Cytology
of the normal central nervous system
INTRODUCTION
The
central nervous system (CNS) is composed of various types of cell including
neurons, glial cells (astrocytes, oligodendrocytes, ependyma and choroid plexus
epithelium), blood vessel elements and microglia. Leptomeninges (pia mater and
arachnoid mater) surround the CNS, and the pachymeninges (dura mater) form an
outer coating which separates the brain from 'the skull and the spinal cord
from the spine.
There are illustrations throughout
subsequent chapters which depict many of the histological and macroscopic
features of the nervous system. This chapter, however, is more concerned with
tlte basic structure and function of the different cell types in the brain and
spinal cord, an under-standing of which is a prerequisite for an appreciation
of the pathology of the nervous system.
NEURONS
Nerve
cells vary enormously in size and con-figuration. The cerebellum presents the
best example of morphological extremes: the Purkinje cells and their impressive
dendritic trees dwarf the neighbouring granule cells and their short dendrites
(Figs 1.1 and 1.2).
The shape of the nerve cell body,
the perikaryon, also varies throughout the central nervous system: pyramidal,
polygonal, round, oval and fusiform forms can be distinguished. Neuronal cell
processes fall into two categories: dendrites and axons. Whilst a neuron
usualJy has only a single axon, the number of dendrites ranges from one to
nearly one hundred. The length and arborisation of processes also display
considerable individual variations. Silver impregnation techniques of classical
neurohistology, electron microscopy, enzyme histochemistry and more recently
immunocytochemistry have all contributed.. to the under-standing of neuronal
morphology and function. The previous morphological classifications of nerve
cells are now gradually being replaced by classifications based on functions:
neurons can be distinguished according to the neurotransmitters they use.
The
nucleus
Nerve
cell nuclei are round or oval, although the
nuclear
profile is occasionally indented. The nucico- or karyoplasm is separated frorn
the cytoplasm by the nuclear membrane which is composed of two laminae
enclosing the perinuclear cisterna: the inner lamina is smooth, whilst the
outer one, studded with ribosomes, is frequently continuous with the rough
endoplasmic reticulum. Finely granular chromatin is evenly dispersed with some
clumping at the nuclear membrane.
The nucleolus is prominent and this
helps to distinguish neurons from astrocytes which contain only a small
inconspicuous nucleolus. Electron microscopy reveals the nucleolus to be
composed of granular and fibrillar constituents. Attached to the nucleolus is
the nucleolar satellite or sex chromatin: a dense, coarsely fibrillated body
which occurs only in females.
Nuclear inclusions of various types,
filamentous, granulofibrillar, tubular and vesicular,' often occur in normal
neurons and can be occasionally discerned in the light microscope. Variations
in nuclear morphology exist according to neuronal type, topography and
functional activity. The large vesicular nuclei of the Purkin)e cells or the
pyramidal cells of the cerebral cortex are in sharp contrast to the small,
dense nuclei of the cerebellar granule cells .
Cytoplasmic organelles
Nissi substance
This
cytoplasmic component is intensely basophilic when stained with cresyl violet
or methylene blue. Electron microscopy shows that Nissl substance is composed
of stacks of the rough-surfaced endoplasmic reticulum (RER) (Fig. 1.4). The
cisternae are usually arranged in parallel arrays and their outer surface is
covered by ribosomes. The amount of RER varies from neuron to neuron and
appears to be related to the length of the axon: it is particularly abundant in
the anterior horn cells of the spinal cord and the pyramidal neurons of the
cerebral cortex. It is present not only in the perikaryon but also in the
dendrites, but it is absent from the axon hillock and from the axon itself.
Since the RER is the site of protein synthesis, its amount is related to the
metabolic
activity
of the cell. The Nissl substance forms an integral component of the neuronal
membrane system which is associated with the transfer of membrane molecules
amongst organdies, the packaging and transport of exportable protein and energy
metabolism.2 In addition to the membrane-bound particles of the RER,
ribosomes are scattered throughout the cytoplasm either singly or in groups as
polyribosomes or ribosomal rosettes.
the
axon and dendrites. Although the precise function of the agranular reticulum is
not known, its widespread distribution within the cytoplasm and its close
spatial relationship to the cell membrane suggest a role in intracellular
transport.
The internal reticular apparatus of
Golgi appears as tortuous anastomosing strands in the light microscope. Located
between the nucleus and the cell membrane or at the base of a dendrite, the
Golgi apparatus is composed of arrays of closely packed, smooth-surfaced
cisternae and numerous associated vacuoles and vesicles (Fig.
1.4).
Its curved configuration allows a convex, external forming face and a concave,
internal mature face to be distinguished. The internal face of the Golgi
apparatus forms an integral part of the GERL
system (Golgi apparatus, smooth endoplasmic reticulum and lysosomes): a
spatially closely related group of organelles which is devoted to the
synthesising and condensing
activity
of the Golgi apparatus.3 This polarisation of the Golgi apparatus,
although not evident in all neurons, is thought to serve the efficient
transport of materials. The demonstration of concanavalin A binding sites on
Golgi membranes strongly suggests that this organelle is concerned with
glycoprotein metabolism.1 The formation of new plasma membranes
during mitosis and the maintenance of normal membrane structure in resting
cells are important functions of the Golgi apparatus.4
The Golgi apparatus itself promotes
the movement of membranes: vacuoles fuse to form a cisterna at the forming
face, while vesicles are budding off at the mature face. The diameter of these
vesicles ranges from 20-60 ~m and they are usually smooth-surfaced, round or
elliptical. Coated or alveolate vesicles of 50-60 ~m diameter are distinguished
by their regu]arly spaced arms or striac. These vesicles are concerned with the
trans-
port
of hydrolytic enzymes to the lysosomal system. Larger alveolate vesicles, lOOnm
in diameter, derived from the cell surface by invagination or pinocytosis,
ingest proteins which then are transferred to the lysosomes to be digested. In
-1addition,
many neurons, particularly those of the supraoptic and paraventricular nuclei,
contain dense-cored, neurosecretory granules up to -150 nm in diameter.
Multivesicular bodies are spherical
structures of 0~5 ~m in diameter which contain vesicles and an assortment of
inclusions. Although frequently associated with the Golgi apparatus, they are
not part of these organelles and occur throughout the perikaryon and cell
processes. Multivesicular bodies sequester material transported to them by
coated vesicles, and hydrolytic enzymes acquired from smaller coated vesicles
convert them into lysosomes.
Lysosomes
Primary
lysosomes are round or oval, uniformly dense bodies up to 1 pm in diameter, bound
by a unit membrane. They contain various hydrolytic enzymes, including acid
phosphatase which serves as a marker enzyme for their identification.3
Secondary lysosomes are larger, more irregular and usually contain various
ingested materials.
Lipofuscin granules
Although visible by light
microscopy, lipofuscin can be better appreciated in the electron microscope: a
single membrane encloses a dense granule and one or two peripheral vacuoles
(Fig. 1.3). Lipofuscin or lipochrome is frequently referred to as
'wear-and-tear' pigment, since the number of granules within the neurons
increases with age. These granules are end products of intracellular
peroxidation and polymerisation of unsaturated fatty acids. Their origin is
controversial, but according to the most widely accepted concept they are
denv~d from lysosomes. The precise function of lipofuscin is unknown.
Degenerated cytoplasmic
constituents contribute to
its formation, which requires intracellular oxidants and antioxidants.
Lipofuscin is thus the byproduct of metabolic activity; it is stored in lyso
somes
and is only disposed of by expulsion during mitosis or by cell death.5 Since
mature nerve cells do not divide, lipofuscin granules accumulate during life.
Appearing as light brown granules in haematoxylin and eosin stained sections,
lipofuscin can be demonstrated by a variety of fat stains including Sudan
black B, Sudan III and Nile blue sulphate. It is acid fast and PAS positive and
reduces ferric ferricyanide to Prussian blue (Schmorl's reaction).
Melanin
This
pigment is present in the leptomeningeal cells and in the pigmented nuclei of
the brainstem. Melanin-containing cells in the leptomeninges are most plentiful
over the ventral aspect of the brain-stem; this is the area in which the rare
primary malignant melanomas most often develop. Two major accumulations of
pigmented neurons occur in the brainstem: in the substantia nigra of the
midbrain and in the much smaller locus ceruleus of the pontine tegmentum. The
cells of the substantia nigra and locus ceruleus are dopaminergic and
noradrenergic respectively. Other
brainstem nuclei, including the motor nucleus of the vagus, also contain
melanin in small amounts.
Mitochondria
These
organelles vary considerably in shape and size, depending upon their location
within the neurons. They are apparently distributed at random in the perikaryon
and also occur in dendrites and axons, including the presynaptic terminals.
Mitochondria have a smooth outer membrane and an irregular inner membrane. The
inner membrane forms complex folds (cristac); the inner compartment of the
mitochondrion is filled with a moderately dense matrix (Fig. 1.3). The inner
surfaces of the cristae are rendered uneven by many minute projections, each
ending in a knob. The inner and outer membranes differ in composition,
structure and function; the electron transport mechanism and many hydrogenases
are located in the inner membrane, whereas the monoamine oxidase system is
found in the outer membrane.1
The cytoskeIeton:-4
The
neuronal cytoskeleton is composed of three elements: neurofilaments,
microtubules and microfilaments.
Neurofilaments. These filaments are 10 nm in
diameter and of indeterminate length. They belong to the class of intermediate
filaments, their diameter being in between that of the micro-filaments (4-6nm)
and that of microtubules (24 nm). Of the five types of intermediate filaments
found in animal cells, two occur in the central nervous system: neurofilaments
in nerve cells and glial filaments in astrocytes. Although the various types of
intermediate filaments appear to be similar morphologically, they are composed
of different proteins.
In cross-section, neurofilaments
display a central hollow core) surrounded by a wall of 3 nm in thickness. The
basic unit of the wall is composed of four globular subunits, each 3.5 nm in
diameter, which are linked together by connecting arms of 2~5 nm in thickness.
These units are stacked one upon another and rotated in the transverse plane by
45~ to each other.6 In longitudinal section, neurofilaments appear
as two dense, parallel lines which enclose the central hollow core. They are
dispersed throughout the perikaryon, but tend to accumulate at the base of
large processes, into which they extend, forming parallel arrays.
Neurofilaments isolated from the
nervous system are formed by three polypeptides with the approximate molecular
weights of 70, 150 and 200 kilodaltons.7 Recent investigation has
revealed that immunohistochemical differences may exist between neurofilaments
in the perikarya, dendrites and axons.
Although the morphology and
biochemistry of neurofilaments have been established,9 very little
is known about their functions. They are intimately related to microtubules,
the other component of the neuronal cytoskeleton, and with them they could play
a role not only in stabilising nerve cells, but also in axonal transport.10
Neurofilaments are also major intrinsic determinants of axonal diameter in
large myclinated nerve fibres: the expression of a single set of
neuron-specific genes encoding neurofilaments directly determines axonal
calibre."
Alicrotubules. Microtubules are
of indeterminate length and 24nm in diameter. Their walls are 6nm thick
and composed of 13 globular subunits, each representing a constituent protofilament.'0
Microtubules are intermingled with neurofilaments both in the penkaryon
and in cell processes: the ratio of neurofilaments to micro-tubules decreases
as the axon becomes smaller, and in thin unmyelinated axons usually only micro-tubules
are present. Tubulin, the major protein component of microtubules.
In an
adult brain this protein comprises 10-30" of the soluble protein'0 and
more recently a group of microtubule-associated proteins has also been
recognised. Many functions have been attributed to microtubules:
skeletal support, maintenance of axonal flow, transport of various substances
and of cytoplasmic vesicles, cellular contraction and a r6le in mitosis.'
Microtubules also play a r6le in the maintenance and function of the Golgi
apparatus."
Microfilaments. These are not obvious in nerve
cells: they have a diameter of 4-6nm and are composed of actin, a globular
protein with a molecular weight of 42000." Microfilaments, in addition to
their stabilising function, also play a r6le in axonal transport.10
Synapses
Synapses
are specialised interneuronal contacts which can be electrical or chemical.
Electrical synapses function by the propagation of electrical impulses and do
not require elaborate structural organisation of the plasma membrane and cytoplasmic
organelles. These synapses, which are rare in matnmals but common in lower
vertebrates, are formed by the close apposition of the cell membranes at a gap
junction. Chemical synapses, in
contrast, utilise various substances-neurotransmitters and
neuropeptides-for intercellular communication:
this chemical transmission requires a sophisticated
subcellular mechanism well demonstrated by electron microscopy. By light
microscopy, using silver impregnation techniques, only the profile of the
end-bulb or bouton terminal of the axon abutting onto the surface of the
perikaryon or dendrite can be seen. A synapse is composed of three
constituents: the presynaptic element, the synaptic cleft and the postsynaptic
component (Fig. 1.5). Both the pre- and the postsynaptic membranes display
densities along the speci~ised stretch of cell membrane and these, together
with the intervening cleft, are referred to as the synaptic junction.
Originally two types of synapse were
distinguished on ultrastructural examination of the pyramidal cells of the
cerebral cortex. The type I junction is usually extensive, the postsynaptic
density prominent, rendering the synapse asymmetrical, and the wide synaptic
cleft contains a dense plaque. In the type Ii synapse, in contrast, the
postsynaptic density is less readily identifiable and the narrower cleft does
not contain a dense plaque; the overall configuration is symmetrical.15
The presynaptic element in most cases is an axon, either at its end (bouton
terminal) or along its course (bouton en passant): the axonal terminal can form
a synapse with any part of another neuron and thus axo-dendritic, axo-somatic
and axoaxonal synapses are distinguished. The presynaptic element, however,
does not need to be an axonal terminal and synapses can be formed between any
part of two neurons, providing a wide variety of synapses.
The presynaptic element contains the
synaptic vesicles which, in turn, are filled with neurotransmitters (Fig.
1.5). The size, shape and content of the vesicles vary, depending upon
the type of synapse. The most frequently Occurring vesicles are apparently
clear, spherical and 40-50 nm in diameter. Elongated or flattened vesicles with
clear centres are 20 nm wide and 50 nm long. There have been many attempts to
correlate vesicular shape with functional activity: spherical vesicles occur in
excitatory, type I synapses, whilst flattened vesicles are associated with inhibitory,
type II synapses. Larger vesicles of up to 60nm in diameter with a dense core
are encountered in areas of catecholamine activity and contain noradrenaline,
dopamine or 5-hydroxy-tryptamine. Another, larger dense-cored vesicle, up to
lSOnm with a spherical dense core of 50-70 nm in diameter, is most frequently
seen in the presynaptic terminals of autonomic ganglia, but also occurs in
various parts of the central nervous system intermingled with clear vesicles.
The presynaptic element, particularly the axonal terminal, also contains other
organelles, including mitochondria, neurofilaments, microtubules, smooth endoplasmic reticulum and glycogen.
The synaptic cleft, separating the
pre- and postsynaptic membranes, is 20-3Onm wide and usually contains a dense
plaque of intercellular material. In addition, filaments appear to traverse the
cleft and contribute to the formation of the plaque.
The
postsynaptic membrane is
rendered conspicuous by an accumulation of dense material on its cytoplasmic
surface (Fig. 1.5). This post-synaptic density, composed of granular material
and an occasional filamentous structure, is more pronounced in asymmetrical,
type I, synapses. The existence of postsynaptic density is dependent upon the
presence of the presynaptic element: if the
presynaptic structures degenerate,
the postsynaptic density will eventually disappear. The postsynaptic
element ~so contains cisternac of the smooth endoplasmic reticulum and the
spine apparatus, which is composed of 2-3 flattened cisternac, separated by
plaques of dense material.
The morphological appearances of
synapses reflect the functional
dynamics of neurotransmission.
Accordingly, in chemical synapses the vesicles contain quanta of the
neurotransmitter which is discharged into the synaptic cleft after the vesicle
has fused with the presynaptic membrane. Thus, the uptake of calcium into the
nerve terminal triggers off a chain of events leading to exocytosis: vesicular
apposition, membrane fusion and fission.16 Although this vesicular
or exocytotic hypothesis of neurotransmission'7 provides a plausible
explanation for the quantal nature of transmitter
release, an increasing body of evidence now suggests that this mav not be the
only mechanism by which chemical signals in the central nervous system are
propagated. Chemical substances may be released from non-synaptic axon
terminals and even from regions of nerve cells other than presynaptic axon.
A parasynaptic system is envisaged
in which so-called neuroactive or informational substances reach specific
target cell receptors by diffusion from release points through the
extracellular space. The mechanism of exocytosis itself remains poorly
understood not only morphologically, but also neurochemically, and various
alternative biochemical mechanisms have been considered to explain the release
of neurotransmitters.
Neuronal processes
AXONS
The
transitional zone between the neuronal perikaryon and the axon is the
cone-shaped axon hillock. In larger, multipolar neurons the axon hillock lacks
Nissl substance, and it is in this region that cytoskeletal elements, the
microtubules and neurofilaments, converge to become parallel as they enter the
axon. The microtubules then form a fascicle in the initial segment of the axon,
a feature 'vhich enables axons to be distinguished from dendrites, in which
microtubules are more evenly distributed. The more distal portion of the axon
may contain various organelles, but granular endoplasmic reticulum and
polyribosomes are usually absent. The internal structure of the axon has
recently been visualised as a three-dimensional lattice: the longitudinally
orientated neurofilaments and microtubules are extensively cross-linked to
each other and to the plasma membrane by thin filaments. Similar bridges also
connect membrane-bound organelles with the components of the cytoskeleton and
with each other .There is axonal transport of material from the perikaryon
towards the periphery (anterograde transport) and to a lesser extent in the
opposite direction (retrograde transport). Since the axon terminal,
the most active site of neurotransmission, can be far removed from the
perikaryon in which proteins are synthesised, this axonal transport is vital
for the proper functioning of the nerve cell. Axonal flow has a slow and a fast
component, 0~2-8~0mm and 50-500 mm per day respectively. Fast axoplasmic flow
carries organelles, including vesicles and mitochondria, and membrane-bound substances
like proteins and neurotransmitters
materials which are essential for synaptic activity. The fast axonal
flow is effected by microtubules, but the possibility that some membrane-bound
proteins are transported by the smooth endoplasmic reticulum Or
another tubulo-vesicular system cannot be excluded. Slow flow,
in contrast, transports high molecular weight and soluble materials which are
involved in the growth and maintenance of the axon. The structural basis of
slow transport is controversial, but the movement of the cytoplasmic matrix
itself may represent the prime force.23 Mechanisms of anterograde
and retrograde transport are similar: both need metabolic energy, have similar
ionic requirements and are sensitive to the same drugs.23 Both
anterograde and retrograde transport can be blocked by low temperatures and
suspended by colchicine and vinblastine, agents which disrupt microtubules. The
factors which determine the direction of axonal transport remain conjectural: alternatives
envisage either two, oppositely polarised transport systems or a single
mechanism in which the direction of movement is determined by the nature of the
material or organelle to be transported. Investigation of the molecular mechanisms
of axonal transport should contribute to our further understanding of this
vital neuronal function.
Dendrites
There
is wide variation in the number and organisation of these cell processes in the
central nervous system. The character of the dendritic tree reflects the
afferent neuronal connections and consequently indicates the functional
activity of the cell. The complex configuration of dendrites is best
appreciated in Golgi preparations; these have allowed the classification of
neurons into three groups based on their dendritic arborisations (Fig. 1.1).
Isodendritic neurons issue straight dendrites which run in all directions,
whilst allodendritic cells are distinguished by shorter, branched dendrites
which are restricted in their wavy course. Idiodendritic neurons have a unique
dendritic tree characteristic of
and determined by
their location.27 A further, more detailed classification of
neurons has been achieved by the dendritic branching pattern.
Although clear
differences exist, it may not be always easy to distinguish a dendrite from an
axon. Dendrites have irregular
contours, taper gradually,
branch at relatively acute angles, are unmyelinated and contain Nissi
substance. Axons, in contrast, display a smoother contour, have a relatively
even diameter along their course, branch at obtuse or at right angles, can be
myelinated or unmyclinated and lack Nissl substance. The smaller axons and
dendrites become, the more difficult is their positive identification; the
presence of clustered ribosomes, particularly in association with cisternac of
rough-surfaced endoplasmic reticulum, remains the most reliable identifying
feature of small dendritic processes.
The dendritic tree represents the
most substantial receptive area of the nerve cells Synapses are formed either
on the dendritic trunks themselves or on the dendritic spines, the specialised
structures projecting from them. The spines are composed of a stalk or neck
which connects the dendrite with the ovoid bulb or head. There is a
considerable variation in their configuration, but their overall length appears
to be relatively constant at 2 um. Spines can also be present on the
perikaryon and they are responsible for 43()() of the total surface area of the
dendrites and the cell body: there are 4000 spines on a single pyramidal
neuron.~ The function of the dendritic spines is still largely unknown.
Although they synapse with axon terminals, their role is not restricted to
increasing receptive surfaces; they may regulate the excitatory input to a
neuron.1
The neuropil and the extracellular
space
The
neuropil is composed of the complex and intricate network of neuronal cell
processes. This intermingling and interconnection of myelinated and unmyelinated
axons and dendrites may appear random, but these processes form the neuronal
circuitry of a particular area. Cell processes of astrocytes, oligodendrocytes
and microglial cells further add to the variety of structures in the neuropil
(Fig. 1.3).
Cell processes and perikarya,
bounded by typical unit membranes, are separated by an extra-cellular space
with an average width of 15-20 run. Electron-dense markers, electrical
impedance and the distribution of radiotracers have conclusivelv demonstrated
the existence of this extracellular space, which constitutes 18~25o() of the total
gre~' and white matter.29
Myelin
Myelin
in the central nervous system is produced by oligodendrocytes (see p. 22-24).
The myclin sheath is formed by the spiral wrapping of the cytoplasmic processes
of the myelinating cells around axons, and the subsequent extrusion of the
cytoplasm leads to a compact, tightly spiralled, multi-layered envelope. Electron microscopy reveals the
characteristic structure of the myelin sheath: major dense lines alternate with
thinner intraperiod lines to form the repeating units. The major dense line
results from the fusion of the thicker, inner leaflet of the oligodendrocytic
plasma membrane, whereas the intraperiod line is formed by the apposition of
the thinner, outer leaflet of this membrane. Unlike Schwann cells (the
myelinating cells of the peripheral nervous system which are concerned with the
myelination of a single axon), each oligodendrocyte may provide myelin for many
axons. From this it follows that the motion by which the sheath is produced by
the myelinating cell may be different in the central and peripheral nervous
Systems. Whilst Schwann cells can, in principle, lay down myelin by rotating
around a single axon, the oligodendrocyte, being in connection with many axons,
cannot perform a revolving motion. Consequently, ensheathment takes place by
the progressive lengthening of the oligodendrocytic process which encircles the
axon completely and an internal mesaxon is formed by apposition of the free
edges of the myclinating process. The outermost lamella of the sheath encloses
the outer tongue and this, together with the internal mesaxon, represents the
tenuous connections which remain between the fully formed myclin and the
myelinating cell.
Nodes of Ranvier occur regularly
along the course of central myclinated fibres; the segment between two nodes is
referred to as internodal myclin, and the region where the lamellae terminate
is the paranode. The thickeness of the myelin sheath is related to axonal
diameter: larger axons are surrounded by thicker myelin sheaths. Evidence from
experimental animals has convincingly shown that the formation of myelin is
preceded by the proliferation of glial precursors which develop into young
oligodendrocytes. The processes of these active oligodendrocytes are frequently
wrapped around axons, but this direct connection is difficult to demonstrate in
later life.' Biologically, the formation of myclin is a two-step process:
first, the oligodendrocyte relates to the axon in response to an axonal
myelinogenic stimulus and, second, it produces myelin, the volume of which is
determined by the internodal axonal surface area. 30
Mvelination in man progresses slowly
and generally proceeds centripetally: it commences peripherally and whilst many
axons, particularly in the spinal cord) are myclinated at birth, the more
central fibres in the frontal and parietal lobes remain unmyclinated well into posmatal
life. Completion of myelin formation is achieved largely during the first two
years after birth.31 Immunohistochemistry for myelin basic protein
confirms earlier findings of the chronological sequence of myelination:
phylogenetically older regions acquire myelin first.32 The
sequential nature of myelination reflects physiological demand: the time and
rapidity of myelination are related to the relative significance of a fibre
system at various periods of cerebral development.
Biochemically, myelin is composed of
alternating layers of proteins and lipids. Of the myelin proteins, a
proteolipid protein constitutes approximately 50 mu, basic protein (the
antigenic agent capable of inducing experimental allergic encephalomyclitis)
30%, and an acidic proteolipid protein 200/0 .3' Myelin-associated glycoprotein
is an acidic, concanavalin A binding minor component which is also present in
the cytoplasm of oligodendrocytes before and during myelination. The various
lipids to be found in the myelin include cholesterol, phosphatidylethanolamine,
phosphatidylserine and phosphatidylcholine, sphingomyelin and
glycolipids. Galactocerebroside
is the main glycolipid component and the cell membrane of the myelinating cell
contains a high percentage of this compound. This suggests that
galactocerebroside may play an important role in the successive layering of
cell membranes, a
process
unique to myelination.
Neurotransmitters and neuropeptides
The
number of putative neurotransmitters has been dramatically increased during the
last decade. In addition to the classical monoamine neurotransmitters
(acetylcholine and catecholamines) and a few amino acids, at least 30 neuropeptides
have been discovered: all these compounds can act as chemical messengers in
the mammalian nervous system. Immunohistochemistry
has allowed the precise localisation of these neurotrarismitters and
neuropeptides and thus contributed to the understanding of how certain regions
of the brain work. This expanding knowledge of the relationship
between structure and function has been accompanied by the realisation that
certain neurological and psychiatric disorders are caused by an imbalance
(overproduction or deficit) of these substances. It is for this reason that the
distribution of neurotransmitters and neuropeptides is of considerable
importance not only to neurobiologists, but also to histopathologists.
Neurochemical analyses of post-mortem brains are affected by various factors
including sampling, precision of dissection, age, sex, medication, the agonal
state of the patient and post-mortem delay.
Of the monoamines, acetylcholine is
found in the motor nuclei of the cranial nerves and in the motor neurons of the
spinal cord; in these locations it serves as the chemical messenger for
neuromuscular transmission. Acetylcholine is also present in the intrinsic
pathways within the central nervous system, and cholinergic neurons project in
a diffuse ascending system from the medial septal nuclei to the hippocampus and
from the nucleus basalis of Meynert to the cerebral cortex.39 The
basal ganglia are rich in this monoamine and the enzymes related to its
metabolism: choline acetyltransferase and acetylcholinesterase, the
synthesising and catabolising enzyme respectively. Large cholinergic neurons
have been recently demonstrated by histochemistry in the human striatum, but
only the isolation, purification and immunohistochemical localisation of choline acetyltransferase have made a more
comprehensive mapping of cholinergic pathways possible.
There are three catecholamines in
the central nervous system: noradrenaline, adrenaline (epinephrine) and
dopamine. The noradrenergic svstem is localised in the brainstem nuclei, the
largest of which is the locus ceruleus, the pigmented column of cells in the
rostral part of the pontine tegmentum: axons originating from these cells
establish extensive connections with the cerebral cortex and hippocampus. The
hypothalamus is also rich in noradrenergic fibres.
The adrenergic system, in contrast,
is more restricted: cells in the pons and medulla project to other brainstem
structures or to the hypothalamus.
The major dopaminergic pathway
originates in the pars compacta of the substantia nigra and ascends to the
striatum: devastation of this system is the underlying cause of Parkinson's
disease.'2 In addition to this nigrostriatal pathway, there is also
a mesocortical and mesolimbic dopaminergic system: cells in the ventral
tegmentum of the midbrain project to the cerebral cortex and to the limbic
areas respectively.
The raphe nuclei form a long,
ill-defined chain in the midline of the brainstem; their nerve cells give rise
to the serotoninergic system which contains 5-hydroxytryptamine (serotonin) and
projects to various sites in the forebrain including the hypothalamus, basal
ganglia and medial forebrain bundle, and descends to the anterior and
posterior horns of the spinal cord.
Gamma-aminobutyric acid (GABA),
glutamate and glycine are amino acid neurotransmitters. GABA is the principal
inhibitory neurotransmitter in the vertebrate nervous system and one-third of
all nerve terminals in the rat brain appears to be GABAergic.36 Moreover,
neurophysiology, auto-radiography and
irrrrnunohistochemistry have all demonstrated that inhibitory synapses of the
cerebellum utilise GABA and that the major efferent pathways of the Purkinje
cells are also GABAergic. GABA is also found in the spinal cord, although
glycine is the maior inhibitory neurotransmitter at this site. Glycine Occurs
in the small inhibitory interneurons of the grey matter and acts upon the large
motor neurons of the anterior horn. An increasing body of evidencc
suggests that glutamate is the universal putativc excitatory neurotransmitter
in the central nervous system. The possibility that some excitatorv synapses
use aspartate instead of glutamate cannot be excluded: the properties of these
amino acids are too similar to allow a clear-cut separation. In the
hippocampus, the major afferent pathways and the local interneurons use
glutamate, as do the granule cells, the principal excitatory intern eurons in
the cerebellum.
The last decade has witnessed the
discovery of a variety of neuropeptides which may act as neurotransmitters or
neuromodulators. The increasing list of these small peptides includes
circulating hormones, pituitary peptides, opioid peptides, intestinal
hormones, hypothalamic releasing factors and a group of miscellaneous
peptides. Some of these compounds have been known to be the products
of the endocrine or the neuroendocrine system, whilst other peptides, like the
endorphins and enkephalins, have been more recently discovered. The
neuropeptides may represent a different mode of intercellular communication
from the fast and point-to-point action of amino acids such as GABA and
glutamate: they have a slower time course, less precise spatial connections and
a wider range of chemical messengers.
There
are now more than 30 regulatory peptides and it is likely that more will be
discovered. Recent developments in neurotransmitter research have confirmed the
view that neurons of the central nervous system are secretory cells and that
the products of this activity represent the chemical signals of interneuronal
communication.
It has been recognised that some of
the neurotransmitters and neuropeptides are abnormally distributed in a
variety of neurological and psychiatric
disorders, including extra-pyramidal abnormalities, Alzheimer's
disease, epilepsy, schizophrenia and
anxiety.
Histological demonstration of
neurons
Nerve
cells can be identified in most cases in sections stained with haematoxylin and
eosin, but various 'special' methods are used in order to demonstrate
particular organelles, cell processes or myelin sheaths. Cresyl violet (Nissl's
stain) displays the coarse granular Nissi substance composed of rough
endoplasmic reticulum and consequently is ideal for detecting chromatolysis in
which Nissi substance is lost.
This
technique, combined with Luxol fast blue for the staining of myelin, is one of
the most valuable methods for revealing topographical details in sections of
brain and spinal cord.
Since neurofilaments are
argyrophilic, there are various silver impregnation techniques for the
demonstration of nerve. The Golgi method has, however, remained superior in
revealing neuronal configuration in its entirety with all the cell processes. Immunohistochemistry
is extensively used in neurocytology, and the various antigens to be found in
neurons and other cells can now be demonstrated by both immunofluorescence and
immunoperoxidase techniques.
Monoclonal
and polyclonal antibodies against these antigens have proved to be valuable in
diagnostic work. Monoclonal antibodies against the protein components of
neurofilaments are also valuable in the study of abnormalities affecting these
organelles9~56 and in the diagnosis of neuronal neop1asms. Of
the three forms of enolase to be found in the central nervous system, the isozyme
occurs in nerve cells (neuron-specific enolase) and thus provides an
immunological marker for neurons.58 However, enolase is widely
distributed in extraneural tissues and is not a neuron-specific antigen.
NEUROGLIA
The
term glia was introduced by Virchow to describe a second cell type
whose function was to bind the nerve cells together in the brain. Later Cala1classified
the glial cells into three categories: the first and second corresponded to the
two varieties of astrocytes (protoplasmic and fibrous), whilst the third
'element' referred to a group of cells which was subsequently shown by del Rio
Hortega to comprise both oligodendrocytes and microglial cells. Del
Rio Hortega introduced the silver carbonate method, which not only distinguished and separated oligodendrocytes from microglia, but also
indicated that they were of different derivation. Thus oligodendrocytes have a
similar neuroepithelial Origin to astrocytes, whereas microglial cells originate
from mesenchyme. Thus, the term neuroglia should not be applied to microglial
cells. Neuroglia includes only astrocytes, oligodendrocytes, ependymal cells,
and the choroid
plexus epithelium derived from ependyma.
ASTROCYTES
Of
the neuroglial cells, astrocytes are the most varied morphologically and the
most versatile functionally. It is traditional and convenient to distinguish
protoplasmic and fibrous astrocytes. The difference between these two types is
based upon the configuration and the number of processes rather than on the
filament content of the cell body. the processes of fibrous astrocytes are
fewer and longer and branch less frequently and at a more acute angle than
those of protoplasmic astrocytes.
Protoplasmic astrocytes are found
mainly in the grey matter (Fig. 1.8) whilst fibrous astrocvtes occur mainly in
the white matter of the brain and spinal
cord. Fibrous astrocytes, however, are also found in the
outer cortical layer, around penetrating arterioles and, together with
protoplasmic astrocytes, in the deep grey matter: the inferior olive contains a particularly large proportion of
fibrous astrocytes.'2 A varietv of fibrous astrocytes, the so-called
marginal glia, sends short, robust processes towards the pial surface, and
contributes to the formation of the external glial limiting membrane (glia
limitans) .
Subdivision
of protoplasmic astrocytes in the grey matter, according to their localisation,
into neuronal satellites, interneuronal astrocytes and vascular satellites
serves little useful purpose. However, astrocytes of the cerebellar cortex,
which are principally of the protoplasmic type, merit separate consideration on
the basis of morphological variation and functional difference. The smooth
astrocyte is found throughout the cerebellar cortex, the velate or lamellar
type chiefly in the granular layer, and the Golgi epithelial cell in the
Purkinje and molecular layer. Golgi epithelial cells are in fact the
Bergmann glia, whose processes cover large areas of the cell body and dendrites
of Purkinje cells. A further type in the cerebellar cortex is the feathered
glia of Fafianas: these cells are more
superficial than Bergmann cells and their short stout processes do not reach
the pial surface. One form of astrocyte which has features of both the
protoplasmic and fibrous astrocytes, but is identical with neither, occurs in the
corpus callosum, basis pontis and spinal cord. This intermediate or mixed
astrocyte has long fibril-containing processes identical to those of the
fibrous type, whilst their shorter processes are more protoplasmic in
structure.~ Astrocytic density varies greatly in various parts of the brain.
The ratio of glial cells to neurons, for example, is 4:1 in the striatum and
100:1 in the globus pallidus, accounting for 20~~(, and ~ of the brain volume
in these areas respectively.~5 These variations in astrocytic
morphology in different parts of the central nervous system are well
established, but information has only recently emerged from immunocytochemical
and tissue culture work regarding the possible differences in astrocyte
physiology and development in various topographical sites in the brain.
Although astrocytes can be identified
in haematoxylin and eosin stained sections, the intricate pattern of
cytoplasmic processes which render these cells star-shaped can be better
demonstrated by the use of special stains (see p.82). The oval or round
vesicular nucleus usually lacks a prominent nucleolus and this feature proves
particularly convenient in identifying cells in the cortex: neurons are
distinguished by their conspicuous nucleoli. Astrocytic processes cover the
external surfaces of the central nervous system, surround blood vessels and
abut upon nerve cells. The external glia limitans is formed by stout astrocytic
processes which, in turn, are covered by a basal lamina beneath the pia (Fig.
1.8). Similarly, end-feet, the expansions of astrocytic processes, envelop
blood vessels and form a limiting membrane around the adventitia of larger
vessels. These astrocytic end-feet or foot processes also invest the
capillaries, and this intimate relationship has provided the structural basis
for the astroglial involvement in the blood-brain barrier. Astrocyte cell
bodies can be in close apposition to neurons, and astrocytic processes are
frequently seen covering the extensive surfaces of neuronal perikarva and dendrites.
Electron microscopy has revealed
that the vesicular astrocyte nucleus contains evenly dispersed, fine chromatin
which is occasionally clumped at the nuclear membrane. The nuclear profiles can
be somewhat irregular with indentations, and the nucleolus, when present, is
small. The cytoplasm is of low electron-density and contains the usual assortment
of organelles. The cisternae of the rough endoplasmic reticulum are short,
ribosomes few and GoIgi complexes not well developed. Mitochondria are present
both in the perikaryon and the cell processes, and the larger, more unusual
forms are likely to correspond to the gliosomes detected by light microscopy.
Glycogen granules are numerous in well-fixed tissues and appear to be
concentrated in areas of high synaptic density and near neuronal perikarya.
Lysosomes are also seen as electron-dense bodies limited by a single unit membrane.
Astrocytic filaments, 10 nm in diameter, belong to the class of intermediate
filaments of the cytoskeleton and are conspicuous in the perikaryon and in cell
processes. The filaments are present in protoplasmic astrocytes, usually in the
form of cytoplasmic bundles (Fig. 1.10), but they are more abundant in fibrous
astro
cytes,
in which they Occur throughout the cell body and extend into the larger
processes .
Microtubules, although present, are
not plentiful in mature astrocytes. Astrocytes often form specialised cell
contacts of the gap junction type (so-called nexus) in which the outer leaflets
of the apposed plasma membranes are separated by an interval of 2-3 nm.
Adjacent astrocytes are also joined by puncta adhaerentia, where the plasma
membranes run parallel, separated by a gap of 25-30nm.
Astrocytic filaments and glial
fibrillary acidic protein (GFAP)
Astrocytic filaments are
10 nm in diameter and indeterminate in length. They are composed of four
globular protein subunits, each measuring 2.5nm, and linked by a cross-arm l.5nm
in thickness. These units are stacked upon each other to produce the
characteristic configuration: in transverse section a hollow central core is surrounded
by a dense wall of 2-5 rim, whilst in longitudinal section two parallel dense
lines enclose the lumen.
The filaments are composed of glial
fibrillary acidic protein (GFAP) which was first isolated in a water-soluble
form from glial scar tissue of burnt out plaques of multiple sclerosis and
from hydrocephalic brains. GFAP has a molecular weight in the range
of 50000 daltons and is well characterised chemically. Immunocytochemistry indicates that
GFAP may exist in two structural states, diffuse in the cytoplasm or localised
to filaments, and these in turn may correspond to the water-soluble and water-insoluble
forms, respectively Metabolic studies of cytoskeletal proteins in cultured
astrocytes have revealed that GFAP is amongst the most actively
synthesised proteins with
a relatively fast turnover, and both the level
of synthesis and accumulation of GFAP can be experimentally manipulated. It
is present in normal, reactive and neoplastic astrocytes and has become the
most reliable immunocytochemical marker by which the identity of astrocytes can
be positively established both in diagnostic work and for research. In
addition to astrocytes, GFAP can be demonstrated by immunocytochemistry in
other cells which contain glial
filaments: in reactive ependymal cells, tanycytes,
reactive Muller cells of the retina, in the pituicytes of the neurohypophysis
and in the glial cells of the enteric nervous system. It is
also present transiently during development in the myelin-forming
oligodendrocytes of the human fetal spinal cord, and in ependymal cells of
human fetuses between the 15th and 40th week of gestation. The
function of GFAP has yet to be determined, but experimental evidence suggests
an important role in fibrillogenesis which, in turn, is associated with
astrocytic differentiation.
Functions of astrocytes
The
concept of astrocytic function has changed profoundly during the last 20 years:
cells which once were thought to be mere physical supporting elements have been
shown to have a wide range of activity in the developing, normal and diseased
central nervous system.
Srructural support. Virchow's original view of neuroglia
as cells which somehow held the neurons together has been supported by
successive observations of astrocytic morphology. First, astrocytic processes
cover the outside surfaces of the central nervous system, forming the glia
limitans, and envelop intraparenchymatous blood vessels, constituting a sleeve
of end-feet (Fig. 1.8). Second, astrocytic filaments provide mature astrocytes
with a cytoskeleton which stabilises the cell configuration and endows
processes with considerable strength. These processes may form bundles which
interweave with nerve fibres, particularly in the white matter. Finally,
astrocytic processes are often joined together by specialised cell contacts
which increase their strength and cohesion. Although microtubules play a role
in process formation in differentiating astrocytes, filaments are the
organelles which maintain and influence the overall morphology of mature cells.
Repair. The ability of astrocytes to produce
abundant filaments is seen in various pathological conditions (see p.50).
Although the formation of glial scar tissue is important in repair of the
central nervous system following injury, fibrillary gliosis may occur
physiologically in certain areas including the olivary nuclei, the floor of the
fourth ventricle, and around both the cerebral aqueduct and the central canal
of the spinal cord.
The blood-brain barrier. The structural proximity of
astrocytic foot processes to capillaries has provided a morphological basis for
the view that astrocytes contribute to the maintenance of the blood-brain
barrier (see p.30).
Isolation of neuronal surfaces.
Astrocytes
establish intimate spatial relationships with nerve cells. They are
occasionally satellites to neurons, but more importantly their processes
regularly cover receptive surfaces of the nerve cells, including the perikarya
and dendrites. Moreover, astrocytic processes often abound in areas of intense
synaptic activity; the most striking example of this phenomenon is to be found
in the thalamus, where the synaptic glomeruli are wrapped in astrocytic sheets
which form capsules several layers thick. Astrocytic processes are thus not
disposed at random, but they conform to a pattern which ensures that receptive
neuronal surfaces are protected from non-specific afferent influences.77 Astrocytes
also play a r6le in synaptic remodelling in the mature, normal brain by
removing degenerating synapses.
Neuronal development. Evidence for astrocyte involvement
in neuronal development originates from both in vivo and in vitro observations.
Radial astroglial fibres appear to guide immature, migrating neurons and to
form a template for the growth of nerve cells.7~ Neurons of the central
nervous system are difficult to grow in primary cultures, unless an astrocyte mono layer is provided or the
medium is conditioned by growing astrocyte cultures. In vitro
studies have also shown that glial cells can influence the protection and
branching patterns of nerve cells and may be instrumental in determining the
neuronal polarity observed in vivo.
Electro physiology and ion transport. Recent investigations have revealed
that astrocytes are not inactive cells: they respond to K accumulation or
release, with changes in intracellular K + concentration. Astrocytes can act
both as spatial buffers, merely redistributing extracellularly accumulated K+,
and as active accumulators of K+. These mechanisms complement each other and
may enable astrocytes to monitor the extracellular ionic milieu and
consequently to control neuronal function.53 Furthermore, release of
potassium ions (K +) from astrocyte end-feet may play an important r8le in
regulating regional cerebral blood flow in response to changes in neuronal
activity. It has been demonstrated in primary astrocyte cultures, which are
presently the best experimental system for ion transport studies, that not only
K+, but also Na+ and Cl- can enter and leave these cells, suggesting that
astrocytes contain significant ion transport pathways.80~85 Under
controlled conditions in culture, astrocytes show both spontaneous action
potentials and action potentials induced by current. These responses indicate
the presence of voltage-dependent Ga2 -channels which may be
important in the regulation of excitability within the central nervous system.8b
Furthermore, both glutamic and aspartic acid directly depolarise rat brain
astrocytes in primary cultures, suggesting that the electrophysiological
effects of excitatory amino acids in situ may not be exclusively a neuronal
property.
Creatine kinase (isoenzyme BB) has
been shown by immunocytochemistry to be present in human astrocytes. The
function of this enzyme in the brain may be related to the increase in
respiration and the fall in both ATP and creatine phosphate levels which result
from exposure to high potassium concentrations or from electrical stimulation.
Neurotransnzitter metabolism.
Astrocytes in culture possess receptors for
neurotransmitters, and exposure to noradrenaline results in increased levels of
intracellular cyclic adenosine mono-phosphate (cAMP). Adrenergic x- and
fl-receptors have also been identified on astrocytes, and dopamine, for
example, enhances cAMP by activation
of fl-receptors.Astrocytic processes surrounding synapses could control
the levels of transmitters by taking up these compounds, but it remains to be
determined whether this uptake is involved in the further metabolism or
inactivation of the neurotransmitter. Primary cultures of astrocytes
indeed take up dopamine, noradrenaline, serotonin, GABA (Gamma-aminobutyric
acid) and glutamate and have some of the enzyme systems required for their
metabolism. Astrocytes in culture also respond to histamine and its agonists,
but the functional role of histamine receptors remains to be elucidated.92
However, the wide range of receptors on astrocytes allows these cells to
play an important and varied r6le in the central nervous system. The role of
astrocytes in the metabolism of glutamate, a putative excitatory
neurotransmitter, is well documented. Glutamate is released by neurons at
synapses and is taken up by astrocytes in which glutamine synthetase catalyses
the reversible formation of glutamine from glutamate and ammonia. Glutamine is
freely diffusible and reaches nerve cells in which glutaminase will produce
glutamate and thus complete this metabolic pathway. The small glutamate pool in
the brain is, therefore, compartmentalised in astrocytes .Immunohistochemical
studies have demonstrated that glutamine
synthetase is confined to
astrocytes, and the amount present in various brain areas correlates well with
sites of presumed glutaminergic activity.
Detoxification of ammonia. From the above description it
follows that as astrocytes possess glutamine synthetase activity, they are also
important in the detoxification of ammonia.
Phagocytosis. There is now little doubt that
astrocytes have pinocytotic and
phagocytic functions and by removing various substances, including
plasma, particulate material and cell debris
from the extracellular space, they contribute to
the maintenance of a controlled internal environment.
Immune response. Recent experimental evidence suggests
that astrocytes may have a physiological function essential for the generation
of immune responses within the brain: they respond to lymphocyte-derived growth
factors and secrete immunoregulatory molecules in culture.~ They can be
activated to release prostaglandin E and interleukins. Rat astrocytes appear to
be able to present antigen to T lymphocytes in a specific manner which is
restricted by the major histocompatibility complex. Astrocytes are
stimulated in the presence of intefferon, produced by T lymphocytes, to
express Ia antigens. Astrocytes and their precursors also respond, in culture,
to glial maturation factor and produce interleukin a1; this, in turn,
stimulates the proliferation of astrocytes.
Histological demonstration of
astrocytes
The
configuration of astrocytes, complete with their cytoplasmic processes, can be
demonstrated by a range of specialised histological stains. The older
techniques include Mallory's phosphotungstic acid haematoxylin (PTAH),
Holzer's crystal violet, Cajal's gold chloride sublimate and del Rio Hortega's
silver carbonate impregnation.
The
most widely used method is Mallory's PTAH but, unfortunately, this technique
also stains myclin sheaths, smooth muscle fibres and extracellular
proteins. The intimate relationship between
astrocytes and capillaries
is best visualised by Cajal's
method: the close apposition of foot processes to the capillary wall is most
convincingly shown.
Immunohistochemical techniques,
developed more recently, offer superior specificity and sensitivity over
conventional staining methods, and are increasingly used in diagnostic and
research work. Astrocytic antigens can be demonstrated by both
immunoperoxidase and immunofluorescence techniques; and whilst
the former is preferred for paraffin sections, the latter is the method of
choice for tissue culture preparations. GFAP is now most widely used to
identify astrocytes, whilst glutamine synthetase, confined to astrocytes in the
central nervous system, offers an alternative marker.
OLIGODENDROCYTES
Oligodendrocytes
can be subdivided according to their location: satellite cells are adjacent to
neurons in the grey matter and
interfascicular oligodendrocytes occur between nerve fibres in the white
matter. In the grey matter, oligodendrocytes are also associated with nerve
fibres or are adjacent to blood vessels. Perineuronal oligodendrocytes are
functionally similar to those in the white matter. Immunocytochemistry for
myelin basic protein and myelin-associated glycoprotein reveals a similar
staining pattern for all oligodendrocytes in the normal brain, during remyelination
and following trauma.'01 Based on ultrastructural features,
particularly on cytoplasmic
density, three subtypes can be
distinguished: light, intermediate and dark.102 This separation is
arbitrary and these forms represent developmental stages through which
oligodendrocytes evolve during postnatal life.
Light oligodendrocytes appear
to be mitotically the most
active and they become smaller and darker as they mature.
In sections stained with haematoxylin
and eosin, oligodendrocytes can be recognised by their round or oval nuclei
which are surrounded by a rim of cytoplasm. Silver impregnation reveals only a
few processes, which are usually long and delicate and radiate from the
polygonal or spherical perikaryon. By electron microscopy, oligodendrocytes
are, in general, darker cells than astrocytes, with an intrinsic density of the
cytoplasmic matrix created by tiny granules which occupy all the space between
organelles. The chromatin in the round) oval or, occasionally, irregular
nucleus often forms clumps, which is another difference
from the evenly dispersed chromatin
of astrocytes. Oligodendrocyte
cytoplasm contains abundant rough
endoplasmic reticulum, many
free ribosomes and a well-developed Golgi apparatus. Mitochondria, lysosomes
and heterogeneous inclusion
bodies are present. Unlike in astrocytes, however, glycogen granules are not
seen in
oligodendrocytes.
There is a striking difference between the cytoskeletons of oligodendrocytes
and astrocvtes in that the ratio of filaments to micro-tubules is reversed:
oligodendrocytes have many microtubules and only an occasional filament. The
microtubules are dispersed at random in the cell body, whilst in processes they
are arranged in parallel bundles. The following features distinguish the
oligodendrocyte from the astrocyte: greater nuclear and cytoplasmic density,
the lack of filaments and glycogen granules, and the abundance of microtubules.
Functions of oligodendrocytes
Two
major functions are usually attributed to oligodendrocytes: the formation and
maintenance of myelin, and the nutrition of neurons. There is now little doubt
that the myclinforming cell in the central nervous system is the
oligodendrocyte, which first appears immediately before myelination begins. in
the developing brain direct connection can be seen between oligodendrocytes
and the myclin sheath as it forms.103 Biochemical studies have shown
that oligodendrocytes, particularly neuronal satellites, can contribute to the
nutrition of nerve cells. The metabolic activities of neurons and oligodendrocytes can complement each
other in a symbiotic fashion, and these two cell types form a functional
unit.'~ Furthermore, mature oligodendrocytes, in vitro, proliferate only in
the presence of neurons, indicating that axons are mitogenic for these cells.105
Tissue culture studies have also shed new light on the functions of oligodendrocytes. Isolated
cells synthesise both galactocerebrosides and sulphatides1~
and contain various enzymes including 2' :3'-cyclic-nucleotide
3'-phosphodiesterase, carbonic anhydrase, glycerol phosphate dehydrogenase and
glucose-6-phosphate dehydrogenase, indicating a high rate of metabolic
activity.107 Oligodendrocytes respond to a variety of lymphokines
and other growth factors and thus may be involved in immunological reactions.
Histological demonstration of
oligodendrocytes
Oligodendrocytes
are usually recognised without difficulty in sections stained with haematoxylin
and eosin , but silver impregnation techniques to demonstrate cell processes
are capricious even in the most experienced hands." Of the
immunocytochemical markers, carbonic anhydrase (isoenzyme II or C) appears to
be the most promising, whilst the surface marker) galactocerebroside, can be demonstrated
in cultured cells and myelin basic protein is present in immature
oligodendrocytes during myelination.
EPENDYMAL
CELLS
- The ependyma lines the ventricular system, the cerebral aqueduct
and the central canal of the spinal cord. These epithelium-like cells are
cuboidal or columnar and show characteristic polarity of cellular organisation (Fig. 1.15). A round or oval nucleus is
located in the basal part of the cell, whilst most organdIes occupy the apical
portion. The cell membrane is specialised according to surface. Thus the
lateral membranes of adjacent cells run parallel and are joined together
by specialised junctions;
the cell membrane at the base of
the cell has an irregular contour and rests upon glial fibres; the apical
surface is studded with cilia. Ependyma lining the ventricular system varies in
its morphology. In general, cells covering the white matter are more flattened
and have fewer cilia than those covering grey matter.
Electron
microscopy shows that the nucleus contains evenly distributed, fine chromatin and
a small eccentric nucleolus. The well developed Golgi apparatus is
supranuclear, and mitochondria tend to crowd the apical portion
of the
cell. The rough endoplasmic reticulum consists of only a few, short cisternae,
but free ribosomes are numerous. Multivesicular bodies, lysosomes and vesicles
of various sizes are also present. The cytoskeleton is composed of 10 nm
intermediate filaments, 4.6 nm microfilaments and occasional 24 nm
microtubules. Intermediate filaments are similar to those found in astrocytes,
but they do not appear to contain GFAP in normal, mature ependyma. However,
ependymal cells in human fetuses contain GFAP transiently between the 15th and
40th week of gestation. The nature of filaments in mature ependymal
cells remains an enigma: they may be antigenically different from astrocytic
filaments or the binding of GFAP within the filament may have resulted in loss
of affinity for the specific antiserum.
Cilia spring from the
basal bodies in the apical cytoplasm which can occasionally be seen as blepharoplasts
with the light microscope in good PTAII preparations. Each cilium is composed
of9 pairs of microtubu]es surrounding a central pair. Between the cilia,
microvilli and simple cytoplasmic protrusions increase the apical surface area
of the cell. The lateral plasma membranes of adjacent ependymal cells often
interdigitate, forming gap junctions (nexus), extensive zonulac
adhaerentes and occasional
tight junctions (zonulae
occiudentes) toward the apices.
Tanycytes
Tanycytes are
modified ependymal cells distinguished by their long, radially
orientated and unbranching basal processes which usually reach subependymal
capillaries These cells, which owe their name to their elongated shape, have a
somatic portion, which lies in the ependyma and contains the nucleus, a neck
portion, which is located in the periventricular neuropil, and a tapering tail.
The apical surfaces of tanycytes have more thin cytoplasmic projections and
fewer cilia than other ependymal cells and, by electron microscopy, their
cytoplasm is somewhat darker and contains fewer filaments and more
microtubules.
Functions
of ependymal cells
The cilia of ependymal
cells beat rapidly during life and this movement may contribute to the
circulation of the cerebrospinal fluid (CSF). The structural organisation of
ependyma, with its interdigitations and specialised junctions at the lateral
cell surfaces, is suggestive of a supportive function, similar to that
performed by astrocytes. As ependyma forms a barrier between the ventricular
CSF and the parenchyma of the central nervous system, it is ideally situated to
influence the transport of substances. When electron-dense markers, such as
horseradish peroxidase and ferritin, are injected into the ventricles they
penetrate into the brain between ependymal cells. In addition, these materials
are taken up by the ependyma and transported in vesicles and multivesicular bodies,
suggesting that other substances in the CSF may follow a similar pathway. As
tanycytes connect the ventricular surface and capillaries, it is thought that
they transport material from the CSF to the brain and into the vascular
circulation, but their major function is structural. Ependymal cells
may perform sensory and secretory functions in various animal species,1 but
evidence for these activities in man is still not available.
MICROGLIAL
CELLS
The profusion of names
which exist for microglial cells is a good indicator of the controversy
surrounding the Origin and morphological heterogeneity of microglia. Hortega
or Robertson-Hortega cell, cerebral histiocyte, phagocyte, rod cell and
mesoglia are synonyms for the microglial cell.
Microglial cells are ubiquitous in
the central nervous system, although they are somewhat more numerous in the
cortex than in the white matter. Most appear to be distributed at random, but
some are preferentially located near neurons and blood vessels. The nucleus is
triangular or elongated and the chromatin pattern is less vesicular than in
astrocytes, although not as dense as in oligodendrocytes. Very little
cytoplasm is visible in haematoxylin and eosin stained sections, but
immunocytochemical techniques and
silver impregnation demonstrate the cell processes, which are
occasionally very long, but less numerous than astrocytic processes. Electron
microscopy reveals the usual complement of organelles in microglial cytoplasm.
Thus, a few, long cisternae of the rough endoplasmic reticulum, some
ribosomes, an active Golgi apparatus, mitochondria, microtubules and a few
filaments are seen. The most prominent feature of the cytoplasm is the presence
of dense inclusion bodies, mainly lysosomes. The nucleus contains coarsely
clumped chromatin, and is often located at one end of the cell, surrounded by a
thin rim of cytoplasm, whilst the organdIes occupy the opposite pole.
Origin of microglia
The Origin of microglial
cells has been the subpect of long controversy and the problem has yet to be
convincingly settled. Various hypotheses have been proposed and include origins
from pial, neuroepithelial, pericytic and monocytic cells. A mesodermal or pial
origin of microglia suggests that primitive mesodermal cells accumulate
beneath the pia and penetrate the brain parenchyma as diverse forms of amoeboid microglia which,
during differentiation, retract their
pseudopodia and develop
branching processes to become mature microglial cells. The opposing
neuroepithelial theory maintains that microglial cells originate from the primitive
cells of the subependymal plate either directly or indirectly through amoeboid
microglial cells. Microglioblasts or amoeboid microglia are, in fact,
glioblasts derived from the matrix cells and thus, according to this theory,
microglial cells would share a common progenitor with astrocytes and
oligodendrocytes.
Pericytes are cells
closely apposed to the capillary endothelium and enclosed by the basement
membrane. Morphological similarities and the phagocytic capacity of pericytes's
have led to the view that they could be a source of microglia; these
cells could migrate into the brain through disruptions in
the basement membrane. However, pericytes have been shown
to remain anchored to the vascular wall in pathological conditions and do not
become transformed into actively phagocytosing microglial cells.
The
theory that microglial cells originate from monocytes has attracted many
supporters,who maintain that monocytes from the blood enter the brain, in
which, conforming to a different environment, they acquire the features of
resting microglial cells. That blood monocytes can permeate the brain has been shown using carbon-labelled
cells; thus, the sequential appearance of carbon particles in monocytes,
amoeboid microglia and microglia strongly suggests that monocytes become microglial
cells. However, the infiltration of blood monocytes into the brains
of experimental animals is age dependent, and the blood-brain barrier may also
play a role in preventing monocytes from entering the brain in large numbers in
mature animals. Moreover, immunological studies have produced
evidence against the monocytic origin of microglia; microglial cells do not
possess monocytic membrane antigens and mononuclear phagocytic markers. However,
monocytes may lose their rnembrane markers on entering the brain and then adapt
to an entirely different macrophage microglia ii similarities
Functions
In conclusion, the
original concept of a pial origin for microglial cells has not been refuted; on
the contrary it has gained new support.123 However, the possibility
of a dual origin, both pial and monocytic, cannot be excluded. It is likely
that occasional monocytes can enter the normal brain, and this has been
demonstrated in experimental animals and in the adult normal human brain; A
the relationship of these cells to resting microglial cells has yet to be
determined.
Functions of microglia
The
function of resting microglia in the normal brain is far from clear. These
cells are thought to maintain a close, functional relationship with neurons,
axons and myelin sheaths, to regulate the ion and fluid balance of the
extracellular space and to transport substances. The presence of Fc and
complement receptors and of HLA class II complex on microglial cells suggests
that they could play a role in the immunological defence of the nervous system. Microglial cells have the
ability to engulf and ingest various substances, including particulate
material, parts of other cells or even whole cells. They are rich in lysosomal
hydrolytic enzymes, which enable them to perform phagocytic activity.
Histological demonstration of
microglial cells
Although silver
impregnation demonstrates cell processes and gives the most comprehensive
picture of microglial cells, the technique only works in expert hands. Enzyme
histochemistry for acid phosphatase and non-specific esterase stains lysosomes
and thus indicates phagocytic activity.
BLOOD VESSELS AND THE
BLOOD BRAIN BARRIER
Cerebral capillaries are
fundamentally similar to those of other tissues, but there are important
differences. There is a paucity of cytoplasmic vesicles in the endothelial
cells, and the tight junctions between the endothelial cells differ from those
of other tissue capillaries. In addition, astrocytic foot processes surround
each capillary. The lumen is lined by endothelial cells which display oval or
elongated nuclei located in the thickened part of the capillary wall. The
cytoplasm contains the usual set of organelles, of which mitochondria are the
most abundant. Complex membrane-bound
structures, the Weibel-Palade bodies, are difficult to find.
Cytoplasmic vesicles are scarce and fewer than in most other capillaries. The
structure of the interendothelial connections is variable, but in general the
adjacent cell membranes are parallel, and, towards the luminal end, the outer
leaflets fuse to form tight junctions (zonulac occludentes).
The endothelial cytoplasm
is richly endowed with enzymes, including
adenosine triphosphatase,
nicotinamide adenine dinucleotide, monoamine oxidase, acid and alkaline phosphatases, various
dehydrogenases, DOPA
decarboxylase and glutamyl transpeptidase. The wealth of these enzyme systems
reflects the unique role played by the cerebral endothelium in the blood-brain
barrier. Moreover, differences in the intensity of various hydroJytic enzymes
at the luminal and abluminal cell membrane strongly indicate the polarity of
endothelial function in the control of the blood-brain interface. Outside the
endothelium lies a continuous basal lamina or basement membrane approximately
40-50 nm thick and composed of an admixture of substances, including type IV
collagen, heparan sulphate proteoglycan, laminin and entactin. Astrocytic
foot processes abut onto capillaries, forming a complete envelope in most cases
:
occasionally other cells
may have direct contact with the basal lamina. Pericytes are completely
surrounded by a duplication of the basement membrane, and are frequently
seen extending their processes around the capillary; their cytoplasm contains
many lysosome-like bodies. The origin and function of pericytes remain to be
established, although the view that they could give rise to microglial cells
has gained some support .
Arterioles and small
arteries differ from capillaries not only in their larger size, but also by the
presence of smooth muscle cells in their walls. Outside the endothelium, one or
two layers of smooth muscle cells are transversely orientated and sandwiched
between thick basal laminae. Venules resemble large capillaries and the
transition between the two types of vessel is difficult to identify
.
Histological
demonstration of blood vessels Van Gieson's technique
alone or combined with an elastin stain gives good results in demonstrating
connective tissue components of vessel walls. The overall pattern of
vascularisation is well demonstrated by reticulin stains. Of the enzymes, the
allaline phosphatase reaction reliably identifies endothelial cells both by
light and electron microscopy. The immunocytochemical demonstration of factor
VII I-related antigen is now routinely used to define endothelial cells.
The blood-brain barrier
The concept of a
blood-brain barrier was first based upon the observation that intravenously
injected vital dyes, like Evans (azovan) blue and trypan blue, entered and
stained various organs, but not the brain.1~ Later, ultrastructural
studies with electron-dense tracers, such as horseradish peroxidase or
lanthanum, demonstrated that tracers do not penetrate the interendothelial cell
junctions in the brain, neither are they carried across the
endothelial cell by
vesicular transport.141 The morphological basis for the
blood-brain barrier appears to reside in two features of endothelial cells: the
presence of tight junctions and the paucity of cytoplasmic vesicles. The
oversimplified single-membrane model, however, is no longer accepted;
intercellular tight junctions, intracellular enzyme systems and the two
endothelial cell membranes all contribute to the barrier effect. It
has been demonstrated in vitro that astrocytic foot processes, unique to
cerebral capillaries, could contribute to the barrier. Astrocytes are essential
for the expression of the endothelial enzymes which play a r6le in transport
mechanisms142 and for the induction of barrier properties in vitro.
The blood-brain barrier
does not pertain in all parts of the mammalian brain: a few, relatively small
and usually periventricular structures are freely permeable to vital dyes and
electron-dense tracers. These structures include the area postrema, median
eminence, subcornmissural organ, pineal gland, subfornical organ, supraoptic
crest and neurohypophysis. The blood vessels in these areas have
ultrastructural, enzymatic and permeability features which are different from
those in other areas of the brain.
It has been realised that
the blood-brain barrier is more a regulatory interface between the blood flow
and the cerebral parenchyma than a simple rigid physical barrier.The passage of
a particular substance across the blood-brain barrier may depend upon various
factors, including its lipid solubility, electrical charge, molecular size,
dissociation constant, affinity for a
carrier molecule and the nature of the substance in relation to the capacity of
the blood-brain barrier for active transport.
The function of the blood-brain
barrier is threefold. First, it prevents or hinders the entry of most
water-soluble substances into the brain; the permeation rates are usually
determined by lipid solubility. Secondly, the blood-brain barrier promotes the
transport of certain materials, including some hexoses and several amino acids,
by stereo-specific carrier transport systems which are present in the cerebral
endothelium. The transport of various materials across the blood-brain barrier has
been recently reviewed. Thirdly, the blood-brain barrier plays an important
r6le in the volume regulation of the central nervous system. This is achieved
by two mechanisms which limit the bulk flow of water across the blood-brain
barrier: these are the low hydraulic conductivity of capillaries and the high
osmotic activity of the major solutes.
THE CHOROID PLEXUS
The
choroid plexus is composed of a vascular fold of the pia mater and an
epithelial layer derived from the ependymal lining. There are four choroid
plexuses, one in the medial wall of each lateral ventricle and one each in the
roofs of the third and fourth ventricles. They have clearly defined attachments
to the ventricular wall and their free edges are invaginated into the
ventricles. The surface area of the choroid plexus is greatly increased by the
many fronds, which in turn consist of tiny villous processes. The arteries
supplying the choroid plexus branch out into capillaries, one for each villus,
which then join to form a vein.
The epithelium consists
of a single layer of cuboidal cells mounted on a basement membrane; in a few
areas, however, pseudostratification or even true stratification may occur.
Choroid plexus epithelium can be
identified immunocytochemically
by the presence of carbonic anhydrase 11(c) in the cytoplasm; this enzyme
probably plays a role in the production of cerebrospinal fluid (CSF). The round
or oval nucleus of the cell is usually centrally located in the cytoplasm,
which has the usual complement of organelles. Mitochondria are particularly
numerous and are mainly in the apical portion of the cell: they provide the
energy necessary for the active transport carried out during production of the
CSF. Smooth and coated vesicles of various sizes are seen throughout the
cytoplasm and these take part in the transport of materials. The apical surface
of the epithelial cell is greatly increased in area by masses of microvilli and
occasional cilia which tend to be grouped together. The lateral plasma
membranes are bound together by complex connections: by tight junctions
(zonulac occludentes) at the apical end, by zonulac adhaerentes, and by
intricate infoldings at the basal end. Occasional phagocytes, the epiplexus or
Kolmer cells, are seen on the surface of the choroid plexus epithelium; they
may play a r6le in keeping this surface free of debris.
The fibrovascular core of
the choroid plexus supporting the epithelium contains arachnoid cells; whorls
of collagen fibres in the fibrous stroma become calcified with advancing age.1~0
Blood vessels of various sizes include small arteries, arterioles, capillaries
and venous sinuses. Capillaries in the villi are
fenestrated and their endothelial lining is very thin.
Functions of the choroid plexus
The main function of the
choroid plexus is the production of the CSF, although a proportion of the CSF, estimated to be 10-20%, is derived
from extrachoroidal sources.In man, approximately 500-700 ml is produced every
day; of this only 140 ml can be accommodated at any one time:
25-30
ml in the ventricles and the rest in the subarachnoid space.Although it has
been disputed whether the CSF is the result of passive dialysis or active
secretion, evidence now favours the latter mechanism. Factors which are
involved in the formation of CSF include pressure, serum osmolality,
temperature, age, innervation of the plexus and prostaglandins. The
enzyme systems of the choroid plexus and various theories of CSF production
have been recently reviewed.
The choroid plexus may
also take part in the absorption of materials as demonstrated in experimental
animals,isi but this function has not been unequivocally confirmed. An
estimated l0% of the CSF may be absorbed by the choroid plexus.
THE MENINGES
The
meninges covering the central nervous system are composed of three layers: the
dura mater, the arachnoid mater and the pia mater. The arachnoid and pia
jointly form the leptomeninges.
The dura mater (pachymeninx) is a
tough, dense membrane which surrounds the brain. It has two extensions: the
faix, between the two cerebral hemispheres,
and the tentorium
cerebelli, separating the contents of the posterior fossa from the rest
of the brain. The cranial dura is closely attached to the skull; its two
layers, the periosteal and meningeal dura, are fused and separate only to form
the venous sinuses. In the spinal canal, however, the dura is separated from
the vertebral periosteum by the epidural space, which contains fibro-fatty
tissue and an epidural venous plexus.
The dura is formed by dense, interlacing
bundles of collagen in which flattened fibroblasts are embedded. The central
part contains more cells and occasional blood vessels. Its outer surface is
covered by thin, overlapping cell processes and its inner border also has a
covering of flattened cells. The subdural space is artefactual, since the dura
and arachnoid are closely apposed in life with no appreciable gap between them.
The arachnoid mater has a variable
thickness, in places being formed by several cell layers. Its outer, dural aspect
is smoother than the inner, pial -aspect from which trabeculae emerge to bridge
the subarachnoid space. The arachnoid cells are joined together by specialised
contacts, including tight junctions, which ensure an effective physiological
barrier impermeable to CSF.
The cells of the pia mater are
similar to those of the arachnoid, but the pia itself is thinner than the
arachnoid. Pial cells form a complete layer joined by desmosomes and gap
junctions.156 The subpial space separates the pia from the glia limitans
of the underlying neural tissue and the pia mater separates the subarachnoid
space from the perivascular (Virchow-Robin) spaces of the brains.
Arachnoid villi are
diverticula of the arachnoid mater and the subarachnoid space which extend into
veins and venous sinuses of the dura. Arachnoid granulations are larger than
villi and are visible to the naked eye, whereas villi are microscopical structures.
Each villus or granulation is coated on its venous aspect by endothelial
cells and is bathed by venous blood. As the villus or granulation penetrates
the dura, it forms a narrow neck which then expands to form a central core
composed of channels and collagenous trabeculac. Towards the apex of the
granulations there is a cap of arachnoid cells with wide channels running
through to the coating endothelium.15~ These structures are a major
pathway for the drainage of cerebrospinal fluid, which percolates through the
cores of the villi or granulations and is transported across the endothelium
into the blood.
THE SUBEPENDYMAL PLATE
The subependymal plate
has long been recognised as a layer of primitive cells beneath the ependymal
lining of the lateral ventricles in the adult human ~ It is the remnant of the
embryonal matrix (the subventricular zone). Studies of the subependymal
plate in various animal species have revealed that in the fetus it gives rise
to both neurons and glia, whilst after birth it is a source of glial cells
oflly.161~162 The cells of the subependymal plate display
ultrastructural features common to primitive cells: high nuclear-cytoplasmic
ratio, dominance of free ribosomes over membrane-bound ribosomes and a general
scarcity of organelles. Mitotic activity persists into later adult
life in various species, including primates.Unfortunately, information on the human
subependyn extrapolation from may be misleading.
Human subependymal plate
is limited, and an extrapolation from experimenta] animals to man may be
misleading.
In addition to the
subependymal plate there are other secondary germinal sites in the mammalian
central nervous system, including the dentate gyrus of the hippocampus, the
olfactory bulb and the external granular layer of the cerebellum. This latter
zone, which has been more comprehensively studied than the other two, is formed
in fetal life and postnatally continues to produce the neurons of the internal
granular layer. The proliferative activity of these secondary germinal zones
and the hormonal, nutritional and pharmacological factors which influence
cellular turnover have been reviewed.'67
The presence of the subependymal
plate with potential mitotic activity in the adult human brain raises the
question of the replacement of glial cells and of their proliferative activity
in the normal brain. The view that cells of the adult central nervous system do
not divide cannot be maintained any longeri68 as there is
convincing evidence that astrocytes and cells of the subependymal plate maintain
mitotic activity throughout adult life. Although oligodendrocytes undergo
mitosis in pathologica] conditions,'69 their ability to divide in
the normal brain has not been unequivocally demonstrated. Similarly, microglial
cells do not appear to be mitotically active in the normal, adult central
nervous system. Neurons, ependymal cells, choroid plexus epithelium and pericytes
do xiot divide after they have become differentiated, whilst endothelial cells
continue to undergo mitosis during adult life.'68 The low turnover
of cells in the adult central nervous system, coupled with the difficulty of
positively identifying dividing cells and the occasional cell which is not
fully differentiated, makes precise assessment of the mitotic activity of a
particular cell type difficult. Moreover, recent tissue culture studies of the
developing rat optic nerve have revealed that glial precursors, depending on
the composition of the culture medium, can differentiate into either astrocyte
or oligodendrocyte even without the influence of other brain cells.170 If
these cells persist into adult life thcy may retain their differentiation
potential and mitotic activity.