoS Med. 2006;3:e265.)

At the time of neural tube closure in the embryonic period, the surface of the brain is smooth, that is, lissencephalic; as development continues, gyri and sulci begin to take shape on the fetal brain surface ( Fig. 7.7 ). The pattern of gyrification follows orderly and defined sequences, such that the brain can be dated to a particular gestational week by its developmental stage of gyrification. Information about these sequences were garnered from 507 brains and serial sections of 207 brains from infants from 10 to 44 gestational weeks of age in the National Perinatal Collaborative Project. The sequential developmental changes of the individual fissures, sulci, and gyri of the cerebral hemispheres throughout the gestational period were tabulated. The period of greatest development of brain gyrification is during the third trimester of pregnancy , a period in which the brain undergoes considerable growth. At midgestation, the brain is lissencephalic, except for the presence of the Sylvian fissure and central sulcus ; between midgestation and birth, all primary, secondary, and tertiary gyri are formed, an explosive period in gyrification. The gyrification index (GI), defined as the ratio between the lengths of coronal outlines for the brain including and excluding the sulcal regions, is a quantitative approach to measure gyrification ; brains with higher degrees of cortical folding give higher GI values. This measure was used to quantify the developmental trajectory of gyrification in humans with the major finding that gyrification increases dramatically during the third trimester and then remains relatively constant throughout subsequent development. This anatomical change is readily followed by advanced MRI measures (see later). Little is known about changes in gyrification during childhood and adolescence, although considering the continuing changes in gray matter volume and thickness during this time period, it is conceivable that alterations in the brain surface morphology could also occur during this period of development. Despite the stereotypical development of gyrification, it should be emphasized that there are structural variations among individuals , although not apparently between males and females. The central sulcus, for example, varies in location by up to 2 cm between individuals. This variability raises important considerations in analyzing structural and functional brain images in neuroimaging studies. Importantly, there are left-right asymmetries of the transverse temporal gyri, sylvian fissures, and planum temporale. In general, the right cerebral hemisphere shows gyral complexity earlier than the left. Such findings have led to speculations about the significance of left-right asymmetry of the brain as it affects speech and language development.

Schematic depiction of gyral development in human brain.

Note the particularly prominent changes in the last 3 months of gestation.

(From Cowan WM. The development of the brain. Sci Am. 1979;241:113.)

The mechanistic basis of cortical folding is complex and likely involves multiple interactive factors (see also Chapters 5 and 6 ). The following different folding mechanisms have been postulated based mainly on experimental animal data and computer-based modeling: (1) tissue buckling from mechanical stress and external constraints as the brain conforms to the confines of the skull; (2) axonal tethering and local connectivity between developing gyri, or in other words, tension-based morphogenesis; (3) localized cellular proliferation relative to localized cortical surfaces; and (4) radial intercalation of new neurons at the top of the cortical plate that causes the cortical plate to expand tangentially more rapidly than the underlying tissue, and as a result, the cortical plate buckles into a series of folds. The size and shape of the lobes of the cerebral hemisphere also involve patterning center(s) of the embryonic forebrain that contains a protomap of the future lobes and involves genetic interactions.

Neurite Outgrowth

Neurite outgrowth refers to the elaboration of dendritic and axonal ramifications of neurons (see Table 7.1 ). While beginning in the first trimester, it becomes a dominant organizational event in the second half of pregnancy, the neonatal period, and infancy. The most significant early studies of neurite outgrowth in human cerebral cortex were made in 1939 by Conel, whose Golgi-Cox preparations of cerebral cortex from birth to 2 years of age demonstrated progressive enrichment of the dendritic and axonal plexus, with much smaller increases in size and no proportionate increases in the number of individual neurons. The remarkable elaboration of dendritic branching that results can be seen in the cerebrum of a normal child shown in Fig. 7.8 . The studies of Mrzljak and co-workers showed similar events in frontal cortex before birth (see Fig. 7.1 ). Accompanying the elaboration of dendritic and axonal ramifications are the appearance of synaptic elements, the development of neurofibrils, and an increase in size of endoplasmic reticulum within the cytoplasm of cells. The biochemical correlates of these changes are increasing cerebral content of RNA and protein relative to DNA. Immunocytochemical studies document parallel expression of a variety of neurotrophins, neurotransmitters ( N -methyl- d -aspartate [NMDA], alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid [AMPA]/kainate, GABA, and glycine receptors), surface glycoconjugates, and cytoskeletal components. The maturational changes occur relatively rapidly in the hippocampus, whereas they occur over a more protracted period in the supralimbic region; the latter, of course, is of great significance, because it is the locus of the major association areas. Dendritic development in the human occurs earlier in thalamus and brain stem than in cerebral cortical regions. These findings were amplified by studies by Purpura, Huttenlocher, Marin-Padilla, Rakic, and other investigators, who used electron microscopic and immunocytochemical methods as well as Golgi techniques.

Golgi preparation (×80) of middle frontal gyrus from a 6-year-old child without known neurological disease. Note the abundant and complex horizontal and tangential dendritic branches.

(From Buchwald NA, Brazier MAB, eds. Brain Mechanisms in Mental Retardation. New York: Academic Press; 1975.)

One study of developing human brain demonstrated the strikingly active axonal development in the cerebrum over the last trimester of gestation and in the early postnatal period ( Fig. 7.9 ). Thus immunostaining with GAP-43, a protein expressed on growing axons, shows exuberant expression in the cerebral white matter to approximately the subplate region at 20 weeks, to the cortical plate at 27 weeks, within the cortex at 37 weeks, and into the first year of life. This differential pattern may reflect, at 20 weeks, growth of axons from thalamus to subplate neurons, and at 27 weeks, from subplate neurons to cerebral cortex. At 37 weeks, the increase in cerebral cortical expression of GAP-43 may reflect the increase in cortical penetration of thalamic ascending fibers no longer waiting at the subplate layer, in corticocortical fibers, and in descending cortical fibers, initially pioneered by subplate axons (see earlier). These findings are consistent with more recent studies with different techniques by Kostovic and co-workers. Although more data are needed on these issues, it is clear that the last trimester of human gestation is a period of rapid axonal development .

GAP-43 expression in developing human parietal white matter and cortex.

Cortex is indicated by an asterisk (*) . Note at 20 postconceptional (PC) weeks (A), evidence for strong expression in subcortical white matter to a region below the cortical plate, possibly the concentration of subplate neurons. At 27 PC weeks (B), the expression begins to enter the cerebral cortex. By 37 PC weeks (C), diffuse expression in cortex and decreased expression in white matter are apparent. At 144 PC weeks (D), that is, approximately 2 years of age, expression is prominent in cortex but not in white matter. See text for interpretations. Sections are ×40; scale bars are 200 µm.

(From Haynes RL, Borenstein NS, DeSilva TM, Folkerth RD, et al. Axonal development in the cerebral white matter of the human fetus and infant. J Comp Neurol. 2005;484:156–167.)

The relationship between neurite outgrowth in cortex and development of functional capacity can be illustrated in human visual cortex during the third trimester ( Fig. 7.10 ). Most impressive are the appearance and elaboration of basilar dendrites and the tangential spread of apical dendrites. This dendritic development is accompanied by the appearance of dendritic spines, or in other words, sites of synaptic contact (see the subsequent section on synaptic development). These anatomical expressions of differentiation are paralleled by the neurophysiological expression of maturation of the visual evoked potential (see Fig. 7.10 ). Such detailed relationships between dendritic structural development and specific details of neurophysiological development have been studied in depth in developing animals.

Camera lucida composite drawings of neurons in visual (calcarine) cortex of human infants of indicated gestational ages. Note the appearance and elaboration of basilar dendrites and the tangential spread of apical dendrites, as well as the accompanying maturation of the visual evoked response (top) .

(Courtesy Dr. Dominick Purpura.)

The progress of dendritic differentiation depends on the establishment of afferent input and presumably synaptic activity . ^a

a References .

The central axon is a smooth, thin process of variable length that extends from the polarized neuronal cell body and propagates action potentials. The neuronal cytoskeleton of the axon is composed of three different types of filaments: actin microfilaments, microtubules, and intermediate filaments (IFs) . A mechanism for transport down the axon involves a system of both anterograde transport from the cell body to the terminal and retrograde transport from the terminal to the cell body. Once produced in the cell body, membrane-bound organelles, including mitochondria and secretory vesicles, are transported down the length of the axon using microtubules and the motor molecule kinesin, which is thought to walk molecules along the microtubule. The growth cone is formed at the tip of the growing axon and is responsible for sensing environmental cues, determining the direction of growth, and guiding the growing axon in the proper direction. The temporal and spatial movement of the axon to its appropriate target is critical to the proper development of neuronal circuits. This movement is controlled by a number of different environmental cues that direct the axons via interactions with the growth cone receptor. Signals include a number of molecules categorized into three families of ligands—cell adhesion molecules (CAMs), ECM molecules, and ephrins—each of which acts through receptor-mediated interactions on the cell surface of the growth cone. Other signaling factors include the netrins, Slits, and semaphorins.

Advanced diffusion-based MRI imaging known as tractography provides further insight into the changes in fiber tract development in the living premature infant (see later). Intracerebral connectivity emerges in a sequential manner, starting from dorsal posterior brain areas, continuing in an anteroventral direction, and ending in inferior temporal and inferior frontal lobes ( Fig. 7.11 ). This observed order follows the same order of normal gyrification and myelination. Corticocortical association fibers have been found in the cortical plate around 23 to 25 gestational weeks. Decreases in ipsilateral corticocortical connections observed after 22 gestational weeks may be due to axonal loss from failure of final targeting or from pruning after initial exuberant over-connectivity.

Fetal fiber tract development.

See text for details.

(From Takahashi E, et al. Emerging cerebral connectivity in the human fetal brain: an MR tractography study. Cereb Cortex. 2012;22:455–460, with permission.)

Synaptic Development

Synapses are the principal sites for communication between presynaptic and postsynaptic neurons via chemical messengers called neurotransmitters (see Table 7.1 ). In the human brain, about 10 ¹⁵ synaptic contacts interconnect the 10 ¹⁰ to 10 ¹¹ neurons. Synaptic formation differs appreciably among brain regions in the human brain. In the brain stem, the number of dendritic spines, the sites of synaptic contacts, in the medullary reticular formation reaches a peak at 34 to 36 weeks of gestation and declines rapidly after birth (see later discussion of disorders of organizational events). In the cerebrum, synapses are observed initially on neurons of the preplate at 4 to 5 gestational weeks and by 10 weeks on neurons in subplate and marginal zones. In the hippocampus, synapses are abundant as early as 15 and 16.5 weeks of gestation ( Table 7.3 ). The earliest synapses in the cerebral cortex are observed around 18 postconceptional weeks and are located on prospective layer V (pyramidal neurons). Shortly after the pronounced ingrowth of thalamocortical axons to the cortical plate around 21 postconceptional weeks, dendritic spines begin to appear on immature pyramidal neurons and interneurons between 24 and 27 weeks.

With Golgi preparations, Purpura and co-workers defined the subsequent progression of dendritic spine development in the human cortex from the fifth month of gestation ( Figs. 7.12 and 7.13 ). Initially, dendrites appear as thick processes with only a few fine spicules. As development progresses, a great number and variety of dendritic spines appear. In visual cortex, synaptogenesis is fastest between 2 and 4 months after term, a time also critical for the development of function in visual cortex, and maximal synaptic density is attained at 8 months ( Fig. 7.14 ). Synapse elimination then begins, and by age 11 months, approximately 40% of synapses have been lost. In frontal cortex, the time course of synaptic formation and of elimination differs somewhat from that in visual cortex; maximal synaptic density is reached at approximately 15 to 24 months, and synapse elimination, although reaching the same loss of 40% is more gradual. In the prefrontal cortex, synapse elimination extends into midadolescence. Elegant studies in the monkey exhibit more uniformity in the temporal features of synaptogenesis among cortical regions, but the basic principles are formation of earliest synapses in the marginal and subplate zones, an increase in synapses in cortical plate to a peak in excess of the adult number, and a subsequent period of synaptic elimination.

Camera lucida drawings of proximal apical dendritic segments of motor cortex pyramidal neurons during development. (A) 18-week-old fetus; (B) 26-week-old fetus; (C) 33-week-old preterm infant; (D) 6-month-old infant; (E) 7-year-old child. Early phase of dendritic spine differentiation in proximal apical segments is associated with the development of long, thin spines and relatively few stubby and mushroom-shaped spines. The latter two types are prominent in the postnatal period and early childhood.

(Courtesy Dr. Dominick Purpura.)

Camera lucida drawings of dendritic segments of motor cortex neurons, rapid Golgi preparations, from a normal and a mentally retarded infant. (A) From a normal 6-month-old infant: 1 and 2 , proximal apical dendritic segments with a predominance of stubby and mushroom-shaped spines; 3 and 4 , distal apical dendritic segment and basilar dendritic segment have many more thin spines. (B) From a 10-month-old infant with mental retardation of unknown etiology: 1, 2, 3, proximal apical and, 4 , basilar dendritic segments. Note the presence in the proximal segments (1, 2, 3) of many long, thin spines, comparable to the appearance of cortex in normal preterm infants (see Fig. 7.14 ).

(Courtesy Dr. Dominick Purpura.)

Synaptic density in layer I and layer II/III of striate cortex.

Open circles represent layer I; closed circles represent layer II/III. Note the striking increase in the first postnatal year and the subsequent decline.

(From Huttenlocher PR, de Courten C. The development of synapses in striate cortex of man. Hum Neurobiol. 1987;6:1.)

Synaptic function results in the development of such neurophysiological measures as cortical evoked responses (see Fig. 7.10 ). Function is dependent on the action of neurotransmitters. These substances are stored in vesicles and are released from presynaptic nerve terminals at the so-called active zone, a restricted area of the cell membrane situated exactly opposite to the postsynaptic neurotransmitter reception apparatus. At the active zone neurotransmitter, containing synaptic vesicles (SVs) dock, fuse, release their content and are recycled. SV components are initially trafficked to the synapse with members of the kinesin motor family. Exocytoxic function is mediated by a docking/fusion core complex that is regulated by several molecules, including Munc18-1 and syntaxin. Vesicles fuse very quickly in response to calcium elevations in the cytoplasm; this fusion event is thought to be mediated directly by the SNAREs. Transport proteins are involved in neurotransmitter uptake into the SVs and are composed of proton pumps that generate electrochemical gradients. Vesicular transporters move recycled neurotransmitters from the cells’ cytoplasm into the SVs.

The development of synaptic specificity begins once a neuron has identified its correct synaptic partner. The initial axodendritic contact is transformed into a functional synapse by the recruitment of presynaptic and postsynaptic components. The factors that stimulate synaptic formation and development in developing brain include initially activity-independent events, (i.e., molecular mechanisms involved in targeting), followed by activity-dependent events occurring after the development of receptors on target neurons and the generation of electrical activity (see previous discussion on neurite outgrowth). Many molecules and signaling pathways are involved in dendritic spine development and remodeling. The two principal themes are modulation of the following: (1) ion channels, especially calcium-permeable channels, by neurotransmitters, especially glutamate; and (2) cell surface receptors by a variety of ligands. The intracellular events lead ultimately to effects on actin-binding proteins and the actin cytoskeleton, with resulting changes in spine shape, size, and motility. The importance of synaptogenesis and synapse elimination in the plasticity of the developing nervous system and in the potential effect of experiential factors on developing neural function, including cognitive function, could be enormous.

Cell Death and Selective Elimination of Neuronal Processes and Synapses

Cell death and selective elimination of neuronal processes and synapses, or regressive events in brain development, are now recognized to be highly critical (see Table 7.1 ). Results of studies in a variety of developing neuronal systems showed that after formation of neuronal collections by the progressive processes of proliferation and migration, cell death occurs. Although variable in degree among neuronal regions, typically about half of the neurons in a given collection die before final maturation. This process of cell death is initiated and sustained by the expression of specific genes and their transcription products that actively kill the neuron. Critical in the final phases of the sequence to cell death is the activation of a family of cysteine proteases known as caspases. The term programmed cell death has been used to emphasize that this is an active developmental process, although more commonly the term apoptosis, a Greek-rooted word referring to the naturally occurring seasonal loss or falling of flowers, is used to refer to this developmentally determined cell death.

The factors that activate this death system appear to relate to competition of neurons for limited amounts of trophic factors, generated by the target, afferent input, or associated glia. This loss of neurons appears to serve two major functions in development: quantitative adjustments (numerical matching) of interconnecting populations of neurons and elimination of projections that are aberrant or otherwise incorrect (refinement of synaptic connections or error correction). ^a

a References .

Neural organization is refined further by a second regressive event, selective elimination of neuronal processes and synapses. This event primarily causes the removal of terminal axonal branches and their synapses, although even larger-scale elimination of a total pathway also occurs. Vivid demonstrations of synapse elimination are apparent in developing brain stem and cortex of the human infant (see earlier). The determinants of selective elimination of neuronal processes and synapses are similar to those described for cell death. Activation of the NMDA type of glutamate receptor appears to be an important step in synapse elimination during development. An additional crucial role of the specific region of cerebral cortex in determining the pattern of selective elimination of its terminal axons was shown by studies with cortical transplants in developing rats. Thus cortical neurons transplanted to another region of cortex (as explants) eliminated their distal axon collaterals in the same way as did neurons of the host cortical region (into which they were transplanted), rather than in the way neurons of the donor region eliminated their distal collaterals.

The observations that cell death and elimination of neuronal processes and synapses occur during the organizational period of development have implications for the frequent demonstration that the plasticity of developing brain decreases as this period is completed. It is likely that the regressive events described in this section are modified when the brain is injured and that neuronal processes and synapses destined for elimination can be retained if needed to preserve function. In addition, new projections may develop in response to injury during the period in which the brain has the capacity to perform organizational events. In favor of one or both of these predictions is the demonstration in both human infants and experimental models that, after neonatal cerebral lesions, ipsilateral corticospinal tract projections can be demonstrated and presumably can ameliorate the functional deficit. The demonstration of an ipsilateral corticospinal projection until early childhood in humans suggests that retention of a normally occurring ipsilateral corticospinal tract, which otherwise is eliminated during development, is the crucial event in this form of plasticity. The possible additional role of assumption of motor functions by ipsilateral cerebrum adjacent to the lesion is suggested by other studies.

Glial Proliferation and Differentiation

Astrocytes, oligodendrocytes, and microglia are the major glial cells of the central nervous system (CNS). Glial proliferation and differentiation are of major importance in developing brain; glial cells clearly outnumber neurons in the CNS. In fact, in human cerebral cortex, glial cells outnumber neurons by approximately 1.25 to 1 and are almost the exclusive cell type in white matter. Astrocytic and oligodendrocytic lineage, proliferation, and differentiation have been the topic of intense investigation in experimental systems in recent years, and initial data also are emerging from studies of human brain. ^a

a References .

Organizational Events Studied In Vivo

Investigation of organizational events in living infants has been based principally on the study of premature infants by advanced MRI methodologies ( Table 7.5 ). Several principal MRI methods have been used and include three structural measures, that is, volumetric MRI, diffusion tensor MRI, and surface-based cerebral cortical measures, as well as functional MRI (fMRI), both task-related (e.g., response to specific sensory input) and resting state (RS) (see Table 7.5 ). Many excellent reviews of the application of these measures are available and are discussed in other relevant chapters in this book (see especially Chapter 10 , Chapter 16 ). This section focuses on studies relevant to normal development in the fetal and neonatal periods . Because of the principal application of these measures to preterm infants, the great preponderance of data involves brain development primarily over the period from 28 to 40 postconceptional weeks. These findings are described briefly next.

Diffusion Tensor Magnetic Resonance Imaging

Diffusion tensor imaging (DTI) measures water diffusion along an axis and thereby is valuable for assessing fiber tracts, especially axons, and less commonly, dendrites (see Table 7.5 ). Preferred directionality of water diffusion, measured by DTI as relative or fractional anisotropy (FA), provides information about white matter microstructure and, particularly, the development of white matter fiber tracts. Increases in FA in cerebral white matter was shown in premature infants over the period 28 to 40 weeks’ postconception, nearly 20 years ago ( Fig. 7.15 ). Many subsequent studies have confirmed this observation and described important regional differences (see later). The anatomical correlate for this increase in relative anisotropy (RA) is likely the increase in axonal size and density as development proceeds. However, an additional anatomical determinant, especially later in the third trimester, may reflect axonal ensheathment by premyelinating oligodendrocytes. As noted earlier the latter process is especially active at this time, and experimental studies have delineated such a period of premyelination anisotropy . Differentiation of axial versus radial diffusivity is useful for the distinction of axonal development versus ensheathment by premyelinating oligodendrocytes. Axial diffusivity increases in fiber tracts with axonal development, and radial diffusivity declines in tracts as premyelinating oligodendroglial ensheathment occurs.

Diffusion tensor MR determination of relative anisotropy (RA) in cerebral white matter of normal preterm (PT) and term (FT) infants (open circles) as a function of postconceptional age. Note the striking increase in RA, indicative of increasing directionality of diffusion, perhaps related at least in part to oligodendroglial ensheathment of axons, with maturation. The lower values of the preterm infants studied at term (closed circles) suggest a deleterious effect of prematurity on this process (see text for details).

(From Huppi PA, Maier SE, Peled S, et al. Microstructural development of human newborn cerebral white matter assessed in vivo by diffusion tensor magnetic resonance imaging. Pediatr Res. 1998;44:584–590.)

Regional differences in development of white matter tracts are reflected by differences in the trajectory of increases in FA. White matter areas that mature early, such as the posterior limb of the internal capsule and the optic radiations, show a correspondingly early rise in anisotropy.

DTI data also provide information about developing fiber tracts, and such diffusion tractography is valuable for visualization of white matter tracts ( Figs. 7.16 and 7.17 ). When combined with fMRI studies, structural connectivity within developing cerebrum can be defined (see later).

Diffusion tractography in two preterm infants studied at term equivalent age.

The optic radiations (green) and corticospinal tracts (pink) are shown.

(From Tao JD, Neil JJ. Advanced magnetic resonance imaging techniques in the preterm brain: methods and applications. Curr Pediatr Rev. 2014;10:56–64, with permission.)

Quantitative diffusion tensor imaging.

Color anisotropy parameteric maps of a preterm infant brain. The color maps display the direction of individual fibers: right to left (red) , anterior to posterior (green) , and superior to inferior (blue) .

(From Tao JD and Neil JJ. Advanced magnetic resonance imaging techniques in the preterm brain: methods and applications. Curr Pediatr Rev. 2014;10:56–64, with permission.)

Interestingly, in contrast to the increase in FA in developing cerebral white matter, FA declines in developing cerebral cortex . The higher anisotropy in early developing cortical gray matter may relate to the preponderance of radially oriented fibers, that is, neurons with simple axons and an underdeveloped dendritic tree, RGFs, and the decline over the period from 26 to 40 weeks may reflect the elaboration of the dendritic tree and axonal ramifications and a regression of RGFs ( Fig. 7.18 ). A graphical representation of data from Smyser and co-workers shows the simultaneous declines in RA in cerebral cortical areas and increases in RA in corresponding cerebral white matter regions ( Fig. 7.19 ). The crossover point between the maturation lines may reflect the relative rates of white and gray matter development (see Fig. 7.19 ). The earliest maturing cerebral cortical and white matter areas, that is, primary motor and visual areas, have earlier crossover points than do the later maturing areas, that is, visual association and prefrontal areas (see Fig. 7.19 ).

Diagram depicting proposed explanation for cortical anisotropy.

On the left are representations of cortical microstructure. On the right are the corresponding diffusion ellipsoids at 26 weeks’ gestational age (A), radial glial fibers and pyramidal neurons with prominent, radially oriented apical dendrites are shown. This organization has the effect of restricting water displacement parallel to the cortical surface more than displacement orthogonal to it, resulting in diffusion ellipsoids which are nonspherical with their major axes oriented radially (arrow s ) . By 35 weeks’ gestational age (B), prominent basal dendrites for the pyramidal cells and thalamocortical afferents have been added. This has the effect of restricting water displacement more uniformly in all directions. As a result, the diffusion ellipsoids are spherical, without a preferred orientation.

(From McKinstry RC, et al. Radial organization of developing preterm human cerebral cortex revealed by non-invasive water diffusion anisotropy MRI. Cereb Cortex. 2002;12:1237–1243, with permission.)

Scatter plots demonstrating fractional anisotropy measures versus postmenstrual age in the white (open squares) and gray (red diamonds) matter of the (A) motor, (B) visual, (C) visual association, and (D) prefrontal areas for very preterm infants. The solid lines depict results from linear regression. Note differences in the crossover point for regression lines for gray and white matter across regions. Values for term-born infants are provided for comparison on far right.

(From Smyser TA, et al. Cortical gray and adjacent white matter demonstrate synchronous maturation in very preterm infants. Cereb Cortex . 2016;26:3370–3378, with permission.)

Surface-Based, Cerebral Cortical Magnetic Resonance Imaging Measures

Cerebral cortical surface MRI analysis is a relatively recently applied approach to the study of the premature newborn (see Table 7.5 ). The major foci of this analysis, which uses data from conventional MR images, are delineation of cortical surface area and folding/gyrification . Because the last 16 to 20 weeks of gestation is the period of most active cortical folding (see earlier), cortical surface analysis is especially relevant to the premature infant. Cortical surface area increases approximately 12% per week during the premature period. Increases in cortical folding, development of gyri and increases in sulcal depth are similarly dramatic ( Fig. 7.20 ). The anatomical determinants were discussed earlier (see Gyral Development ). It is likely that the migration of a full complement of neurons to cortex, including late migrating GABAergic neurons, with expansion especially of superficial cortical layers, followed by elaboration of dendritic and axonal ramifications are critical. However, in addition to the forces generated by development of cerebral cortical surface area, cortical folding likely depends also on the mechanical tension exerted by axons in rapidly developing subcortical white matter.

Plots of individual mean sucal depth and gyrification index versus postmenstrual age (PMA) for term (black circles) , uninjured preterms (blue squares) , and injured preterms (red diamonds) . Note the striking increase in the preterms (uninjured more than injured) as a function of increasing PMA.

(From Shimony JS, et al. Comparison of cortical folding measures for evaluation of developing human brain. Neuroimage . 2016;125:180–190, with permission.)

Functional Magnetic Resonance Imaging

Whereas the MRI techniques described earlier assess structure, fMRI evaluates neural activity (see Table 7.5 ). As described in more detail in Chapter 10 , the method is based on the fact that neural activation causes local changes in oxyhemoglobin and deoxyhemoglobin levels, changes that result in a detectable MRI signal, and therefore are referred to as the blood oxygenation level dependent (or BOLD) signal. The neural activity may occur in relation to sensory stimulation (visual, auditory or sensorimotor) or performance of a motor task (active or passive), that is, task-related, or with the infant at rest, that is, RS functional connectivity . These methods provide in vivo correlates of functional connectivity in the neonatal brain, and thus are dependent on fiber tract development, neuronal differentiation, synaptic development, and neuronal activity.

Cortical neural activation has been shown by fMRI after visual, auditory, and somatosensory stimulation in the newborn, including the premature newborn . A particularly informative report described the maturation of sensorimotor functional responses in the preterm brain. The evolution of the responses induced by passive movement of the wrist is shown in Fig. 7.21 . The localized functional activity is seen initially in the contralateral primary sensorimotor cortex at 31 to 32 weeks’ postmenstrual age (PMA), then progresses to include the midline supplementary motor area at 33 to 34 weeks, the ipsilateral peri-Rolandic cortex and thalamus at 35 to 36 weeks, and by term equivalent age, an adult-like pattern is seen with activation also of basal ganglia and contralateral opercular cortex/secondary somatosensory cortex. Notably, interhemispheric functional activity increases rapidly during the preterm period to a peak at 36 weeks PMA, but interestingly, then declines at term equivalent age. This decline in functional connectivity could result from increasingly specific functional connectivity.

Evolution of sensorimotor functional responses induced by passive movement of the right wrist. Following right wrist movement, localized functional activity was identified in all infants in the contralateral (left) primary somatosensory cortex. Functional responses can be seen to progress from a contralateral only pattern in the youngest infants at 31 to 32 weeks postmenstrual age (PMA) ( top row , n = 9), to include the midline supplementary motor area (SMA) at 33 to 34 weeks ( second row , n = 13), and the ipsilateral peri-rolandic cortex and thalamus at 35 to 36 weeks ( third row , n = 10). At term equivalent age (37 to 44 weeks; fourth row , n = 15), a mature adultlike activation pattern is seen in the bilateral peri-rolandic regions, basal ganglia, SMA, and contralateral opercular cortex/secondary somatosensory cortex.

(From Allievi AG, et al. Maturation of sensori-motor functional responses in the preterm brain. Cereb Cortex. 2016;26:402–413, with permission.)

A second approach to the investigation of neural connectivity involves RS connectivity (see Chapter 10 ). In this circumstance, the infant is either asleep or resting, and low frequency BOLD signals are studied. Temporal correlations within regions and between connected regions are identified. The two principal methods involve either seed correlation analysis (i.e., a specific region of interest, seed , is identified and signals from other regions that correlate temporally with the seed region are sought) or independent component analysis (i.e., analysis of signals from the entire brain to identify areas of correlation). Both approaches have been used effectively in the study of premature infants. Evolution of highly connected cortical regions, that is, hubs , especially involving various early maturing cortical areas and thalamus, has been identified from early in the premature period. Short corticocortical connections are apparent initially, and longer corticocortical connections develop toward term. These so-called resting state networks (RSNs) incorporate cortical gray matter regions located initially in primary motor and sensory (somatosensory, visual, and auditory) cortices. The foundations of these networks are identifiable as early as 26 weeks PMA. Thalamocortical connectivity also is especially prominent. The rate of development of the various networks appears to correlate with the rate of development of the regions involved and, presumably therefore, the establishment of connectivity. Thus RSNs involving primary motor and sensory areas are established by term PMA. However, RSNs involving association cortices (e.g., those mediating attentional functions) mature more rapidly after term. These features reflect the development of axons, fiber tracts, myelination, and cortical dendrites. Combining these studies with diffusion tensor tractography provides an insight into the fiber tracts likely involved in interconnecting various components of the developing brain.

Disorders of Subplate Neurons

Clearly, the time periods when the functions of the subplate neurons must be operative in the developing human brain correspond closely to the times of occurrence of a variety of periventricular hemorrhagic and ischemic lesions (see Chapters 16 and 24 ). If these lesions disrupt the subplate neurons or their axonal collaterals to subcortical or cortical sites, the functions described earlier would be impaired, and the impact on cortical neuronal development and on a variety of crucial projection systems could be enormous. Indeed, the major portion of the striking increase in cerebral cortical volume in the last trimester of gestation consists of the extensive elaboration of the afferent fibers, previously “waiting” in contact with subplate neurons, as they enter the cerebral cortex. As noted later, this increase in cerebral cortical volume is blunted by injury to cerebral white matter in the premature infant. The role of subplate neuronal injury in the encephalopathy of prematurity is discussed in Chapters 14 and 15 . In addition, subplate neuronal pathology has been suggested in a variety of other neurological disorders, including epilepsy, autism, and schizophrenia beyond the neonatal period. Drug-resistant epilepsy is often accompanied by severe cortical dysplasias, in which large groups of cells are also abnormally located within the cerebral white matter. It has been postulated that this excess of interstitial white matter neurons is the result a failure of programmed cell death in subplate cells, although a failure of migration of cortical plate neurons to their final addresses has not been excluded. Excessive numbers of interstitial white matter neurons have been reported in autopsy analyses of the brains of patients with schizophrenia and autism. In effect, subplate neurons are the link between developing and mature circuits in the cerebral cortex, and dynamic disorders in their number, position, and molecular and genetic functions can have a major impact on the development of human cognitive and affective processing with far-reaching implications beyond the fetal, premature, and perinatal periods.

Disorders of Lamination

Many malformations of cortical development underlying epilepsy, cerebral palsy, global intellectual disability, and neuropsychiatric disorders include aberrant laminar patterns. These abnormal patterns include focal cortical dysplasia and the four-layered types of lissencephaly and polymicrogyria that are discussed in detail in Chapter 6 . Layer-specific markers have provided insight into the neurobiology of these patterns. A major goal in developmental neuropathology is to develop a panel of markers to analyze all neuron types and their distribution in the human neocortex to elucidate abnormalities in the position and number of each neuron type in cortical malformations, as well as in metabolic and degenerative diseases that involve selective loss of specific neuron types.

Disorders of Gyrification

Abnormalities in gyrification in the cerebral cortex are associated with various genetic and acquired malformations, including pachygyria, lissencephaly, and polymicrogyria, as reviewed in Chapter 6 . Other gyral abnormalities include maldevelopment of individual gyri and/or lobes, resulting, for example, in small superior temporal gyri in Down syndrome.

Disorders of Dendrites and Synaptogenesis

Dendritic pathology occurs in multiple human disorders in early life, of which several major examples are highlighted here for illustration.

Mental Retardation With or Without Seizures

Several studies in which the Golgi technique was used have shown abnormalities of development of dendritic branching and spines in children with idiopathic mental retardation with or without seizures. The children in these studies had no anatomical evidence for destructive disease, metabolic disorder, or other developmental aberration (see Table 7.6 ). Huttenlocher, using the Golgi technique and quantitative estimation of dendritic branching, initially studied 11 brains from individuals with severe mental retardation of unknown cause. In six of these brains, severe defects in the number, length, and spatial arrangement of dendritic branching and in dendritic spines, the sites of synaptic contacts, were demonstrated. The relative sparsity of horizontal and tangential dendritic branches is shown clearly in Fig. 7.22 . Four of the six affected children with marked dendritic abnormalities had, in addition to the severe mental retardation, histories of infantile myoclonic seizures and hypsarrhythmic electroencephalograms (EEGs). Moreover, Purpura demonstrated, in a cerebral biopsy specimen of a severely retarded infant, marked abnormalities of dendritic spines, characterized principally by a marked reduction in short, thick-necked spines (see Fig. 7.13 ). This finding was the only detectable defect in the biopsy specimen and after extensive clinical and laboratory studies yielded negative results.

Golgi preparation (×80) of middle frontal gyrus from a 10-year-old child with severe mental retardation of unknown origin. Note the relative sparsity of horizontal and tangential dendritic branches (compare with Fig. 7.10 ).

(From Buchwald NA, Brazier MAB, eds. Brain Mechanisms in Mental Retardation. New York: Academic Press; 1975.)

Insight into a major mechanism in the production of such defects was provided first by the work of Purpura and co-workers. Golgi studies of cerebral cortex from five children with mental retardation and seizures (two in one family) demonstrated striking dendritic abnormalities, the most prominent of which was the formation of distinct varicosities along the dendritic processes ( Fig. 7.23 ). Ultrastructural studies showed an aberration of microtubules with loss of the usual parallel array of these structures. These findings indicated that a disturbance of cytoskeletal structures, so critical for maintenance of cell shape and for outgrowth of dendrites and axons, can cause a severe dendritic abnormality and marked neurologic disturbance.

Camera lucida drawings of distal dendritic segments from an infant with mental retardation and seizures. Note the irregular varicosities of the dendritic segments.

(From Purpura DP, Bodick N, Suzuki K, et al. Microtubule disarray in cortical dendrites and neurobehavioral failure. I. Golgi and electron microscopic studies. Brain Res. 1982;281:287–297.)

More recent work has begun to delineate the molecular bases for at least some of these cases. X-linked genes have been shown to be particularly important, perhaps accounting in part for the higher incidence of mental retardation in male than in female patients. The greatest insight into X-linked mental retardation disorders involves fragile X syndrome and Rett syndrome (discussed subsequently). However, many other genes have recently been identified. Prominent among these are four X-linked genes found mutated in families with mental retardation that encode proteins known as Rho guanine nucleotide exchange factor 6 (ARHGEF6), oligophrenin-1, p21-activated kinase, and guanine dissociation inhibitor 1. These proteins are involved in signaling pathways that regulate the actin cytoskeleton, so critical for neurite outgrowth, dendritic spine formation, and morphology and neurotransmitter release. This rapidly evolving field may lead to insights into potential therapies.

Rett Syndrome

Rett syndrome is a complex disorder observed in full form only in females; it constitutes one of the most common causes of mental retardation in girls and women. The disorder is characterized clinically by onset of deceleration of rate of head growth in the first months of life, loss of purposeful hand movement near the end of the first year, and development of stereotypical movements with repetitive hand wringing, autism, ataxia, microcephaly, seizures, and global intellectual disabilities before the age of 5 years. Rett syndrome patients also demonstrate a spectrum of sleep disturbances and autonomic and respiratory dysfunction, including erratic breathing while awake with periods of alternating hyperventilation, cyanosis, apnea, abnormal heart rate variability, and sudden death. The course is typically progressive until early childhood when it becomes essentially static. The neuropathology consists of a small brain with an apparent disturbance of neuronal development, characterized by dendritic spine abnormalities (decreased spine density, simplified branching) and small, densely packed neurons ( Fig. 7.24 ).

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