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Development oC the spinalcord

S. Climent Peris

Summarizing neural development has its perils.Because so many factors are involved, syntheses areofien more intimidating than helpful. In spite of thedifficulty, attempts at distillation are probably usefulbecause ideas that seem attached to particular aspectsof development emerge as recurrent themes. We havecollected sorne of these ideas and have arranged themin roughly the order of their manifestation during neuraldevelopment, underscoring the themes that have shapedour current thinking about neural development.

1. EARLY EVENTS

Primary neurulation

In vertebrates, gastrulation creates an embryo having an internal endodermal layer, an intermediatemesodermallayer, and an external ectodermo In addition, a cord of mesodermal cells, the notochord, liesdirectly beneath the most dorsal portion of the ectoderm. The interaction between the mesodermal cellsand their overlaying ectoderm is one of the most important interactions of all development, for it sets inhand a chain of events, the outcome of which is theformation of the hollow NEURA~ TUBE, which willdifferentiate into the brain and most of the spinal cord.The action by which the mesoderm instructs the ectoderm to become neural tube is called PRIMARYEMBRYONIC INDUCTlON, and the cellular responseby which the flat layer of ectodermal cells is transformed in a hollow tube is called PRIMARY NEURULATlON.

Despite the central role of this inductive process,the mechanism remains obscure. It is often assumedthat a single event underlies its initiation and that succes or failure of neural induction can be assesed adequately by monitoring the progress of events such asthe formation of a neural tube, but this assumption maybe falseo In fact, there is evidence that neural induc-

tion is a more complex process. In Xenopus embryos,by comparing transcription from three regional markergenes (anterior, XIF3, posterior, XIHbox6, and generalneural, XIF6), SHARPE & GURDON (1990) have shownthat the normal induction process requires interactionsbetween ectoderm and mesoderm that persist throughgastrulation into the late neurula stages.

Neurulation involves a number of signals, each ofwhich can be separately manipulated by choosing theappropiate experimental conditions. Thus, lithium ionsaffect morphogenesis and patterning, but no neuronaldifferentiation. Inhibiting the sodium pump preventsneuronal differentiation but no morphogenesis or patterning. Blocking gap-junctional communication interferes with patterning but not morphogenesis orphenotypic differentiation. This rather strongly suggests that mesoderm cells induce dorsal ectoderm cellsto form the nervous system either by generating morethan one signal or by initiating completely separateevents with a single signal. However, the identity ofinducing signal(s) and the mechanism whereby they initiate morphogenesis, patterning, and phenotipic differentiation remain to be elucidated.

Secondary neurulation

The future lumbosacral region of the spinal cordforms during SECONDARY NEURULATlON by cell rearrangements and canalization in the tail bud. The tailbud is derived from the remains ofHensen's node andthe prirnitive streak following gastrulation. The cellsin the dorsal midline of the tail bud aggregate to formthe medullary cord. The cells of the medullary cordbecome further divided into a peripheral population anda centrally located population. Cavities subsequentlyappear at the boundary of the two cell populations.

. These enlarge while the central cells apparently mergewith the lumen of the primary neural tube (GRIFFITH& WILEY, 1990).

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Development of the spinal cord

During neurulation, the original ectoderm is dividedinto three sets of cells:

- the neural tube, internally located- the epidermis of the skin- The neural crest cells, which migrate from the

region that had connected the neural tube and the epidermal tissues.

How does neurulation occur?

The first indication that a region of ectoderm isdestined to become neural tissue is a change in cellshape. The elongation of dorsal ectodermal cells causesthese prospective neural regions to rise aboye the surrounding ectoderm, thus creating the NEURAL PLATE,which includes as much as 50 % of the ectodermo Ectodermal cells elongate as the randomly arrangedmicrotubules of these cells align themselves paralle1 tothe lengthening axis. This stage can be blocked by colchicine, an inhibitor of microtubule polimeryzation(BURNSIDE, 1973).

Shortly thereafter, the edges of the neural platethicken and move upward to form the NEURAL FOLDS,while a U-shaped NEURAL GROVE appears in the centre of the plate, dividing the future right and left sidesof the embryo. The neural folds migrate toward themidline of the embryo, eventually fusing to form theneural tube beneath the overlying ectodermo Thesechanges involve the apical constriction of cells, whichis coordinated by a ring of actin microfilaments encircling the apical margins of the cells. The contraction ofthese microfilaments produces a «purse-string» effect,constricting the apical end of each cell. When embryosare cultured in the presence of cytochalasin B, theneuroectodermal cells can elongate but cannot constrictto form the neural folds (BURNSIDE, 1971; KARFUNKEL, 1972). The actin gets the energy for contraction from myosin, to which is linked (NAGELE & LEE,1980), and is also linked to the apical membrane througspectrin, and integral protein of the plasma membranethat can bind to microfilaments inside the cell (SADLERet al., 1986).

In a recent review article, SCHOENWOLF & SMITH(1990) evaluate the traditional viewpoint of howneurulation occurs, and formulate the following fundamentals:

- neural plate shaping and bending is a multifactorial process resulting from forces both intrinsic andextrinsic to the neural plateo

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- forces for cell shape changes are generated byboth the eytoske1eton and other factors.

The cells at the junction between the outer ectodermand the neural tube become the NEURAL CRESTCELLS, which will migrate through the embryo andwill give rise to several cell populations, including pigment cells and the cells of the peripheral nervoussystem.

The formation of neural tube does not occursimultaneously throughout the ectodermo Neurulationin the cephalic region is well advanced while the caudalregion of the embryo is still undergoing gastrulation.Regionalization of the neural tube also occurs as a resultof changes in the shape of the tube. In the cephalic end,the wall of the tube is broad and thick, and there area series of swellings and constrictions that define thevarious brain compartments. Caudal to the head region,however, the neural tube remains a simple tube thattapers off toward the tail. The two open ends of theneural tube are called the ANTERIOR NEUROPORE andthe POSTERIOR NEUROPORE. These openings allowamniotic fluid to flow through the neural tube for a time.

Failure to close the anterior neuropore at day 21-22results in a lethal condition, ANENCEPHALY. Here, theforebrain remains in contact with the amniotic fluid andsubsequently degenerates. Fetal forebrain developmentceases, and the vault of the skull fails to formo Thisabnormality is not rare occurring in about 0,1% of allpregnancies. However, failure to close the posteriorneuropore at day 27 (or its subsequent rupture shortlythereafter) results in SPINA BIFIDA, the severity ofwhich depends upon how much of the spinal cord remains open. Neural tube closure defects can now bedetected during pregnancy by various physical andchemical tests (THORNTON et al., 1991; WIECHEN etal., 1991).

2. DIFFERENTIATION

The early mammalian tube is a straight structure.However, even before the posterior portion of the tubehas formed, the most anterior region balloons into threeprimary vesicles: PROSENCEPHALON, MESENCEPHALON, and RHOMBENCEPHALON. This expansion is veryrapid (30-fold between days three and five of incubation in the chick embryo) and is thought to be causedby positive fluid pressure inside the neural tube. Thisfluid pressure does not arrive nor is dissipated by thespinal cord because a constriction forms at the


base of the brain as well in the human (DESMOND,1982) as in the chick embryo (SCHOENWOLF & DESMOND, 1984; DESMOND & SCHOENWOLF, 1986). Theoccluded region reopens after the initial rapid enlargement of the brain ventricles.

The original neural tube is composed of a germinalneuroepithelium, one celllayer thick. This is a rapidly dividing cell population. The central nervous systemis made up of an extremely large number of cells. Thehuman spinal cord alone has as least one hundredmillion (108

) nerve cells, and over one billion (109)

glial cells!SAUER (1935) was the first that shown that all of

these cells are continuous from the lurninal edge of theneural tube to the outside edge but that their nuclei areat different heights, thereby giving the superficial impression that the wall of the neural tube has numerouscelllayers. The position of the nucleus depends on thestage of the cell's cycle. DNA synthesis (S phase) occurs while the nucleus is at the outside edge of the tube,and the nucleus migrates lurninally as mitosis proceeds.Mitosis occurs on the luminal side of the cell layer.

During early development, all of the neural tubecells incorporate radioactive thymidine into the DNA(FUJITA, 1964). Shortly thereafter, certain cells stopincorporating this DNA precursor, thereby indicatingthat they are no longer participating in DNA synthesisand mitosis. These are the young neuronal and glialcells that migrate to the periphery of the neural tubeto differentiate. Subsequent neural differentiation isdependent upon the position these NEUROBLASTS occupy once outside the region of dividing cells(LETOURNEAU, 1977; JACOBSON, 1978).

As the cells adjacent to the lumen continue todivide, the migrating cells form a second layer aroundthe original neural tube. This layer becomes progresively thicker as more cells are added to it from the germinal neuroepithelium. This new layer is called theMANTLE ZONE and its cells differentiate into bothneurons and glia. The cells make connections amongthemselves and send forth axons away from the lumen,thereby crating a cell-poor MARGINAL ZONE. Eventually, glial cells cover many of these marginal zoneaxons in myelin sheats, giving them a whitish appearance. Hence the mantle zone, containing the cellbodies, is often referred to as the GRAy MATTER; andthe axonal, marginallayer is often called the WHITEMATTER.

There is a ventral-to-dorsal gradient of proliferation (NORMES & DAS, 1974). As development pro-

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ceeds, proliferation diminishes in the ventral cord, andby day 15 ofthe gestation in the rat only the most dorsal portion remains active.

Although nonproliferative events may contributeto the later regional variations in motoneuron numbers,QpPENHEIM et al. (1989) showed that there are initialdifferences created early on by regionally specific proliferative events.

2.a. Segmentation and patterns

Segmentation of the nervous system is a subject ofconsiderable interest to embryologists. There are reallytwo aspects to neural segmentation in vertebrates: howthe peripheral nervous system becomes segmented, andwhether the central nervous system is intrinsicallysegmented.

Qne of the simplest forms of neural organizationin vertebrates is the repeating segmental pattern of theperipheral nerves as they emerge from the spinal cord.The pioneering studies by LEHMAN (1927) and DETWILER (1934), removing or grafting somites inurodeles, showed that segmentation of the peripheralnerves is secondary to segmentation of the mesodermoRecent studies by KEYNES and STERN (1984), rotating180. o rostro-caudally a portion of neural tube or a portion of segmental plate mesoderm prior to axonoutgrowth, allowed to conclude that axons growthrough the rostral half-sclerotome, regardless of itsposition relative to the neural tube or to the rostrocaudal axis of the whole embryo.

STERN et al. (1988), studied the lineage of cells inthe ventrolateral region of the neural tube of the trunkof the chick embryo by injecting single cells withrhodamine-lysine-dextran at various rostrocaudallevels. They found that the clone derived from eachinjected cell, regardless of rostrocaudallevel, is alwaysrestricted to a region of the spinal cord equivalent to thelength of one somite, or, at the most, one and a halfsomites. The clones produced by a single injected futureventral horn cell, 48 hours after injection, comprisedabout 30-40 cells, suggesting that the cell cycle duration of ventral horn progenitor cells is about 10 hours.

As cells proliferate in the ventricular zone andmigrate locally to more superficiallayers, the lateralwalls of the embryonic spinal cord become thick. Incontrast, the dorsal and ventral margins remain thinand are referred to as the roof plate and the floor plateoA longitudinal grove appears in each lateral wall of

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the neural tube and divides it into dorsal (alar) and ventral (basal) plates. These plates give rise to the dorsaland ventral horns of the grey matter of the spinal cord,that are made up by functionally distinct dasses ofneurons. For instance, motor neurons begin to differentiate in the ventral horn and send their axons out byway of the ventral root. Sensory neurons develop in thedorsal horno

It is not clearwhich factors stablish this basic patterno Recent studies indicate that the floor plate, adistinct structure at the ventral midline of the neuraltube, which consists of specialized neuroepithelial cells,may contribute to the establishment of pattern andpolarity in the developing spinal cord by influencingmotor neuron differentiation and acting as an intrinsicorganizer (HIRANO et al., 1991). The floor plate appears to be the first structure to differentiate, under theinflu.ence of the notochord (PLACKZEK et al., 1988) andreleases a diffusible factor that may attract cornmissural

. axorls to the ventral midline of the spinal cord and guidethese axons afier they cross the midline through the ventral . cornmissure (TESSIER-LAVIGNE et aL, 1988;BOVOLENTA & DODD, 1990).

The roof plate may be a barrier to axon growth.This region is comprised of primitive glial cells andundergoes a gradual change in morphology from awedge shape to a long, thin septum-like structure at thedorsal midline of the spinal cord. Both the early ventral cornmisural axons as well as primary afferents fromthe dorsal root ganglia come in close proximity to theroof plateo Even though both axon systems have potential targets or pathways in the contralateral spinal cord,they do not cross the roof plate to reach them. Keratansulphate, a GAG present in several noninnervate regionsas the outer epidermis, developing cartilage, tectalmidline and dorsal septum, alone or in combinationwith other molecules expressed by the roof plate, maybe responsible for the inhibition ofaxon elongationthrough the roof plate in the embryonic spinal cord(SNOW et al., 1990).

During subsequent development, there is surprisingly líttle change in the internal organization of thespinal cord. The spinal cord remains divided into a dorsal sensory region and a ventral motor region. Neuronsmediating sensation or motor control become organized into cell columns (or thin sheets for sorne sensoryneurons) that run in the rostrocaudal direction of thespinal cord. Initially the gray columns are uniform alongthe entire length of the spinal cord. However, as thelimbs develop the columns at the corresponding axial

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levels become significantly enlarged. The enlargementsoccur in the cervicothoracic and lubosacral regions andare called intumescences. The presence of a greaternumber of neuronal cell bodies in these regions is theresult of degeneration of immature neurons in nonlimbinnervating regions. Most of these cells die because theyfail to form viable contacts with developing muscletissue. AIso, in the thoracic level immature neuronsmigrate dorsomedially out of the ventral motor columnand form the intermediate gray column. This containsthe neuronal cell bodies of the sympathetic neurons.

The major change in the morphology of the spinalcord is reflected in its length in relation to the lengthofthe vertebral canal. Early in development the spinalcord fills the vertebral canal. As development progresses, the vertebral column lengthens more than thespinal cord so that an increasingly smaller portion ofthe vertebral canal is occupied by the spinal cord. Atbirth the caudal end of the spinal cor<! líes at the levelof the third lumbar vertebra, and in the adult, the spinalcord extends only to the caudal margin of the first lumbar vertebra. The dorsal and ventral spinal rootletsrelated to the lumbar and sacra! segments must thereforetravel a long way within the vertebral canal before theyjoin the spinal cord (or after they leave it). These spinalrootlets are collectively called the cauda equina and,together with the spinal cord, are wrapped in meníngealcoverings.

The space around the cauda equina is part of thesubarachnoid space, which surrounds the entire central nervous system. Cerebrospinal fluid accumulateshere in the region called the lumbar cistern and canbe sampled for clinical examination without risk ofdamaging the spinal cord.

2.b. Migrations

A cell's position in the embryo is important becausedifferentiation is ofien dictated by location, and the central nervous system is not an exception. The final location in the nerve cells is particularly important becauseneural function depends on the precision of connectionsbetween neurons and their targets. The migration ofnerve cells in vivo are highly directional. What tell thecells where to go and when to 'stop?

Inherent directional preferences. Because cell division duplicates, and therefore polarizes, cell organelles,the motilíty apparatus in daughter cells is mirrorsymmetric, and they move appart in mirror-images pat-


terns. This may be important in the development ofbilateral symmetry and in directing the initial extension of neurites (ALBRECHT-BuEHLER, 1978).

Chemotaxis. The direction of cell movement is influenced by diffusible agents, which may act eitherlocally as neurotrophic factors (neurite-promoting factors, survival and maintenance factors, etc.) or overrelative1y longer distances as chemotropic agents.

Differential adhesion. Cells choose a directionalresponse to directional properties within a solid because of differential adhesiveness to different surfaces. This response can also be mediated by nondiffusible molecules, which may be either extracellularmatrix molecules (collagen, proteoglycans, laminin,etc.) or plasma membrane molecules (NCAM,NgCAM, etc.). This molecules may function asspecific cell-cell recognition signals and as axonguidance cues.

Galvanotropism. Voltage gradients are commonplace in developing embryos. In general, are generatedby local electrogenic membrane pumps, and the circuit is completed by adjacent regions which are leakyto the particular ion involved. This electrical fields canindeed influence cell movement and axonal growth(Jaffe, 1981). Neurites grow more rapidly toward thenegative pole than toward the anode, and this cathodalinfluence can be observed in electrical gradients assmall as 7 mV/mm! Under the influence of fields ofthis magnitude, membrane proteins such asacethylcholine receptors can move rather dramatically. This finding is important because it implies that theelectrophoresis of particular molecules at the growingtip of an axon might affect the direction of movement.

2.b.1. Migration of neuronal precursors in thedeveloping central nervous system

Primary migration.

Following mitosis at the ventricular lining, at thetime that motor axons first grow out of the spinal cordof a 3-day-old chick embryo, the cells lack dendritesand sometimes retain a basal process that extendstoward the ventricular epithelium. A day later mostmotor neurons have migrated to their definitive position in the lateral motor column. As in the genesis ofcortical structures, the cells born last migrate throughregions occupied by older cells to reach a more superficial position (Ramón y Cajal, 1929).

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Secondary migration

Is a very important process in the development ofthe mammalian cerebellar cortex or the avian optic tectumo In the chick spinal cord, a secondary migrationtakes place after a uniform column of motor cells hasformed by the initial migration of neuronal precursors.In the thoracolumbar region, from the fifth to the eigthday of incubation, sorne of these cells undergo a secondary migration in a mediodorsal direction to form thepreganglionic visceral motor nucleus (the column ofTerni).

2.b.2. Migration of neurons in the peripheralnervous system

Most studies of nerve cell migration have examinedthe vertebrate neural crest, the embryological sourceofperipheral nerve cells. This tissue is remarkable for:

- provides precursors to a large assortment of centypes, including sensory and autonomic ganglion cellsand glia

- migrates long distances along predictable trajectories

- their movements are very robust and still occurafter a variety of perturbations.

Prior to migration, neural crest are located at themargins of the neural plate and remain as a distinctgroup just dorsal and lateral to the neural tube. Theyusually migrate laterally in two streams, one proceedingdorsolaterally and the other ventrolaterally. Whateverdictates the initial route, their subsequent course isunder environmental control. This has beendemostrated by transplantation experiments. In thechick, parasympathetic neurons originating from thecrest at levels corresponding to somites 1 through 7colonize the entire gut; crest cens migrating from caudalregions (somites 28 and more caudany) give rise onlyto ganglia of the postumbilical gut as well in the chickas in the mouse embryo (POMERANTZ et al., 1991;SERBEDZIJA et aL, 1991). Neural crest cells from theintervening regions (somites 8 to 27}do not normallycontribute to the enteric ganglia at an, but rather colonize the adrenal medulla and form the sympatheticchain ganglia (for a review, see Le Douarin, 1982).When segments of neural crest are transplanted fromone level to another, the transplanted cells adopt theroute characteristic of their new location (Noden,1975).

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2.e. Stablishing eoneetions

2.e.l. Extension of proeesses

A unique feature of developing neurons is the extension of processes. This special form of cell movement occurs at growth canes, which advance in amanner similar to the advance of the entire cells. It isattractive to suppose that the same cues that determinethe migratory path of neuronal precursors direct thesubsequent outgrowth of neuronal processes. The formation of orderly connections between neurons andtheir appropiate targets, as found in the adult animal,is increasingly thought to be dependent on epigeneticfactors.

Most work has focused on the extension of motorneurons to muscles rather than the ingrowth of sensoryaxons. Axon outgrowth and functional innervation ofmuscle occurs very early: almost as soon as motorneurons arise, they send axons 10 the periphery. Thus, motor neuron pools are still in process of segregating whenaxon outgrowth begins. When motor axons frrst enter thelimb, they bear no obvious relation to one another. Subsequently, the axons distribute themselves within the limbsin patterns that gradually become recognizable as thoseof the adult nerves. During this process, the limb budcontains a central core of precarlilage that separates twomesenchymal condensations called the dorsal and ventralpremuscle masses. Thus, axons do not actually growto particular muscles, but to regions of what appearsto be homogeneous mesenchyme. However, retrogradelabelling with horseradish peroxidase in chick and mammalian embryos shows that the adult pattern of nerveprojections is established at very early stages.

2.e.2. Formation and rearrangement of synapses

The work on synapses dominates present-dayneurobiology, and most of our knowledge about themorphology, biochemistry, and physiology of synapseformation comes from work on the relatively simpleneuromuscular junction.

Synaptogenesis begins very early in development,as soon as axons reach the vicinity of postsynaptic cells,and it continues for a long time. It seems unlikely thatthere are specialized postsinaptic sites that attract innervation before nerve contact. Once formed, however,the postsynaptic specialization persists and provides acompelling attraction to regenerating axons.

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The presynaptic axons have a strong influence onthe differentiation of postsynaptic specializations, butthe postsynaptic cell, in turn, has a similar infiuenceon the presynaptic cell. So, the formation of synapsesappears 10 be the result of a prolonged two-way conversation between presynaptic and postsynaptic elementsduring development, and in this sense is a particularmanifestation of the trophic interactions that will bedescribed latero

In the stablishment of patterned inervation (specificity of the neuronal connections), the influences that playa major part are:

- cell surface recognition molecules- specific responses to trophic factors- competition for trophic support- modulatory effects of neural activity on target

cells.

2.e.3. Trophie effeets of targets on neurons: NGFand others

There is a profound dependence of neurons on thetargets they innervate. In two specific parts of the nervous system, the sympathetic chain and dorsal rootganglion cells, there is good evidence that exists atrophic interaction mediated by the protein nerve growthfactor. This NGF is foulld in high concentrations in thesubmaxilary gland of the adult male mouse (not in thefemale, nor in the gland of closely related marnmalssuch as rats and rabbits) and in the guinea pig and bovineprostate. Why? It remains a mystery.

NGF binds to specific receptors on axons terminalsand is taken up and retrogradely transported to nervecell bodies. In addition to its infiuence on survival, ithas local effects on growing neurites, including both

~

an influence on the direction of neurite growth and localmaintenance oi terminal arbors.

The system that might be expected to operate inclosest analogy with NGF-dependent sympatheticganglion cells is the system of parasympathetic gangliaof vertebrates. Several laboratories have exploredneuronal death and survival in the avian ciliary ganglionduring the course of normal development, and havecharacterized components in conditioned media or tissueextracts that may ultimately qualify as trophic factorsfor ciliary ganglion cells. Two distinc components fromeye tissues appear to have different effects on ciliaryganglion cells (NISHI & BERG, 1981). One componentstimulates neuronal growth (assayed by cell size and


protein synthesis), whereas a second, specificallystimulates the cholinergic development of these neurons(as measured by choline acetyltransferase) but has noeffect on neuronal growth.

While it is well established that NGF is essentialas a survival and maintenance factor for developingand adult sympathetic neurons (LEVI-MONTALCINI& ANGELETTI, 1968), and that the vast majority (atleast 80 %) of neurons within spinal sensory gangliarequire NGF as a survival factor during a limitedperiod of development, it is now evident that NGF isnot a universal neurotrophic factor for all sensoryneurons.

NGF is clearly a key peripheral target-derived survival factor for neural crest-derived sensory neurons.Given that each sensory neuron projects not only toa peripheral target but also in the CNS, there is nowconsiderable interest in the possibility that specification of sensory neurons during development may alsobe influenced by trophic factors derived from their central target. The first indication carne from studies whichshowed that conditioned medium from a rat C-6 gliomacellline and from adult rat brain astroglial cells contained neurotrophic activity which supported the survival and outgrowth of neurites from dissociatedneuron-enriched cultures of chick embryo dorsal rootganglia. In both cases, the ínability of anti-NGF antibodies to neutralize the CNs-derived neurotrophic activity has been taken as an indication that this factoris distinct from NGF. The purification of a brainderived neurotrophic factor (BDNF) from pig brain,has provided the most compelling evidence that sensory neurons may require a peripheral (NGF) and acentral (BDNF) target-derived neurotrophic factor.

2.cA. Neuronal death

Death of neurons is a prominent feature of embryonic development in vertebrates. Normally occurring neuronal death has been found in spinal motorneuron pools, spinal preganglionic neurons andautonomic ganglia. In all of these systems, the extentof cell death is considerable (30-75 %).

Although there are sorne exceptions (mammaliansympathetic ganglia or olfactory epithelium), the timing of cell death is usually quite sharp and ofien occurs at approximately the same time that axons reachtheir targets. In the chick embryo spinal cord, dorsalroot ganglia and ciliary ganglion, the period of cell

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death spans just a few days in early development (days5 through 12 of incubation).

For many of these cells, death is not a result oflineage but occurs because of competition betweennerve cells at the level of the target they innervate. Theconventional view is that the neurons innervating atarget compete with one another and that the losers die.The major purpose of this competitive phenomenon ispresumed to be matching the size of innervating populations to the capacity of their targets (HAMBURGER &

QpPENHEIM, 1982). Present evidence supports the ideathat vertebrate neurons commonly die because of failurein competition for a trophic factor or factors produced bay the target.

Synapses are not immutable in maturity but changein a least two ways: the number of synaptic contactsand its efficacy. The synaptic connections in adultsrepresent a balance between a tendency ofaxon terminals to sprout and a tendency to withdraw. A second way concerns changes in the function of individualsynaptic contacts with use or disuse, and there is nodoubt that this occurs both at the neuromuscular junction and in the mammalian CNS. It is this aspect ofsynaptic modification that has attracted most attentionas a possible basis for learning.

In old age, neuromuscular junctions show more terminal branching and a greater number ofterminal buttons. In spite of this increased complexity, agedneuromuscular junctions are more easily depressed,contain fewer sinaptic vesicles, and are less capableof sprouting.

2.c.S. Acquisition of transmitter properties,generation of action potencials, and differentiationof neuronal formo

A number of differentiated neuronal properties aresufficiently important for the function of mature nervecells to have become the objects of study in their ownright. Qne series of such studies has focused on thefactors that infiuence the choice of neuronal transmitter. It is well known that neural crest cells give riseto sympathetic and parasympathetic ganglia, and onegeneral rule is that most sympathetic ganglion cells areadrenergic (and therefore contain the synthetic enzymesfor norepinephrine), whereas parasympathetic ganglioncells are cholinergic and thus have a different set oftransmitter enzymes. A natural question is whether different parts of the crest are regionally differentiated

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from an early stage or whether their differentiation isinflueneed by destination or rnigratory route. Whensympathetie trunk erest is transplanted to the vagalregion, it gives rise to eholinergie ganglia that arriveat their destination by a rnigratory route eharaeteristieof the vagal trunk erest. The most important lesson ofthis work is that sorne aspeets of neuronal fate are clearly regulated by the eell's environment.

Another aspeet of neuronal differentiation that hasbeen studied in sorne detail is the aequisition of theeharacteristic ion ehannels that support signaling. Veryshortly afier the neural tube closes, appear differentiated neurones, capable of generating action potentials.Studies of vertebrate neurons in culture show that sornenerve cells are initially activated by voltage-sensitiveCa2+ channels; this early property is then graduallyreplaeed by the sodium conductance mechanismcharaeteristic of most mature nerve cells.

Much of the work of classical anatornists involvedcategorizing neurons according to their shappe.Although each neuron has a unique branching pattern(think of individual trees), different classes of neuronstend to have similar geometries (think of oaks andmaples), as it has been shown by placing dissociateddeveloping neurons in culture.

3. REGENERATION

Studies ofaxon outgrowth during regeneration provide additional information about the behavior of growing axons and raise important clinical issues.

In experiments on salamander limbs, it has beendemonstrated that most classes of peripheral axonsregenerate readily after they are severed and ofien regaintheir normal targets (muscles or sens0ry receptors, forexample). However, especially in mammals, they havelittle luck finding their original targets within the largercontext of a limb or sorne other body parto It seemsto be that the tissues of the mature limb are unable toprovide interpretable eues that enable the axons to findtheir way. Thus, when motor axons regenerate, they appear to innervate deprived muscles or skin on a firstcome, first-served basis. Despite the enormous capacityfor regeneration of both peripheral nerve and muscle,clinical results of muscle reinnervation are ofien disappointing (IRINTCHEV et al., 1990; WASSERSCHAFF,

1990).In the central nervous system ofmammals (including

man), an additional phenomenon is apparent: severed

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central axons are loath to grow at all, extending onlyshort distances beyond a lesiono In a series of simple,yet powerful experiments, A. 1. Aguayo and his colleagues showed that central axons can actually extendup to 35 mm when they are confronted with the distalstump. of a peripheral nerve, but are prevented fromdoing so by sorne aspect of the central environment(Aguayo et al., 1982). The most likely villain in thispiece is the central glial cell, but the fact that centralaxons can extend long distances offers sorne promise,in principIe, of adressing spinal an other lesions (of theoptic nerve, for example) where injured axons have togo a long way to re-establish their normal connections.

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