Below are the first 10 and last 10 pages of uncorrected machine-read text (when available) of this chapter, followed by the top 30 algorithmically extracted key phrases from the chapter as a whole.
Intended to provide our own search engines and external engines with highly rich, chapter-representative searchable text on the opening pages of each chapter.
Because it is UNCORRECTED material, please consider the following text as a useful but insufficient proxy for the authoritative book pages.
Do not use for reproduction, copying, pasting, or reading; exclusively for search engines.
OCR for page 273
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.
OCR for page 274
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
OCR for page 275
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
OCR for page 276
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
OCR for page 277
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 norepinephrine—play 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.
OCR for page 278
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)
OCR for page 279
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~.
OCR for page 280
Representative terms from entire chapter:
neural tube
