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he Developmental Neurobiology of the Central Nervous System The developing nervous system has a unique sensitivity to interference by exogenous agents, including environmental agents, specific cytotoxins, and ionizing radiation. Several structural abnormali- ties that occur in the prenatal CNS (e.g., anencephaly, spine bifida, hydrocephalus, and anophthalmia) can be produced in labor- atory animals with the appropriate choice of species, test agent, and stage of in- trauterine development. This chapter provides background and a conceptual base for demonstrating that the vertebrate nervous systems develops through distinct processes and the estab- lishment of neurochemical systems. The morphogenetic processes include: Cytogenesis. Transformation of neuronal precursors (neuroblasts) from a mitotic population into a population of irreplaceable, non- mitotic neurons. Morphogenetic migration of this neur- on population to its appropriate position in the neuronal architecture. Death of selected members of the pri- mordial neuron population ("morphogenetic cell death," according to Saunders, 1966) that contributes to the final makeup of neuronal groups. Overt cytodifferentiation in the 273 cytoplasm and on the cell surface, leading to the formation of specialized cells (glia and neurons) and processes (axons, den- drites, and synapses). The chapter also reviews the complex array of endogenous neurochemicals that lead electric impulses across the synaptic junctions between neurons. Neuronal com- munication has several important fea- tures, including: The complex cellular architecture of the neuronal system, which involves multiple connections, redundancies, and positive and negative feedback loops. The synthesis, storage, release, and takeup of multiple neurochemicals in many neurons. Almost complete dependence of neuro- chemical synthesis on peripheral availa- bility of amino acids. BASIC MORPHOGENESIS The purpose of this selective review is to demonstrate that dysgenesis of the CNS can be understood in terms of two devel- opmental events: neuron death and neuron migration. The two events and their bio- logic consequences can be considered as biologic markers of neuronal development.

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274 If known, these events in the development of the CNS could serve as an effective model for the analysis of normal and abnormal development in other organ systems. Cell death is important in the development of the limbs, oral cavity, and secondary palate; cell migration is essential for normal development of the gonads, hemato- poietic system, and immune system. Several other components of normal dif- ferentiation could also serve as useful and effective biologic markers. These include: Ontogeny of the neural cell adhesion molecule. Patterns of axoplasmic flow and axon growth. Ontogeny of dendritic patterns. Expression of neuron transmitters. However, cell death and cell migration are concentrated on here, because they represent basic developmental events that are readily monitored and are known to be associated with normal morphogenesis. Neuronal death and migration can be manipu- lated to cause abnormal development within the CNS, and experimental produc- tion of cellular derangements can cause behavioral alterations in animals exposed to toxicants at specific periods of CNS cytomorphogenesis. The initial event of vertebrate CNS de- velopment is an alteration in the embryonic surface ectoderm by the chorda-mesoderm or its structural analogue. The altera- tion, referred to as the primary inductive stimulus, is apparently chemical. The region of the ectoderm that receives the stimulus becomes committed to the expres- sion of the neuronal phenotype. It is called the neural plate, and it is formed on the nineteenth day of intrauterine life-embryonic day 19 (ED 19)in the hu- man, on ED 7 in the mouse, and on ED 9.5 in the rat (Hoar and Monie, 1981~. Coinciden- tally with formation of the neural plate, the neural crest is recognized as a dis- tinct group of cells at the junction of the neural plate and the remainder of the sur- face ectoderm. The neural crest is the primary source of a wide array of neurons and mesodermal NEURODEVELOPMEN7AL TOXICOLOGY cells. A series of complex, well-organized alterations in the cytoskeleton of the cells in the neural plate, now properly called the neural epithelium, lead to an elevation of the plate that results in the formation of the neural groove. The raised sides of the neural groove fuse in the apical midline on the dorsal surface of the embryo to form the neural tube. At first, the interior of the neural tube is in direct communication with the fluid- filled amniotic cavity. Separation of the neural tube from the amniotic cavity occurs with the closing of the anterior and posterior neuropores of the tube. The closing occurs rapidly in mammals, being completed on ED 25-27 in the human, ED 9.0- 9.5 in the mouse, and ED 10.5-11.0 in the rat (Hoar and Monie, 1981~. BASIC CYTOGENESIS The closing of the neural tube starts a period of rapid proliferation followed by discrete waves of migration and cytodif- ferentiation. Capacity of the cells of the neural epithelium to proliferate oc- curs in a time-dependent, orderly sequence that results in the presence of a mitotic gradient from the cephalic to the caudal end of the embryo. However, specific re- gions of the CNS (e.g., the cerebellum and the cerebral cortex) give evidence of pro- longed proliferative capacity. The capa- city for cell division is retained well beyond birth in some parts of the brain in humans. Almost all neurons originate in the de- scendant cells of the neural plate that form the primitive neural tube. The cells line the central canal of the neural tube and give evidence of a characteristic pro- liferative pattern. Nuclei near the cen- tral canal can be observed in the various phases of mitosis. At this point, the mi- totic cells are connected by tight junc- tions and form an internal limiting mem- brane. These cells form a similar attach- ment, the external limiting membrane, on the lateral surface of the neural tube (Kaufman, 1966~. A precise pattern of DNA synthesis, in- terkinetic nuclear migration, mitosis, and postmitotic nuclear migration occurs

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DEVELOPMENTAL NEUROBIOLOGY OF CNS (Sidman et al., 1959; Fujita et al., 1964; Kauffman, 1966; Langman et al., 1966~. The neural epithelium is now referred to as the primitive ependymal zone (Sidman et al., 1959) or the matrix cell layer (Fu- jita et al., 1964~. Most cells enter the S phase of DNA synthesis when their nuclei are in the periphery. On completion of the S phase, the cells round up with their sur- face membranes still held at the internal limiting membrane. The nuclei (now at the 4c stage of mitosis) migrate within the cytoplasm to the medial surface of the layer of cells, where they complete mito- sis. The posttelophase nuclei migrate within the cytoplasm of the new daughter cells and again arrive at the periphery. This in-and-out nuclear (interkinetic) migration, with an interspersed S phase and a period of mitosis, contributes to a large increase in the size of the neural tube. The neuroblasts that are programed to differentiate lose contact with the central canal and, presumably as postmi- totic cells, migrate from the neural epi- thelium and come to populate the mantle layer. On completing their migration, these cells begin processes that allow them to complete their differentiation. Development of Major Subdivisions The anatomic disposition of the CNS is the result of a series of developmental events within the neural tube. The basic morphology of the brain and, in particular, the formation of its major subdivisions telencephalon, diencephalon, mesencepha- lon, metencephalon, and myelencephalon- arise from differential mitosis and selec- tive cell death (Bergquist and Kallen, 1954; Bergquist, 1964~. The basic pattern also occurs in the developing spinal cord, where neuroblasts in the anterior (basal plate) region have a higher initial mitotic index than those in the dorsal (alar plate) region (Corliss and Robertson, 1963~. Migrating neuroblasts in the anterior region therefore become postmitotic neur- ons earlier (Langman and Haden, 1970~. In general, neuroblasts formed in the cere- bral cortex conform to the basic pattern, albeit with some exceptions-for example, the Cajal-Retzius cells in layer I. How- 275 ever, in the cortex, cells closest to the central canal migrate, as neuroblasts, out of the primitive ependymal zone earlier than more peripheral cells. This "inside- out" pattern occurs as a result of specific spatial and' temporal gradients that cause large neurons to be produced before small ones (Hicks et al., 1961; Langman and Welch, 1967; Jacobson, 1978~. Cell death is another part of normal development of the CNS. It occurs through- out neurogenesis (Kallen, 1965) and plays a necessary role in the formation of sever- al regions of the CNS, two of which deserve special mention. In the limbs and axial musculature, the two components of the complex of motor nerves and striated muscle develop independently, and lack of effective contact between the two cel- lular elements leads to the degeneration and death of both (Jacobson, 1978; Vrbova et al., 1978~. In addition, the nuclei of motor neurons are characterized by over- production of cells; cells whose peripher- al processes fail to make contact with developing myotubes undergo a normal se- quence of degeneration and death. In the developing eye, cell death also plays a prominent role. During development, the neural retina exists as typical neural epithelium. However, unlike the cerebral cortex, this structure does not develop ~inside out.~ The largest and most peri- pheral neurons (the ganglion cell layer) are formed first on ED 11 in mice, whereas bipolar and photoreceptor cells are formed on ED 13 (Sidman, 1961~. Retinal neuron formation continues postnatally, ceasing on postnatal day (PN) 6 (Sidman, 1961; Young, 1985~. After formation of central connections by the optic nerve, selective postnatal death of neurons formed earliest in development, the ganglion cells, occurs throughout the retina (Sengelaub et al., 1986~. Specific Development of Neuronal Type Altman ( 1986) classified neurons into three principal types on the basis of their developmental origin: macroneurons, meso- neurons, and microneurons. Typically, macroneurons are large cells with long axons; the motor neurons of the spinal cord

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276 are macroneurons. Mesoneurons function primarily as relay cells, such as the relay neurons of the dorsal column nuclei. Mi- croneurons are local elements that con- tribute to the fine circuitry of a given brain region; examples are the granule cells of the olfactory bulb, the cerebellar cortex, and the hippocampal dentate gyrus. The three kinds of neurons differ in ontogeny. Macroneurons tend to form and differentiate early, during the embryonic period. Microneurons, at least in the rat, are formed and complete their development during the postnatal period (Altman, 1966; Pellegrino and Altman, 1979~. Mesoneurons are intermediate in this context. Neuronal ontogeny is best documented in the development of the cerebellum. The macroneuron component, the Purkinje cell, forms first and during embryonic devel- opment-ED 11 in the mouse (Uzman, 1960) and ED 14-15 in the rat (Altman and Bayer, 1978~. The Golgi cells form next. The cere- bellar microneurons form from a prolifera- tive population on the surface of the cere- bellar cortex, i.e., the external granule cell layer. The external granule cells are intensely mitotic in the first 7- 10 days after birth in rats. The postmitotic neuroblasts formed in this region then migrate centrally through the molecular layer and come to reside as the internal granule cell layer, or granule cells, be- neath the layer of Purkinje cells. This pattern of proliferation and migration occurs sequentially, so subpopulations of the granule cells are formed between PN 10 and PN 25 in rats (Pellegrino and Alt- man, 1979~. The pattern is similar in the ontogeny of the granule cells of the dentate gyrus, which make up the microneuronal compart- ment of the hippocampus. These cells also originate in the primitive ependymal zone surrounding the lateral ventricle. They migrate and continue their mitotic activi- ty over the first 2 weeks after birth in rats and take up their position as postmi- totic neurons in a specific pattern. The oldest cells are deposited as the top row of granule cells in contact with the super- ficial plexiform layer, and the younger cells end up in the basal layer (Altman and Das, 1966; Jacobson, 1978; Cowan et al., 1980~. NEURODEVELOPMENTAL TOXICOLOGY NEUROCHEMISTRY OF NEURONAL COMMUNICATION Biochemically, the nervous system func- tions as sets of connecting pathways of cells that send and receive information by releasing specific chemicals that translate changes in the electric proper- ties of cell membranes into intracellular activity of enzymes. These chemical events usually occur within very small spaces that separate most nerve cells and their effecters, i.e., the synaptic clefts. Many cell-cell connections are short and involve only cells near each other. However, others are very long and connect the releasing cells with distant organs or targets by long cellular proc- esses or indirectly through the circula- tion. The cellular architecture of the nervous system includes multiple connec- tions, redundancies, recurrent pathways, negative and positive feedback loops, autoreceptors, densely and often highly arborized projections, and a variety of structures and cell types. In only a few instances have the connections between regions of the CNS been comprehensively mapped; in most cases, the efferent and afferent networks in even a fairly well- defined region (such as the locus cerule- us) are known only very incompletely. The term "neurotransmitters" is used here to include all chemical substances that carry signals between cells, includ- ing neuromodulators and other cell-cell signaling chemicals. Only a small frac- tion of the very large number of cells in the human brain release the few well-char- acterized neurotransmitters. That is, the neurotransmitters released by most neurons are either unknown or poorly char- acterized. Moreover, many neurons are now known to contain and release more than one neurotransmitter; that greatly increases the complexity of information processing between cells. The old concept of the brain as a computing machine, in which cells or nodes in the system were only "on" or Noff,N has been replaced by an awareness of gradations in neural states. In addition, events can persist and influ- ence the outcome of later events. Communication between cells is affected by linked biochemical events involving

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DEVELOPMENTAL NEUROBIOLOGY OF CNS cascades of second and third messengers, such as the cyclases, phosphodiesterases, calcium-binding proteins, and other en- zymes and proteins (Snyder, 1984~. RNA- directed protein synthesis can also be part of the messenger sequence of neuro- transmission (Kandel and Schwartz, 1981; Gusella et al., 1984~. These events take place within cells, so their biochemical products might never be released into com- partments other than the immediate synap- tic milieu. Other neurons regulate more distant events, such as the release of trophic hormones from the pituitary, up- take of nutrients from the gut, and func- tion of smooth muscle in the peripheral vasculature. These products of neuro- transmission can be measured physiolog- ically as changes in circulating hormones, intestinal absorption, or venous blood pressure, for instance. In addition to mediating intercellular communication, endogenous neuroactive substances play an important role in devel- opment. In the early stages of brain devel- opment, some of the classic neurotransmit- ters-such as gamma-aminobutyric acid (GABA) and norepinephrineplay a trophic role, guiding the formation and fixation of axonal projections and synaptic connec- tions (Black et al., 1984~. The latter neurochemical-dependent processes of enervation have been elegantly demon- strated in the model of neuronal develop- ment of Hoffer et al. (1987), in which fetal brain is transplanted into the chamber of the eye in rodents and its development is monitored chemically, morphologically, and electrophysiologically. Synthesis of transmitters (T) 2 Release (secretion) 3 Binding to receptor (R) 4 ~ 1 Removal or destruction of T | ~ ~ R l T ~ T | 277 As shown in Figure 25-1, the fundamental neurochemical cycle of neurons and glia in the nervous system comprises the proc- esses of uptake, transport, synthesis, storage, and release. Neuronal uptake, which is kinetically highly efficient and saturable at low concentrations, can serve several functions, including termination of cell stimulation by removing the neuro- active compound from the receptor, resup- ply of intracellular pools for later re- lease, and provision of precursors for the synthesis of neurotransmitters, such as choline for acetylcholine, tyrosine for dopamine and norepinephrine, and tryp- tophan for serotonin. Transport is a cri- tical process in neurons, particularly those with long axonal projections and extensive dendritic arborizations, in which enzymes and other materials synthe- sized in the cell body must be moved to the terminals. Synthesis of neurotransmit- ters involves highly regulated pathways. Some of the pathways can be used in other metabolic processes, in which case the synthetic pathway in neurons is usually distinguished by kinetic properties, rate-limiting cofactors, or compartmenta- tion. Other synthetic pathways involve RNA-directed synthesis and enzymatic cleavage of large polypeptide precursors for the formation of neuroactive peptides, such as the enkephalins and so-called gut peptides. Storage in neurons involves compartmentation and intracellular trans- port of precursors and products by mechan- isms that protect these substances from enzymatic degradation or hydrolysis. Storage can also provide a dosimetric func- FIGURE 25-1 Four biochemical steps in synaptic transmission: synthesis of neurotransmitter ~, re- lease of transmitter synaptic cleft, binding of transmit- ter to postsynaptic receptor, and removal or destruc~ tion of transmitter substance.

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278 lion by packaging neurotransmitters in minimal releasable amounts (or quanta). The dosimetric function can be important for maintaining trophic relations between cells, as has been demonstrated for cholin- ergic pathways in the peripheral nervous system. Release is the exocytotic process in which neurotransmitters are secreted by cells into the extracellular space. The release is usually ion-dependent and hinges ultimately on changes in intracel- lular free-calcium concentrations, which appear to control the fusion processes necessary for exocytosis. After release, neurotransmitters act by binding to receptors on cells. Most receptors in the nervous system are mem- brane-bound and react to substances that reach the outer membrane of the receiving cell. Neuronal receptors can be grouped in complexes, such as the interrelated set of benzodiazepine, GABA, and chloride ionophore receptors in the GABA-ergic pathway. Activation of neuronal receptors translates into biochemical events in the receptive cell (such as activity of adenyl cyclases) that are then linked in a func- tional cascade of phosphorylation reac- tions that can stimulate or inhibit other enzyme activities, alter the nature and permeability of the cell membrane, and manifest other functions (Kandel and Schwartz, 1981~. The problems of obtaining access to the essential biochemical processes so as to use them as markers of neurobiologic function are exemplified in amino acid neurochemistry. Some amino acids-such NEURODEVELOPMENTAL TOXICOLOGY as glutamate, aspartate, and glycine are neurotransmitters in brain and spinal cord. However, the largest quantities of these amino acids in the body are in- volved in general intermediary metabol- ism; only a small fraction is reserved for the specific role of cell-cell communica- tion. The brain does not synthesize amino acids for neurotransmission, nor are they derived from catabolism within the brain. Neurons obtain amino acids for neurotransmission by removing them from the circulation through previously de- scribed high-affinity uptake processes. Building-block amino acids, such as tyro- sine and tryptophan, are required for syn- thesis of other neurotransmitters; the brain must obtain these amino acids from the circulation. Because of that absolute dependence, changes in the peripheral availability of amino acids might be ex- pected to alter the concentrations of neurotransmitters and, consequently, affect the function of some neural pathways in the brain. Conversely, high amounts of some amino acids in the diet, such as the excitatory neurotransmitters gluta- mate and aspartate, might be expected to be neurotoxic. The potential neurotoxi- city of dietary amino acids, particularly during development, has received some attention recently, because of the in- creasing use of aspartame, a simple deriva- tive of aspartate, as a sweetening agent (Sved, 1983~. A body of evidence from neuropharmacologic research (Sved, 1983) indicates that alterations in circulating TABLE 25-1 Biochemical Markers of Development and Cell Injury in the Nervous System Biochemical Marker Indicator For Central Spinal Fluid Marker Protein I D2 (neural tube) B50 PSD 95 Myelin basic protein (MS) Myelin-associated glycoprotein GFAP Brain Tissue Markers Protein III Synapsin I Status of synaptic membranes of CNS neurons Status of synaptic membranes of CNS neurons Status of synaptic membranes of CNS neurons Postsynaptic receptors Status of oligodendroglia and myelin sheath Oligodendroglia Astrocytes (gliomas) Cell loss (nerve terminals) Cell loss (nerve terminals)

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DEVELOPMENTAL NEUROBIOLOGY OF CNS concentrations of some amino acids (such as tryptophan) can affect CNS neurochemis- try. However, the implications of the biochemical modifications for functional changes, such as neuronal activity in sero- toninergic pathways, are not yet clear. Reliable inferences regarding the sta- tus of function in pathways that use amino acids cannot be drawn from measurements of peripheral amino acid metabolism. Some research attempted to develop an index of CNS cholinergic function with arterio- . 279 ferences that could be correlated with major changes in cholinergic function were ever found. Thus, chemical indicators or biologic markers of neuronal function (see Table 25-1 for a partial list) are difficult to obtain, particularly outside the nervous system itself. However, other cell proc- esses might be investigated, such as cell death, turnover of membranes, and cellular differentiation (O'Callaghan and Miller, 1983; Bondy, 1985~. The utility of chemi- venous difference in blood choline con- cat indicators was demonstrated in studies centration as a marker (E. Silbergeld, of neurotoxicity in animals exposed to Environmental Defense Fund, personal the neurotoxicant trimethyltin (O'Calla- communication, 1987~. No consistent dif- ghan and Miller, 1984; Harry et al., 1985~.

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