The development of cholinergic neurons

261

Brain Research Reviews, 13 (1988) 261-286 Eisevier BRR 90085

The development of cholinergic neurons Ken Vaca DepartmentofNeurology,Program in Neuroscience, Baylor College ofMedicine,Houston, TX 77030 (U.S.A.) (Accepted 14 June 1988) Key work Cholinergic neuron; Development; Ceil lineage; Innervation; Synaptogenesis; Ceil death; Neuromuscular junction; Growth factor

CONTENTS 1. Introduction

............................................................................................................................................

2. Initial expression of choiinergic properties

......................................................................................................

262 262

3. Ceil lineage of cholinergic neurons ................................................................................................................ 3.1. Exogenous markers used to trace ceil lineage ............................................................................................ 3.2. Ceil fates in chime& transplants ............................................................................................................ 3.3. Predisposed traits and secondary inductions in culture . ................................................................................ 3.4. Antigenie expression and ceil type ..........................................................................................................

263 263 264 264 264

4. Innervation of target tissue .... ...................................................................................................................... 4.1. Migration and axon extension ............................................................................................................... 4.2. Topographic sorting .... ....................................................................................................................... 4.3. Giia may facilitate innervation ...............................................................................................................

265 265 265 265

5. Synaptogenesis ........................................................................................................................................ 5.1. Initial contacts are functional ................................................................................................................ 5.2. Ultrastructural specialization ................................................................................................................ 5.3. Physiologic~ changes .......................................................................................................................... 5.4. Acetylchoiine receptor clustering ........................................................................................................... 5.5. Extracellular matrix ............................................................................................................................

266 266 267 267 267 268

6. Ceil 6.1. 6.2. 6.3. 6.4.

269 269 269 269 270

death ............................................................................................................................................... General features of normal ceil death ...................................................................................................... Competition for target ......................................................................................................................... Roieof activity in target ... .................................................................................................................... Role of afferent input ..........................................................................................................................

7. Synapse emanation .................................. ................................................................................................ 7.1. Competition and dependence on target activity .......................................................................................... 7.2. Sprouting and poiyneuronai innervation .................................................................................................. 7.3. Constraints of a critical period ............................ ................................................................................. 7.4. Synapse elimination in culture ...............................................................................................................

270 270 271 271 272

8. Modulation of choiinergic expression in culture ......................................... 8.1. Target tissue factorsand conditioned medium ............................................. 8.2. Effects of electrical activity .....................................................................

272 272 273

9. Effects of NGF on cholinergic neurons . . . . . . . . .. . .. . . . . ..~..~.....~.........~.........~...........~............~.....~.~.~.~.~..............~.... 273 Correspondence: U.S.A.

K. Vaca, Department

of Neurology, Program in Neuroscience, Baylor College of Medicine, Houston, TX 77030,

0165-0173/88/$03.50 @ 1988 Eisevier Science Publishers B.V. (Biomedical Division)

262 10. Maturation ofcholinergic function 10.1. Increased synaptic reliability

_, . .._.

10.2. Latephenotypic transformations 10.3. Continued dependence on target 11. New directions

_, ,_.

._.

.,

.

274

_, ._, ._,

_.

,.

_, _. ., _. _.

Acknowledgements

_. _.

References

_.

1. INTRODUCTION

has seen

276

_, _. _,

27s 275 275

_. _. _, _. _.

Summary

Nature

274 _.

276

. .... .. ...... ... ...... ... .... .. ... ......... ....._.......................

277

general reviews of motoneuron development may be consulted for additional historical background15,67.72. fit to conserve

acetylcholine

(ACh) as principal neurotransmitter of the somatic and autonomic motor systems in vertebrates. As such, the mechanisms which control the ontogeny of these cholinergic systems must necessarily be adapted to achieve functionally successful connections in a wide variety of anatomical venues. In this review, I attempt to provide a conceptual overview of the cellular mechanisms involved at each of the stages of development of cholinergic neurons. Technical advances have made it possible to address issues which were unapproachable a decade ago. Labeling methods have been used to follow the fate of undetermined or pluripotent cells and the initial journey of extending axons. Immunochemical markers have made it possible to recognize type-specificities of cells and potential signposts in the extracellular matrix which formerly were indistinguishable. Tissue culture systems have provided a relatively well-defined environment to look at developmental mechanisms, sometimes over a time course of minutes or seconds. These and other innovations, together with the dogged and skillful application of classical approaches, have made it possible to provide a more or less temporally integrated view of the development of the cholinergic neuron (at least, the motoneuron, see Fig. 1). Recent intense interest in cholinergic neurons in the brain makes it desirable to have a bench mark for comparative purposes. Because the scope of this review is so broad, certain exemplary systems will be presented as prototypic cholinergic neurons. There will be an emphasis on more recent work, particularly where technical improvements have made more definitive interpretation of earlier data possible. Several excellent earlier

‘09. I have chosen to omit reference to fragmentary observations on cholinergic systems which are not easily integrated into a broad context. Regenerating systems, which often recapitulate developmental phenomena, will not be covered. My aim is to outline each of the important phases in the transition from a totipotent, undifferentiated cell to a mature cholinergic neuron. 2. INITIAL EXPRESSION

OF CHOLINERGIC

PROPER-

TIES

Cholinergic systems are widespread throughout embryonic development but the functional roles of ACh at very early stages have not been established. ACh, choline acetyltransferase (CAT, EC 2.3.1.6) and acetylcholinesterase (AChE, EC 3.1.1.7) are present in spermatazoa of a variety of species from sea urchin to human, and sperm motility can be modulated by nicotinic drugs 275. Muscarinic receptors are present in human oocytes”, and the human placenta is a rich source of CAT’25. A careful study established the presence of naphthylvinylpyridine-sensitive CAT, true AChE, sodium-dependent high affinity choline uptake and synthesis of ACh from exogenous choline at the primitive streak (16-18 h) stage of the chick embryo3’*. Filogamo and Marchisio’* have reviewed the earlier literature suggesting an almost ubiquitous but usually transient presence of AChE in early neuroblasts. It has been hypothesized that ACh and other neurotransmitters play important regulatory roles with regard to cell division and morphogenetic movements in the early embryo’84~206. In any case, it should be clear that non-synaptic functions for cholinergic enzymes and receptors may be present and

263 that the presence

of these biochemical

not imply the formation

oorsaI

markers need

of cholinergic

synapses

but

requires corroboration by anatomical and physiological techniques if the inference of synaptic function is

“eg

m

Polyclonal Indeterminate cell llneaae

A”

cell+3

eo~~pus 16 contams motoneuron precursors

9

3

to be made.

512 cellsto e xenopus contains 0% out 15 mOtone”rOn precursors

3. CELL LINEAGE OF CHOLINERGIC

NEURONS Primary neural lnductlon

Cell lineage analysis is aimed at determining

how a

Prlmltwe streak chtck expresses low level copactty for ACh synthesis

cell’s embryonic ancestry influences its developmental fate and its subsequent relationship to other cells. With a few exceptions,

this approach

new insights into developmental trolling cholinergic neurons.

mechanisms

Termtnol mltows of motor neuroblasts

4

512-cell stage and followed the fate of their share some ancestors with progeny 13’. Motoneurons Rohon-Beard (sensory) neurons at early (up to 256cell) stages but become totally segregated by the 512cell stage. All primary motoneurons were derived from 2 or 3 blastomeres at the 16-cell stage and about 15 blastomeres at the 512-cell stage (see Fig. l), although these progenitors also gave rise to non-neuronal cell types ‘38. It was calculated that the daughter cells of these potential ancestors were biased, with a probability of about 0.7, toward remaining in the motoneuron lineage after each successive division. The entire population of primary motoneurons was estimated to undergo a terminal cell cycle at about the 16th generation in late gastrula (but much later in aves and mammals), consistent with observations using tritiated thymidine autoradiography’@. At later stages, when motoneurons have extended axons to the periphery, there was a highly significant preferential association of HRP-labeled motoneurons with clonally related labeled myotubes2”. This suggests that specific matching of motoneurons with their targets may occur very early in development. Most neuronal precursors of the brain segregate early into different compartments than that containing motoneuron precursors13’. This implies that the cholinergic phenotype arises independently in cells

G-i (2% Axon extenson to target Secondary Inductions

Irn

50

A Maturation

Ventricular ZO”e Mlgratlon to form lateral motor column Exprewon of neuronal phenotype

con-

3.1. Exogenous markers used to trace cell lineage Jacobson and colleagues have injected single blastomeres of Xenopus embryos with horseradish peroxidase (HRP) for histochemical marking at the 2through

w

has not

been widely applied to vertebrate neurons, but recent methodological advances hold great promise for

Synoptogenesls CornpetitIon for survival Increased chollnerglc exprewon

of motor endplate 17

222

Mod+atlon act1wty

by

Fig. 1. An overview of motoneuron development, with emphasis on the early stages. Fate maps of early Xenopus embryos indicate that a small group of cells at the blastula stage becomes segregated into a distinct compartment which gives rise to motoneurons and other positionally related cell types’38.2’1.Low level expression of the capacity for ACh synthesis is detectable in the primitive streak stage embryo”*“, although localization is unclear. Sometime after primary neural induction, the motor neuroblasts undergo their terminal mitosis. In Xenopus this occurs in the 1l- 12 h embryo during gastrulationi6s while in the chick it comes at 2.5-4 daysi3’ and in rat at 11-14 days3, long after neurulation. Initial axon extension is autonomous, but further survival and increased expression of the cholinergic phenotype requires secondary inductions from the target muscle. The functional capacity of the maturing motoneuron is fine-tuned consonant with appropriate levels of activity.

of divergent lineages. Application of similar lineage analysis to cholinergic neurons in the brain may require use of a second, postfixation label, such as antibody to CAT, in cases where there are mixed populations of neurons. In species with a longer embryogenesis, a more metabolically stable exogenous label may be required. Recombination of a gene for a histochemical marker with replication-incompetent retroviral vectors holds great promise as a stable and innocuous genealogical marker25’*277.

264 3.2. Cell

fatesin chimeric

An endogenous has an obvious

transplants

marker

advantage

that is readily in following

tended periods of development. leagues have made extensive

visualized

cells over ex-

Le Douarin

and col-

use of quail-chick

chi-

enzymes 1”1.347 . Thus, departure from the cell cycle allows increased expression of certain predisposed traits,

and this expression

is greatly

meras, in which the nucleoli of the two species can be

tion cultures suggests that predisposed expressed

in the absence of inductive

stochastic

manner,

it is possible to follow the

fates

of precursor

of discrete

groups

cells trans-

to various regions of the embryo.

If postmi-

infiuences

gratory

neurons

ganglion,

er, cholinergic

which

have

characteristics,

already

begun

are transplanted

to acquire

cholinergic

to the adrenomedul-

lary or trunk level of the neural axis in younger embryos, they can again migrate, become incorporated into sympathetic ganglia or adrenal gland and acquire adrenergic properties”‘. Conversely, truncal level neural crest cells, which are normally destined to become adrenergic, can acquire cholinergic properties when transplanted to the vagal region or the hindgutt87.2N. When transplants from a wide variety of neural crest levels were compared, it was found that each was capable of differentiating into cholinergic or adrenergic cells irrespective of origin but dependent exclusively on their localization in their hosts’sg.

3.3. Predisposed traits and secondary inductions in culture In spite of the target tissue-dependent plasticity of neural crest, cell culture experiments suggest a level of phenotypic commitment in the premigratory crest. Head and trunk neural crest cultures contain dopamine /%hydroxylase and develop CAT and the ability to accumulate both ACh and catecholamines after several days in culture in the absence of presynaptic spinal input, target tissue or conditioned medium factors’41*202. Mesencephalic neural crest, which gives rise to the ciliary ganglion2r6 and perhaps other cholinergic cells, contains CAT and can synthesize and store ACh from the onset of its migration2’t. Fauquet et al.” report cholinergic traits precede adrenergic ones in both mesencephalic and trunk crest cultures. However, it is clear that culture conditions which favor proliferation of neuroblasts tend to suppress neuronal differentiation including transmitter synthetic

as different

traits may be influences

in a

cells within a clone

exposed to identical culture conditions may express different characteristics287. In the presence of such

planted

of the ciliary or Remak

de-

pending on interactions with appropriate target tissues. Clonal analysis of neural crest in limiting dilu-

readily distinguished by staining with the FeulgenRossenbeck reaction, in the study of the neural crestis6. With this method,

enhanced

as heart cell conditioned differentiation

crest cells while melanin

medium,

howev-

is promoted

in neural

and catecholamine

synthesis

are blockedzs8. The ability of sympathetic neurons in culture to acquire cholinergic properties and form functional cholinergic synapses with cardiac or striated muscle has been extensively reviewed 27.28,‘3s. This cholinergic induction, along with a concomitant reduction in adrenergic traits, is triggered by a recently purified soluble glycoproteins4, secreted by heart and some other non-neuronal cells. Conditions which may induce transmitter plasticity in cultured thetic neurons have been less firmly Chick ciliary ganglia exhibit tyrosine

parasympaestablished. hydroxylase

and phenylethanolamine ~-methyltransferase immunoreactivity which is enhanced by coculture with notochord, but they maintain CAT activity and do not synthesize or store detectable cate~holamines under the conditions

used’34~3’0.

3.4. Antigenic expression and cell type With antibodies of sufficient affinity, it is possible to follow the appearance of differentiated traits in the context of cell lineage. Several laboratories have raised monoclonal antibodies to CAp2s70*191. The postnatal development of CAT immunocyto~hemistry has been studied in rat spinal cord242, but the sensitivity of the method is not yet adequate to reliably stain cells prior to embryonic day 17 (P. Phelps, personal communication) even though measurable enzymatic activity is present earlier. The lE6 antibody’* has been useful in following the development of cholinergic neurons in mixed populations of cultured cells including basal forebrain nuclei, striatum The preparation of higher and spinal cord 2oo+215,294. affinity antibodies may be necessary to detect cholin-

ergic neurons very early in development. The preparation of antibodies to celt surface antigens of specific subpopulations of neurons is in increasingly wide use”. One such antigen, Chol-1, is a polysialoganglioside, conserved from fish to mammals, which may be specific for cholinergic nerve terminals26’, although the antibody has not yet been exploited for developmental study. Similarly, a monoclonal library to Torpedo cholinergic synaptosomes holds promise for useful developmental probeslti. Monoclonal antibodies to membrane fractions of embryonic spinal cord suggested certain antigens common to neurons and gha may appear earlier than those specific for neurons307. Some specific antigens are shared by developing motor and sensory but not other neuronsE6. Four highly specific monoclonal antibodies were raised to chick ciliary ganglion neurons, two of which interacted with the choline uptake system i3. One was shared with a small percentage of spinal cord cells, possibly motoneurons, and two others were shared with a small percentage of mesencephalic neural crest, possibly the potential ancestors of the ciliary ganglion. Strategies for exploring development immunochemically are becoming increasingly refined, and examples will be discussed in reference to appropriate stages of development. 4. INNERVATION OF TARGET TISSUE

4.1. Migration and axon extension While a neuron can begin expressing cholinergic properties with some degree of autonomy very early in development, it must then extend its axon to innervate an appropriate target tissue. Motoneurons of the chick spinal cord lateral motor column provide the most thoroughly investigated example of the innervative process (cf. ref. 3 for rat). In their final cell cycle, neuroblasts begin making neurofilament proteins, briefly coexpress neurofilaments and vimentin in the postmitotic neuron, and then rapidly lose vimentin immunoreactivity3~. Some neuronal cell surface-specific antigens are first expressed upon cessation of DNA synthesis301. The postmitotic neurons make a medial to lateral migration from the ventricular epithelium and begin extending their axons to the periphery. The conclusion of migration may be determined by components present in extracellular matrix, as is the case in neural crest cell migration217.

4.2. Topographic sorting The rules governing the to~graphic arrangement of motoneurons into discrete pools and their selection of specific pathways have been reviewed in detai115*128~176~177. Only a few salient points will be recapitulated here. While radical ablation of the limb bud causes the outgrowing nerves to end in a neuroma”*, partial lesions of the limb bud allow them to sort out in the nerve plexus in a relatively normal fashion, even when their appropriate target was removed316. After specific lesion of muscle by X-irradiation of premigratorj somitic mesoderm, the main nerve trunks and their cutaneous branches entered the muscleless wing and developed normally, but the nerve branches which in a normal limb would lead to individual muscles were absentr9*. The growth cones of extending motor nerves are capable of taking widely divergent trajectories, but sort out in ‘decision regions’ (the nerve plexus and regions where muscle nerves diverge) to make appropriate and highly specific projections317~318.The extending nerve fascicles appear to passively maintain their topographic relationships in the spinal nerves, main nerve trunks of the limb and finally in individual muscle nerves but actively respond to local environmental cues in the ‘decision regions’ to cross over and rearrange themselves, often in complex ways. Mistakes in this sorting process appear to be very rare, as both the initial projections detected by orthograde and retrograde labeling and initial functional innervation are essentially identical to the adult pattern175*178.317. Even when muscles are left uninne~ated after deletions of their motoneuron pool, adjacent motoneurons will pass them by to reach their normal target17*. Relatively extensive target displacements, such as limb duplications or dorso-ventral rotations, lead to some abnormal matches, however, indicative of a partial distortion of cues in the limb129~338-340. 4.3. Glia may facilitate innervation Neural crest cells, postulated to be Schwann cell precursors, have been observed to precede the growing axon into the limb and may possess some pioneering function 22i. In embryos where the neural crest was excised, the outgrowth of ventral root axons was only slightly delayed 262but major deficiencies of innervation of the limb musculature are apparent laters45. suggesting that Schwann cell precursors may

either

guide

or otherwise

facilitate

innervation’““.

Some crucial change in the environment bud takes place to allow the invasion

5. SYNAPTOGENESIS

of the limb

of the axons. as

5.1. Initial contacts arefunctional

limb buds from earlier embryos do no? support motor neurite outgrowth in vitro”‘. Semi-intact prepara-

The earliest stages of synapse formation are not easily studied in vivo due to the small size of the em-

tions of the limb bud in culture look promising

bryonic cells and the paucity of structural

amine

the molecular

mechanisms

involved

outgrowth

and pathway selec?ion’77.““2.

The

matching

ontogenetic

to exin early

tion. In the periphery, propriate

mechanism

between

specializa-

the first motor axons enter ap-

regions of the dorsal or ventral muscle mass

prior to muscle cleavage

or myoblas?

fusion.

ACh

nerve and muscle must to a large extent be phyloge-

sensitivity

netically

apse formation 29. in cultures of Xenupus neurons, spontaneous pulses of ACh release could be detected from growth cones prior to encounter with the target

conserved,

selectively

innervate

as chick and quail motoneurons appropriate

ras formed from limb-bud evidence

implicates

shared positional

muscles

transplants3”“.

in chimePreliminary

a shared cell lineage2”

origin of motoneurons

and/or a

and myoge-

nit target cells”l as determinants of the selectivity of innervation. The specific pattern of preganglionic projections to sympathetic ganglia is similarly biased by segmental

level of origin26”.

is detectable

cellssJ6 and evoked

in the myotome

release

prior to syn-

was reported

later”“‘.

Similarly, release could be evoked from growth cones of isolated ciliary ganglion neurons, albeit with long and variable latency’“‘. At the onset of contact. no synaptic potential was detected when only the tips of the growth cone filopodia touched the muscle

Fig. 2. Developmental changes in the ultrastructure of motor nerve terminafs. A: at the onset of transmi~ion, terminals are remarkably unspecialized, with relatively few synaptic vesicles includ~g those of the pleiomorphic and dense core type. The apposition with muscle is often irregular. Schwann cells and axons in passage may be seen nearby. B: by late in the eel1 death period, terminals appear more organized, with occasionai deposits of dense material in the pre- and postsynaptic membranes, as well as some signs of basal lamina in the cleft. C: successful terminals acquire or increase all major structural elements, including active zones, mitochondria and locally concentrated vesicles as elimination of polyneuronal innervation progresses. Basal lamina fill the cleft. D: mature terminal has large complement of mitochondria and often a huge reservoir of vesicles. It is fully sheathed by a Schwann cell. AZ, active zone; ax. axon; BL, basal lamina; DCV, dense core vesicle; mit, mitochondria; mt, microtubufe; PSD, postsynaptic density; SC, Schwann cell; ser, smooth endoplasmic reticulum. Based on observations in Pilar et al. 248,with recognition of similar findings in other systems in vivo and in vitro’“~LM~21J.

267 membrane, but e.p.s.p.‘s were evoked just a few minutes later as the varicosity of the growth cone arrivedls5. This initial release appeared to be quanta1 in nature. Thus, in tissue culture, the minimal requirements for synaptic function are already present at the time of contact, as had been suggested for in vivo development’64~17g.

The earliest synaptic contacts exhibit minimal ultrastructural specialization (see Fig. 2A). In vivo, spontaneous m.e.p.p.‘s are first observed in muscle at about 1 day in Xenopus embryo’@, 4-5 days in chick embryo178 and 14 days in the rat6’. At that time, vesicle clusters, dense-staining presynaptic active zones, basal lamina with associated AChE in the synaptic cleft, and AChR receptor clusters on uninne~ated myotubes are all generally rare or absent. Occasional 50-nm vesicles, pleiomorphic vacuoles, and densecore vesicles are observed, while terminal mitochondria are rare. Vesicle clusters, presynaptic and extracellular dense material, and postjunctional ridges become apparent over the next 24 h in Xenup~1~,3~ and 2-4 days in chick and [email protected],z~ (Fig. 2B-D). Similar delays between onset of function and ultrastructural specialization occur in cultured rat synapses214 but not with Xenopus 237*337.With freezefracture, intramembranous particles are initially randomly located and oriented and later organized into rows at the active zones15’. Most commonly used culture conditions favor the spontaneous appearance of AChR clusters or ‘hot spots’ on uninnervated myotubes. Innervation in culture induces the formation of new AChR clusters beneath the neurites and results in the gradual dissipation of uninnervated clusters6*81,165.266. Coculture of ciliary ganglion neurons with myotubes results in an accelerated appearance of synaptic vesicle-specific antigens and over the next 4-5 days an eventual confinement of these antigens to sites of nerve-muscle contact25.

5.3. Physiological changes Newly formed junctions exhibit a skewed distribution of m.e.p.p.‘s with a preponderance of small amplitude events 17~t61 , Many of these small events have slow rise times indicative of a distant site of activation (as in the case of multiple junctions or electrotonic coupling to adjacent fibers), however large events

with slow rise times are also seen151*164. M.e.p.p. frequency is initi~ly very low (0.1-5 min in chick and rat) and very gradually increases toward adult values (about l/s) over approximately two weeks. Because of the high input resistance of embryonic muscle fibers, the small currents induced with evoked e.p.p.‘s are often sufficient to elicit muscle contraction’7*6g. These evoked potentials have a low quanta1 content. The skewed distribution of the individual events composing these early e.p.p.‘s is non-Gaussian but may be fit by a gamma function, which describes randomly distributed, unitary events and can accomodate the skewness155. It is reasonable to suppose that the skewed distribution of quanta may reflect the morphological heterogeneity of the vesicle population and some unevenness in the spacing of the synaptic cleft. The estimated probability of release at these virgin synapses (0.5-0.6) is comparable to mature values, but the number of quanta available for release (typically 2-3) is very 10~“~ as is also seen in newly regenerated junction$j. Implicit in the relatively high values for release probability is the presence at the newly formed synapse of competent voltage-sensitive calcium channels, which have indeed been observed in growth cones32,57,105. There appears to be competence for the formation of neuromuscular synapses between widely divergent species. Cultured chick and rat spinal cord cells readily form functional synapses with muscle of the other taxonomic clas@’ as do Xenopw spinal cord cells with the rat L6 muscle cell 1ine’54.Long-term cultures of mouse spinal cord explants with human muscle result in structurally mature endplates between different orders23g. Spinal cord transplants between chick and quail embryos result in chimeras which can hatch and behave normally for several weeks, until immune attack sets in’56. 5.4. Acetylcholine receptor clustering The localization of a high level of ACh sensitivity at the site of contact between nerve and muscle represents a structural specialization, AChR clustering, that is part of synapse formation. interference with synaptic function fails to prevent the longer term formation of these structural speci~izations which become functional once the blocking treatment is removed. Paralysis with tetrodotoxin (TTX) or inhibition of ACh synthesis with naphthylvinylpyridine or

268 hemicholinium or innervation receptor

did not affect either ACh sensitivity of muscle in cultureh’.222.““‘. Similarly.

agonists

and antagonists

chol, ACh with physostigmine, gallamine

and atropine

including curare,

carba-

Naja toxin,

fail to prevent the neurite-in-

type with larger unitary conductance and shorter mean open time. Using bovine muscle mRNA expressed in Xenopus oocytes, channel

properties

the y-subunit

these changes in single

can be reproduced

by replacing

of the ACh receptor with a distinct gene

duced localization of ACh sensitivity and subsequent transmissions”~222~301. In vivo paralysis with TTX or

product encoding the &-subunitzos. Differences in the extent of post-translational modification of AChR

curare permits the appearance

subunits

clusters but reversibly

of junctional

diminishes

receptor

the growth of the

have also been observed

The induction of junctional clusters may be mediated by proteins released by the nerve. A 42 kD

clusters, the appearance of AChE, and the decline of extrajunctional ACh receptors~4,98.“4. /Yenopus cells

peptide

develop

nor-

capable of increasing

To a

receptor

with substantial

mal electrophysiology

autonomy,

expressing

in single cell cultures”4.

limited extent (about 30% of controls),

they can form

in development’.

has been purified clusters

from chick brain which is

receptor

insertion

and inducing

on myotubessz2.

An aggregating

factor (or complex of 4 antigenically

similar proteins)

functional synaptic connections in Ca-free mediurn12j. Xenopus motoneurons can induce proper localization of AChRs and AChE in the presence of TTX, high MG, zero Ca or even >lOO mM I@. In a broader context, continuous paralysis of Xe~~pu~ embryos with chloretone or lidocaine from neural’ fold stage to hatching failed to produce any obvious disruption of motoneuron morphology or the gross

termed agrin, purified from Torpedo electric organ, causes the formation of patches of ACh receptors, AChE, and butyrylcholinesterase220. Agrin resides in the basal lamina of the synaptic cleft and its maintenance is neuron-dependent2’s. Of interest are 3 cell lines which synthesize and release ACh but lack large dense-core vesicles in their neurites and the ability to induce AChR clusters on cultured muscle”‘. They

pattern of physiological activity upon removal of the drug’i7. It is suspected that the great autonomy of Xenopus is at least partly due to the abundance of

form few or no synapses on muscle unless cocultured with neuroblastoma which may lack ACh as long as they contain receptor aggregating activity. On the other hand, myotubes of the L6 cell accept cholinergic synapses even while they are incompetent to form

yolk and intramitochondrial granules in the neurons which supply essential nutrients and may otherwise stabilize the internal milieu26,‘69. Junctional development in chick and rat is more sensitive to the external environment, inciuding calcium concentration. Calcium is required for the organization of AChR clusters and the accumulation of AChE”.*6.‘7’. The asymmetric form of AChE characteristic of the junction declines rapidly after denervation or blockade of activity38~2”8. Details of the developmental changes controlling nicotinic AChRs are reviewed elsewhere7”*1s2~273?83. There is an increase in the metabolic stability of junctional receptors from a half-life of about 24 h to greater than one week after l-3 weeks of functional innervation, which appears to be related to a reorganization of the subsynaptic cytosk~leton~5.3~‘. There is also a developmental decrease in mean channel open time and a smail increase in single channel conductance in mammalian and amphibian muscle. This is due to a gradual shift of two populations of channels, from an embryonic type with smaller unitary conductance and longer mean open time to an adult

large receptor clusters Is4. Primary cultures of noncholinergic neurons are unable to induce localized ACh sensitivity or AChR clustering54~2”.

The extracellular matrix (ECM) may play an organizational role in the formation of AChR clusters and may influence presynaptic differentiation as well. In rat, patches of basal lamina appear within a day of synapse formation and synapse-specific antigens, including AChE, appear shortly thereafter”‘. The endplate region becomes a site of increased adhesiveness of the muscle ce112’2.Accumulation of basal lamina by muscle is modulated by activity and inhibited by paralysis27h. This may be dependent on a soluble factor from nerve273. One major component of ECM, a heparan sulfate proteoglycan. appears in plaques at the sites of nerve-induced AChR aggregates, as well as adjacent to AChR clusters in aneural muscle cultures7*8.‘4. As motoneurons approach the muscle, they initially cause a local reduction in this

269 apparently by proteolysis, followed by uroteo&can, -* the deposition of dense plaques at the synaptic regions. 30th synaptic and extrasynaptic proteoglycan plaques attained metabolic stability at early stages when AChR clusters remain labile to denervation’. Some molecular signpost, of which these proteoglycan plaques and agrin may be a part, is formed in the extracellular matrix and induces regenerating nerves to reform synapses at the original synaptic si?e146* 199274,These regenerate nerve terminals form on the basal lamina sheath even in the absence of muscle fibers and differentiate to a considerable extent although they do not fully mature94.

death is primarily used to eliminate aberrant connections or defective neurons. Neuronal ceil death appears to be an autolytic process, first manifest by a marked dilatation of endoplasmic reticulum, mitochondrial degeneration and later pyknosis and ribosomal disaggregation51.z4~~Once begun in a cell, the degenerative process is rapid and irreversible.

6.2. Competition for target Removal of target tissue does not affect the time at which cell death begins, but it does abbreviate the time course over which increased numbers of neurons degenerate. In the limbless mutant of the chick, formation of the lateral motor column is normal, but most motoneurons are lost to cell deathi83. Grafting 6. CELL DEATH of wing limb-bud precursor tissue from normal donors to ~~~~~~~~ hosts increases motone~ron survival 6.1. ~~ne~a~fE~~ur~~ ~f~orrn~~ celldeath to greater than 40% on the graft side, similar to surAt a critical period in development coinciding with vival in normal chicks 182.Experimental provision of synaptogenesis, most neurons become dependent on extra target tissue, as with a supernumerary limb, successful interaction with their target for continued leads to a partial reduction of cell deatht3’. Conversesurvival. While the nature of this interaction is not ly, when one or more of several distinguishable popprecisely understood, its experimental interruption ulations of neurons ~nne~ating a common target are by ~otomy or target removal inevitably results in the Iesioned prior to cell death, neuron survival is indeath of more than 90% of the neuronal population creased in the remaining population246. Increases in involved. Yet even without interference, a large fracaxon diameter, conduction velocity and glial ention of the neuronal population, typically 40-60%, sheathment are accelerated in the neurons subject to dies at this time. The phenomenology of neuronal cell death has been reviewed extensively’g~~~111~2z9~ reduced competition 246.These obse~ations support the theory that motoneuron survival is dependent on 23i; the essential features will be summarized and successful competition for some limited resource more recent observations discussed here. It is clear available from target tissue. It is clear however that that most motoneurons that eventually die have sent motoneuron survival is not simply related to the mass axons to the immediate proximity of the appropriate limb muscles, as evidenced by retrograde transport of muscle available. When Xenopus embryos were hormonally modified to increase their muscle mass 3of HRP74,L75,and that many of these have made functional synapses, observed with EMG or focal extrato 4-fold, there was no effect on motoneuron survival cellular recording i7*. Failure to interact with a very and only a modest increase in motoneuron soma size297. specifically matched target does not account for most cell death, as forced interaction of motoneurons with 6.3. RoIe ofactivity in target foreign muscle, such as after large spinal cord reverThe activity of the target muscle plays an essential sals, need not result in excess cell deathlT3. In the role in regulating motoneuron death. Chronic blockchick cihary ganglion, all the neurons receive functional preganglionic inputs prior to cell death’@).Moade of neuromuscular activity during the normal cell toneurons are increasing their levels of CAT and death period rescues essentially all of the motoneuAChE at this time49*31iand this autonomous increase rons63~115,i67.22s,2~. Effective agents include the snake is unaffected by limb-bud removal until degeneration a-toxins, curare, hemicholinium, botulinum toxin begins u2 . The ultrastructure of all the neurons of a and tetrodotoxin. Paralytic agents which enhance population appears uniform right up to the onset of ACh receptor activation such as carbachol or eserine cell death51<52.Thus, there is no evidence that cell increase cell death231as does chronic electrical stimu-

270 lation of the hindlimb at the onset of the ceil death periodz3”. The blockers effective in preventing cell death do so in spite of the fact that they decrease the size of the target by preventing production of secondary myotubes 113.2”5 . Paralysis appears not to act directly on the motoneurons, for it was ineffective in preventing loss of motoneurons in embryos subject to varying degrees of limb-bud amputation’h7. Paralyzed embryos undergo a delayed cell death when administration of the blocking agent is stopped and motility return$“. It has been suggested on the basis of cell counts that the number of primary myotube clusters in a motor pool at the time of cell death determines the number of motoneurons which will survive204.As a limiting mechanism, this proposal was supported in chickquail chimera experiments where there was a strong correlation between the number of myotube clusters at the onset of cell death and subsequent motoneuron survivaf, although neuron rescue beyond the normal number was not seen306. In quail-duck chimeras formed by mid-brain transplants, cell death in the trochlear nucleus was normal, even though there were increased target muscle fibers available to the quail motoneurons and an increase in the number of endplates formed 295. While the inabiIity of supernumerary grafts or various chimeric combinations to more substantially prevent cell death may be due to experimental limitations, it is possible that muscle activity at a critical period inevitably leads to an epigenetic cell death which can be limited but not totally avoided. If however, the motoneurons survive to hatching through continued paralysis with curare, the resumption of activity does not result in delayed cell death230, indicating an end to the critical period. 6.4. Role of afferent input In addition to the role of target tissue in regulating cell death, afferent input to a neuron also plays a part. Pha~aco~ogic bIockade of transmission or deafferentation of the chick ciiiary ganglion prior to the cell death period results in the eventual loss of almost 90% of the neurons*5*344,When all supraspinal and sensory input to the spinal cord are removed early in the embryo, there was a 37% enhancement of neuronal loss during the cell death period227. If either descending or sensory inputs alone were removed, there was a 20-Z% increase of cell death, but it was

delayed until after the normal cell death period and uninfluenced by blockade of neuromuscular transmission, suggesting a different cellular mechanism was involved. These results are consistent with the proposal that cell death occurs in cells which fail to achieve an appropriate balance of input and target contactHa Such systems matching may facilitate evolutionary change by allowing adjustments in the size of interacting populations during development14’. The use of culture systems to explore the molecular mechanisms which control normal developmental cell death remains both promising and probIematicI”,“38,ZX9.““1

7. SYNAPSEELIMINATION During the cell death period, the surviving neurons continue to expand their terminal fields such that there is considerable overlap of motor unit territories. Sometime after the cell death period, the cholinergic neuron still has to undergo a radical reorganization of its terminal field to achieve its normal size. An example is illustrated in Fig. 3 where the decline in neuron number in the chick cihary ganglion (open squares) precedes the decline of axon branches in the cihary nerve (filed squares} which continues for about a week after the completion of cell death. The topic of synapse elimination has been reviewed in detai122~1M~1g8~22”~256~329. The rules governing synapse elimination appear to be somewhat flexible, varying from system to system. At the neuromuscular junction, typically 2-6 terminals from distinct motoneurons innervate a single muscle endplate shortly before birth or hatching. Over the course of a week or more, this situation gives way to a single terminal, the others being retracted24*[email protected] is a similar overproduction and subsequent retraction of cholinergic terminals in autonomic ganglia246, although multiple innervation may remain and is not so focal as at an endplate. In fact, synaptic rearrangements, including elimination, may be relatively common events even in the mature CNS58,207. 7.1. Competition and dependence on target activity The process of synapse elimination shares two main elements with cell death: competition between neurons and a dependence on target activity. If a muscle is partially denervated prior to the period of

271 stimulation of one of two nerves which innervate a single muscle results in a larger motor unit size for axons within the stimulated nerve263.

i 4

200

# 100

0

E7

9

II

13

15

17

I9

P2

9

Fig. 3. Physiological, biochemical and population changes in developing chick ciliary ganglion neurons and nerve terminals in the iris (replotted from refs. 49, 180,248). Functional transmission begins on E8 and is complete by El2 (circles). CeH death occurs over days E8-El3 (open squares) even as the number of axon branches in the ciliary nerve is initially increasing up to El0 (filled squares). The number of axon branches gradually decreases, roughly corresponding with the period of synapse elimination, to reach adult values at about hatching (arrow). During synapse elimination, basal ACh synthesis (open triangles) is low and there was no stimulation of ACh synthesis by conditioning depolarization (filled triangles). This changed dramatically after hatching. CAT activity (inverted triangles) increases about lOOO-foldfrom E? to maturity. At E7, CAT activity is about IO-fold in excess of the actual capacity to synthesize ACh from exogenous choline but gradually increases to 1000-fold excess. The immature terminals are highly susceptible to fatigue during repetitive stimulation but become much more reliable when mature (hatched bars). This may be due at least in part to the ability to accelerate ACh synthesis in response to a challenge (filled triangles, expressed relative to basal levels of ACh synthesis). The approximate timing of the morphology of the terminals depicted in Fig. 2 is given for comparative purposes (letters A-D).

synapse elimination, the motor units which emerge following this period will be somewhat larger than otherwise, meaning a higher percentage of each motoneuron’s terminals has been retained42. In developing rat lumbrical muscle as an extreme example, it is possible to cut all but one axon to the muscfe. With all competition withdrawn, there is apparently no synapse elimination for this remaining motoneuron23. Chronic pharmacologic blockade of neuromuscular transmission or decreased activity caused by spinal transection or tenotomy impedes synapse elimination. Conversely, electrical stimulation of motor nerves accelerates it. The effectiveness of activity is dependent on the pattern of stimulation with bursts of high frequency enhancing elimination more than continuous low frequency stimulation314. Repetitive

7.2. Sprouting andpolyneuronal innervation It has been suggested that sprouting in the adult is a recapitulation of polyneuronal innervation and the developmental competition for synaptic sites39. It is noteworthy that pres~aptic sprouting in partially denervated sympathetic ganglia is increased by electrical stimulation of residual axons, an effect abolished by hexamethonium, which blocks ganglionic ACh receptors 196. Competition between reinnervating motor nerves favors active neurons and blockade of activity in one nerve of a competing set, with locally applied tetrodotoxin, results in repression of its terminals*5g~*~.Yet, direct etectricat stimulation of muscle suppresses sproutingm. Apparently, there are distinct effects of activity in the pre- and postsynaptic cells. In the postsynaptic cell, normal patterned activity results in stabilization of appropriate functional synapses and repression of ineffective or inappropriate synapses. Prolonged inactivity delays synapse ehmination during normal development and elicits sprouting in the adult. Activity in the presynaptic neuron is necessary for an effective response to postsynaptic requirements for terminal expansion. 7.3. Constraints of a critical period While useful analogies may be made between sprouting in the adult and synaptic competition in development, there are important differences in the repertoire available to the neuron in the two cases. Rat intercostal motoneurons which have been axotomized shortly after the cell death period are able to reform endplates only within a narrow time frame, another critical period, during which poiyneuronai innervation is maximalzs. When axotomy was postnatal (during the synapse elimination period), the motoneurons regenerated their axons but were unable to reform endplates until at least 3 weeks later6*. If however neonatal muscle is surgically reduced in size, there is an age-dependent reduction in the final number of motor units obtained, indicative of cell death’07*282.When axotomized neonatal motoneurons are prevented from contacting target muscle, they die after about two weeks’42. If as little as l-2% functional reinnervation of a foreign muscle was ob-

272 served, survival of the axotomized

motoneurons

was

preserved lA2. Thus, there is a special period when synapse reformation tends to he repressed but a close proximity to a target remains

Modulation of synapse elimination tion or electrical activity has also been in culture. Multiple innervation of NG108-IS hybrid celts was reversibly

vital. These observations

suggest a synapse-indepeIl-

50 to 5% by chronic depolarization

dent trophic dependence

of motoneuron

substantially

survival on

by depolarizademonstrated myotuhes by reduced from

with veratridine.

an effect blocked by TTXs”. Phasic (bursting]

etectri-

muscle.

cal stimulation

Clearly, the motoneuron is subject to a number of peculiar constraints during the critical period of syn-

whereas tonic stimulation

at 3 or 6 Hz for a week al-

apse elimination.

Iowed continued

innervation’“’

Yet there

is some special

resil-

of ciliary ganglia

tubes in co-culture

caused most myo-

to be mononeuronally multiple

innervated recapitular-

iency. When one of two adjacent ventral roots which have overlapping territories is cut prior to synapse

ing in vivo rest&s of Thompson”r’. If only one of two ganglia in a culture is phasically stimulated, the unsti-

elimination,

mulated

the remaining

loses its overlapping

territory

nerve,

which normally

in the competition

for

ganglion

loses nearly

all functional

con-

tacts”‘.

soleus innervation, now retains its field and has a motor unit size 4 times normal’Ys. The e.p.p. quanta1 content of its terminals is normal If the same cut is made later in development, the nerve can increase its motor unit size as before, but quanta1 content is reduced by about half’“‘. Thus early in development, motoneurons can adapt their function to increased

The extracellular environment can be manipulated to advantage. In vitro stimulation of the sciatic nerve in high calcium solution accelerates the loss of polyneuronal innervation in the soleus muscle over a matter of hoursz2*. The protease inhibitor leupeptin and calcium chelators prevented this effect and later were shown to slow synapse elimination in viva”’ implicat-

peripheral

ing the action of a calcium-activated neutral protease in the synapse retraction processZH’, Thus many properties of synapse elimination seen in vivo have been reproduced in vitro and some new aspects revealed.

demands

more

effectively,

or at least

more rapidly.

True synapse elimination, beyond the trivial case of cells dying off, has also been observed in tissue culture. Cholinergic neurons from embryonic chick retina can form functional synapses with myotubes: neurons from day-8 embryos have mean synapse lifetimes of 53 h, while neurons from day-13 embryos have mean synapse lifetimes of only 7 h25”. This points to a developmental change in some neuronal capacity as well as synapse repression based on a type-specific mismatch. Ciliary ganglia can maintain functional synapses with myotubes, but when co-cultured with ventral spinal cord cells, the ciliary neurons initially form synapses but become non-functional with about 80% of the myotubes after 6 days in culturetxO. The repression was partially blocked by curare, but dorsal cord or dorsal root ganglion cells were without effect on ciliary neuron-myotube synapses. These experiments, which involved unequal plating densities, demonstrate the existence of competition between functionally competent neurons, although they did not clearly show type-preference between spinal and autonomic motoneurons, which remains a possibility.

8.1. Target tissue factors and conditioned medium Co-culture of spinal cord or ciliary ganglion cells with muscle specifically induces a neuronat increase in CAT activity, ACh synthesis and capacity for ACh reIease”3-96.320. Mus&le-conditioned medium or muscle extract can produce the same effect. Similarly, coculture of medial septal explants with hippocampal target tissue increases septal CAT activity’6s. Fractions substantially enriched in CAT-enhancing activity have been prepared from conditioned medium and from extracts of eye and skeletal muscle~~2.2’X.2yZ.ZY~.~~~. These soluble factors can stimulate cholinergic development under culture conditions where neuronal survival is already maximal or can produce effects exceeding any increase in survival. Other soluble fractions obtained from the same tissue extracts had general growth-promoting activity which could act in concert with the ~hoIinergi~-siimulating fractions. There is also a circulating factor present in serum

273

increase ACh syr~thesis~‘~.Hormonal infhrences such as ~~~~~at~ngglu~o~orti~~ds have akso been shown to. infhrence the Ievef of chohnergic expression {see Table I). The bottom line is that multiple growth factors are likely to influence cholinergic development, In addition, lysed muscle membranes with assodated matrix stmulate ACh synthesis and CAT acfv-

which can

ity in diwy

~~~l~~n ~e~ro~~~~* an effect also ob-

served on sympatbeti~ neurons with fixed heart mus-

clert8 and some types of disrupted cell membranesr. A soluble heparin-binding growth factor that stimulates chohnergic and peptidergic development has been isolated from human, rat and bovine brains14g. This factor acts synergisticaZly with a pfasma membrane fraction to increase CAT. A ch~lio~rg~c.stjrn~lating protein ideutified as basic fibroblast growth factor (also a heparin-binding factor} has been isolated from human muscle and a similar factor can be liberated from the extracellular matrix of detergentlysed cultured chick muscle by treatment with high salt or heparinase 323*327 . The heparan sulfate proteog&an which co-localizes with the ACB receptor at the neuromuscular junction’, and which is remodeled during synaptogenesis5, may function as a repository of growth-modulatory activity. The proteolytic enzymes released from growth cones161*16z~24g and from denervated muscle76 could mobilize the growth factors at appropriate times.

Electrical activity and its attendant lot& fluxes have an important infiuence on the level of cholinergic expression Chronic blockade af activity in spinal

cord cuftures with tetrodatoxin resulted in reduced CAT activity withuut any effect on the GABAergic neuronss7. ilfnder these conditions, large spinai neutons become hypopiastic in size and the cuftures show a deficit in neuronspecific binding sites for tetanus and scorpion toxins, suggesting a decrease in neuronal survival_ The deficits can be counteracted by adding conditioned medium from cu&rres grown in the absence of tetr~otox~~~. The positive role of activity can be mimicked in part by growing cells in a high K* medium which partially depolarizes the celXsr3”.This treatment increased CAT activity and protein synthesis in ciliary ganglion neurons, at least partly by increasing calcium influx21g. Conversely, high K’ reverses the effect of conditioned medium which induces s~~patbeti~ neurons to become chohnergic and causes them to revert to the adrenergic phenotype, also through a calcium-dependent mechanism334.This suggests that activity may tend to perpetuate and enhance an earlier inductive event, perhaps related to cell lineage [see Table I).

The first identified and best characterized frophic factor, nerve growth factor (NGF), has direct effects an some cholinergic neurons, indirect effects on others, and is still without measurable effect on others. NGF is best known for its effects on sensory and sympathetic neurons 103S312. Cultured neurons rtf sympathetic lineage induced to become ~ho~i~erg~~ by heart-conditioned medium require NGF for survival and maxima1 expression of CAT activity2”“, NGF is not required for survival but stimulates neu-

TABLE I AC%, ACh synthesis or CAT activity: NE, uorepj~~~h~~~ synthesis or its s~~b~tje enzymes; SF, substance P. 11-1 -.I,Neuron&source Mtisefe CM” NC+ Activitf GlucocoTlicoi&” Spinal cord -” Parasympathetic Sympathetic Medial septal nucleus Corpus striattrm Retina

TACh f ACh TACh JNE’

TACti

--

0 0 or TACh TACh TNE TACh $4Ch ?

fACh TACh fNE JACh &Ps TAChi ?ACh J,SPk fACh=

-~--

_,“~_I_-

TACh’ O? fNE lAChh (ACTH) lAChi ? TACh=

a Mu~~e-~nd~fioned medium, see text for references. bNerve growth factor, see text for references. ‘Activity, inferred if blocked by TTX or enhanced by high K’ OF veratridine, dWydrocortisone, dexamethasone or prenisolone. Torda and Wolff, 19523t5. ‘See Patterson, 1978”s5. a Walicke et al., 19773”, Black et al., 198428. hMcLenttan et al., 19Sdo3, Fukada, 1980s3. I.I.R. Bostwtck, perm sonal communicatkrn. i Botticelli and Wurtman, lPt132~‘.‘Kessler, 1986”*u3,‘Pure et al., 198025’. lnBetz, 1981”. n Puro, 19832%.

274 rite outgrowth and CAT activity in the PC12 cell line, also of neural crest lineage1”h~“7. Anti-NGF sera eliminate the sympathetic cholinergic innervation af rat sweat glandsis” . Neither the survival or CAT activity of somatic or parasympathetic motoneur~ns are enhanced in culture by NGF, although an effect on

sat

ciliary ganglia

Schwann NGF

has been reported

cells begin to produce

receptors

after

NGF

in vivo’37. and express

neurotomy”6.3L)K, the signifi-

tary contractions,

motor units fire at rates of about

8-3Ois

and can go as high as 120/s during ballistic movements8’. Yet young synaptic terminals fatigue rapidly at frequencies of Iess than 1 Hzb9,iRi and cannot sustain moderate frequencies even Iate in developmentZ4”. In the ciliary nerve terminals, there is a relatively rapid reduction in the susceptibility to fatigue over a period of a few days at hatching. This increase in reliability

of transmission

is coincident

with

cance of which is not yet clear.

the acquisition

of the abitity to accelerate

Preganghonic cholinergic neurons of the sympathetic ganglia increase the number of axons in the

Afh synthesis Fig. 3).

in response

nerve trunk, synapses and Schwann ceils per postsyn-

In choiinergic terminals, the machinery for transmitter synthesis has generally been found to increase

aptic cell, and CAT activity after neonatal

NGF treat-

ment’7Y. Antibodies to NGF, postganglionic axotomy or treatment with 6hydroxydopamine prevents the normal increase in CAT and decreases the number of preganglionic axons. Cell death in the chick column of Terni is reduced by daily treatment with NGF but only at levels which innervate sympathetic ganglia but not at parasympathetic levelsl”‘~““‘. In each of these cases, the primary effect appears to be on the size and number of the sympathetic postganglionic neurons. Thus, NGF provides indirect, secondary support to the preganglionic cholinergic neurons and perhaps other neurons in brain = NGF and its specific mRNA have been found in rat brain, with levels being highest in regions innervated by magnocellular cholinergic neurons, including hippocampus and cerebral cortex’s”. NGF increases CAT activity in cultures of rat telencephalon’““, medial septat nucleus3J~“0, and corpus striatum”‘. Injections of NGF into the brain aIso increase CAT in the medial septal nucleus”“. str~atum2~~ hippocampus, neocortex and basal forebrain”“. When these large cholinergic neurons have their axons transected. application of NGF renders them more resistant to cell death”‘.“4’. It has been widely speculated that the degeneration of Alzheimer’s disease could be due to a trophic deficiency and might be ameiiorated by applied NGF’.‘“~“““. 10. MATURATION

OF CWOLlNERGIC

FUNCTION

IO.1. increased synaptic reEiabiEify Correct anatomic organization of appropriately matched ~pu~ations is not sufficient for MI physiologic function of chohnergic neurons. During volun-

the rate of

to evoked releaseZ’i8 (see

biphasically. CAT activity is present at low, barely detectable levels which start increasing at the time of synapse formation”‘~“i~‘i”. After some delay, there is an increase in high affinity chotine uptake, as well as CAT, leading to a dramatic increase in the capacity for acetylcholine synthesis’2~24*~2”4~2*tr.This Iater phase is accompanied by an increased efficiency of coupling between choline transport and acetylation as well as more differentiated structural properties, including a large accumulation of synaptic vesicles. This may represent a switchover from a growth mode where fusion of vesicles with the plasma membrane contributes to a net expansion to a transmission mode where vesicle fusion and recycling ideally approach a steady state. In the adult. ACh synthesis is acutely regulated by an acceleration of the high affinity choline transport system during electrical activity’J”.‘24.‘2”

The target tissue plays a roie in regulating chotinergic differentiation as does electrical activity, but the contribution of each has been difficult to sort out in vivo. The terminals of fast motor nerves contain more CAT activity225 and appear to release more transmitter than do slow motor nerve terminals even when cross-innervating a slow musclei”~‘. Embryonic neuromuscufar blockade with curare increased the CAT activity in both fast and slow muscles, probably by sustaining polyneuronal innervation”“. However, embryonic injections of snake u-toxin at a level which produced muscle atrophy markedly reduced CAT activity in the leg9i, and flaxedil prevented the normal developmental increases in CAT”. Pharmacologic destruction of ~stgangIionic sympathetic neurons prevents the normal developmental increase

275 in CAT in the pr~gangl~oni~ nervei3’. Denervation of the aduh ciliary ganglion for I2 days produces a major loss of ACh from the ciliary nerve’@ and a 60% reduction of CAT in the cell bodies and 30% in the iris nerve terminalss9. This tram-synaptic modulation of cholinergic properties is likely due to activity although other influences of afferent fibers have not been ruled out.

There are instances where certain cholinergic properties emerge relatively late in normal development, supplanting earlier properties. The sympathetic innervation of rat footpad sweat glands is initially adrenergic but becomes cholinergic over the first few postnatal weeks as the target tissue matures:“*‘a5. These cholinergic nerves retain a catecholamine uptake system and can be eliminated by neonatal treatment with the adrenergic neurotoxin &hydroxydopamine or antisera to NGF. Neuromuscular transmission in chick ciliary nerveiris is initially mediated by muscarinic receptors with a slow contractile response typical of smooth muscle and a corresponding ultrastructure~4~. There is a gradually increasing nicotinic component of the synaptic response with age, leading to a fast twitch response of the newly organized sarcomeres. Eventually the subsynaptic receptors are exclusively nicotinic while extrasynaptic muscarinic receptors are retained on the same f&e@. Cryptic or eon-funet~onal muscarinic receptors have also been reported early in the development of the chick hearts7. Upon denervation of the iris, muscarinic activation again predominates with a slow contractile response and sarcomeres are replaced by a dense filamentous cytoplasm 325. The exposition of similar and more novel transformations at cholinergic synapses in the brain may be expected. After all, the pattern and frequency of electrical activity in fast and slow motor nerves exert a compelling influence on phenotypic expression in musc1e30,194.240 10.3. Conrimed dependence on target Once the cholinergic neuron has achieved its mature functional state, it remains susceptible to many of the same influences that shaped its development although at a rather slower rate of response. Even under normal conditions, there is continuous synap-

tic remodeling in the adult neuromuscular junction1M.“2’,spinal cord’s’, and throughout the brainsir, Disruption of the trophic influence of the target organ by axotomy, nerve ligation or nerve crush leads to the chromatolytic response accompanied by decreased synthesis of cholinergic enzymes, altered excitability. decreased axon diameter, and often detachment of presynaptic inputs which together represent a ded~fferent~ation of the neuron4g,~,~~3.Some of the changes typical of axotomy are observed if axoplasmic flow is blocked by local colchicine application244 or if impulse flow is blocked with ‘ITX65. Reformation of functional nerve-muscle connections completely reverses the effect of axotomyYg, If only a portion of the nerve is able to regenerate, it is only those axons which recover. Increasing the atrophy of a muscle by tenotomy decreases the ability of a crushed nerve to reinnervate it*. Although neonatal nerves die more rapidly iu response to nerve section, permanent disconnection in the adult can lead to cell deathr4’. The pathology of cholinergic neurons may perhaps be best understood in terms of a breakdown in developmental regulatory rne~hanisms9.~~~. I 1. NEW DIRECTIONS

Application of molecular biological techniques will lead to new insights about cholinergic development, a few of which can be anticipated here in brief. Complementary DNAs for ~r~s~~~~~~‘~ and porcine CATi have been cloned and reported to have 32% sequence identity. Because nucleic acid probes can be prepared with very high specific radioactivity, the transcription of relatively few copies of the CAT gene can be detected with in situ hybridization at very ear ly stages of choline&c expression. The cDNAs for nicotinic ACh receptor subunits~~~~~muscarini~ receptor(s)3’~‘63,and AChEMs have also been cloned. Two classes of genetic control elements have been the subject of much interest, namely the homeotic genes and the proto-oncogenes. It may be expected that both impinge on cholinergic development in important ways. The homeotic genes are suspected of a role in the spatial organization of the early embryo and may infhrence the pattern of segmental connectivity@. The late expression of the homeobox genes, which is mostly limited to the CNS, may be involved in synaptic reorganization at critical periods in devel-

276 opment.

Proto-oncogene

products

are involved

growth control, signal transduction tion”.“‘. NGF stimulates transcription c-myc nuclear

proto-oncogenes

well as increasing

in PC12 cells”‘? as

CAT activiry. Some of these con-

trol elements may be essential in the ontogenetic trol of the cholinergic lineage-dependent

in

and differentiaof the c-fos and

phenotype

con-

and some may be

modulators.

properties

have been derived

from the

septaf nuclei of the brain”“” and from embryonic cinoma*“. common

These may be useful in determining genetic

elements

in control

thesis,

electrical

carthe

of cholinergic

development. SUMMARY

~otoneuron precursors acquire some principles of their spatial organization early in their eel! lineage, probably at the blastula stage. A predisposition to the chalinergic phenotype in motoneurons and some neural crest cells is detectable at the gastrula to neurula stages. Cholinergic expression is evident upon cessation of cell division. Cholinergic neurons can synthesize ACh during their migration and release ACh from their growth tunes prior to target contact or synapse formation. Neurons of different cell lineages can express the cholinergic phenotype, suggesting the importance of secondary induction. Early cholinergic commitment can be modified or reversed until later in development when it is amplified during interaction with target. Motoneurons extend their axons and actively sort out in response to local environmental cues to make highly specific connections with appropriate muscles. The essential elements of the matching mechanism are not species-specific. A certain degree of topographic matching is present throughout the nervous system. In dissociated cell culture, most topographic specificity is lost due to disruption of local environmental cues. Functional cholinergic transmission occurs within minutes of contact between the growth cone and a receptive target. These early contacts contain a few clear vesicles but lack typical ultrastructural speciafizations and are physiologi~aI~y immature. An initial stabilization of the nerve terminal with a postsynaptic

activity,

by blocking ACh syn-

or ACh

receptors,

but

AChR clusters are not induced by non-cholinergic neurons. After initial synaptic contact, there is increasing deposition of presynaptic active zones and synaptic AChE,

vesicies,

extracellular

and postjunctional

days to weeks.

In addition to the PC12 and neuroblastoma cell lines of sympathetic origin, cell lines which express cholinergic

AChR cluster is not prevented

m.e.p.p.

There

frequency,

basat

lamina

ridges over a period of

is a concomitant

increase

mean quanta1 content,

ic stabilization of AChRs, channel properties.

and

and n~atu~ation

in

metabolof single

At the onset of synaptic transmission, ceti death begins to reduce the innervating population of neurons by about half over a period of several days. If target tissue is removed, almost all neurons die. If competing neurons are removed or additional target is provided, ceil death is reduced in the remaining population. Pre- or postsynaptic blockade of neuromuscular transmission postpones cell death until function returns. Functional afferent input is also necessary for maximal survival and optimal matching of innervating and target populations. Individual neurons continue to expand their terminal fields as they compete for survival. Polyneuronal innervation reaches a maximum shortly after the cell death period. Synapse elimination commences dependent on activity in the target tissue. Synchronous bursts of impulses favor retention of the active neurons and speed the elimination of the inactive terminals. The ability to remodel existing terminals and to form nerve terminals is retained in maturity. Functional maturation occurs after a transition from a rapid growth mode, where cytoskeletal and membrane expansions predominate, to a transmission mode, after successful interaction with target tissue induces increases in the capacity to synthesize and release ACh. This induced increase is mediated by trophic factors released by the target tissue and contact with extracellular matrix and target. Activity continues to play a modulator role on cholinergic expression throughout life. The adult neuron remains ultimately dependent on trophic support from the target.

I am very grateful to Dr. St&ey

H. Appet for con-

277 tinuous support and encouragement, Haverkamp for thought~l criticism script, and Dr. Guillermo

Dr. tanny J. of the manu-

Pilar for encouraging

me to

REFERENCES 1 Adler, J.E. and Black, LB., Membrane contact regulates traasm~tter phenotypic expression, Dev. Brain Res., 30 (1986) 237-241. 2 A&ken, J.T., Sharman, M. and Young, J.Z., Maturation of regenerating nerve fibres with various peripheral connexions, 1. Anat., 87 (1947) l-22. 3 Altman, J. and Bayer, S.A., The Devefopment of the Rat Spinal Cord, Springer, New York, 1984. 4 Anderson, D.J., Molecular biology of the acetyicholine receptor: structure and regulation of biosynthesis. In M.M. Saipeter (Ed.), The Vertebrate Neuromuscular Ju~cr~un, Liss, New York, 1987, pp. 285-315. 5 Anderson, M. J., Nerve-induced remodeling of mu&e basal lamina during synaptogenesis, 1. Ceil Bial., RX?(1986) 863-877. 6 Anderson, M.J. and Cohen, M.W., Nerve-induced and spontaneous redistribution of acetylcholine receptors on cultured muscle cells, J. Physiof. (Land.), 268 (1977) 751-773. 7 Anderson, M.J. and ~ambrough, D.M., Aggregates of acetylcholine receptors are associated with plaques of a basal lamina heparan sulfate proteoglycan on the surface of skeletal muscle fibers, 3. Cell Biof,, 97 (1983) 1396-1411. 8 Anderson, M.J., Klier, F.G. and Tanguay, K.E., Acetylcholine receptor aggregation parallels the deposition of a basal lamina proteoglycan during development of the neuromuscular junction, f. Cell Biol., 99 (1984) 1769-1784. 9 Appel, S.H., A unifying hypothesis for the cause of amyotrophic lateral sclerosis, Parkinsonism, and Alzheimer disease, Ann. Neural., 10 f 1981) 499-505. IO Atsumi, S., Development of neuromuscular junctions of fast and slow muscles in the chick embryo: a light and electron microscopic study, J. ~eurocytof., 6 (1977) 691-709. 11 A&urger, G., Heumann, R., Hellweg, R., Korsching, S. and Thoenen, H., Developmental changes of nerve growth factor and its mRNA in the rat hippocampus: comparison with choline acetyltransferase. Dev. Bioi., 120 (1987) 322-328. 12 Bader, C.R., Baughman, R. W. and Moore, J.L., Different time course of development for high-~fin~ty chofine uptake and choline acetyltransferase in the chick retina, Proc. Natf. Acad. Sci. U.S.A., 7.5 (1978) 2525-2529. 13 Barald, K.F., Monoclonal antibodies to embryonic neurons: cell-specific markers for chick ciliary ganglion. In N.C. Spitzer (Ed.), Neuron& Development, Plenum, New York, 1982, pp. 101-119. 14 Bayne, E. K., Anderson, M.3. and Fambrough, D.M., Extracelfular matrix organization in developing muscfe: correlation with acetylchoiine receptor aggregates, .I. CefI Biol., 99 (1984) 1486-1501. 15 Bennett. MR., Development of neuromuscular synapses, Phyxiof. Rev., 63 (1983) 91.5-1048. 16 Bennett, M.R. and Florin, T., A statistical analysis of the release of acetylcholine at newly formed synapses in

write this review. The author was supported by the Robert Foundation

J. I&berg

and Helen

in part

C. Kleberg

and EY07001-13,

striated muscle, J. Phy~~~f. ILond.1, 238 (1974) 93-107, 17 Bennett, M.R. and Pettigrew, A.G., The formation of synapses in striated muscle during development, J. Physiof. (Lond.f, 241(1974) 515-545. 18 Berg, D.K,, New neuronal growth factors, Annu, Rev. Neurosci., 7 (1984) 149-170. 19 Berrard, S., Brice, A., Lottspeich, F., Braun, A., Barde, Y.-A. and Mallet, J., cDNA cloning and complete sequence of porcine chohne acetyltransferase: in vitro translation of the corresponding RNA yields an active protein, Proc. NC&. Acud. Sci. U.S.A., 84 (1987) 9280-9284. 20 Betz, H., Choline acetyltranskrase activity in chick retina

cultures: effect of membrane depoiarizing agents, Brain Res., 223 (1981) 190-194. 21 Betz, H., Bourgeois, J.-P. and Changeux, J.-P., Evolution of cholinergic proteins in developing slow and fast skeletal muscles in chick embryo, J. Physiol. (Lo&.], 302 (1980) 197-218. 22 Betz, W.J., Motoneuron death and synapse elimination in vertebrates. In M.M. Salpeter (Ed.), The Vertebrate Neuromu.wularJunction, Liss, New York, 1987, pp. 117-162. 23 Betz, W.J., Caldwell, J.H. and Ribchester, R.R., The effects of partial denervation at birth on the development of muscle fibres and motor units in rat lumb~~al muscle, J. Phj~siol. {LLand.), 303 (1980) 265-279. 24 Bixby, J.L., Ultrastructura~ observations on synapse etimination in neonatal rabbit skeletal muscle, J. Neurocytol., 10 (1981) 81-100. 25 Bixby, J.L. and Reichardt, L.F., The expression and iocahzation of synaptic vesicle antigens at neuromuscular junctions in vitro, J. Neurosci., 5 (1985) 3070-3080. 26 Bixby, J.L. and Spitzer, N.C., Early differentiation of vertebrate spinat neurons in the absence of voltage-dependent Ca’* and Na’ influx, Dev. Biof., 106 ff984) 89-96. 27 Black, LB., Stages of neurotransmitter development in autonomic neurons, Science, 215 (1982) 1198-1204. 28 Black, LB., Adler, J.E., Dreyfus, CF., Jonakait, GM., Katz, D.M., LaGamma, E.F. and Markey, K.M., Neurotransmitter plasticity at the molecular level, Science, 225 (1984) 1266-1270. 29 Biackshaw, S. and Warner, A., Onset of acetylcholine sensitivity and endplate activity in developing myotome muscles of ~e~o~~, Nature (toad.), 262 (1976) 217-218. 30 Blau, H.M., Pavlath, G.K., Hardeman, E.C., Chiu, C.-P., Silberstein, L., Webster, S.G., Miller, S.C. and Webster, C., Plasticity of the differentiated state, Science, 230 (1985) 758-766. 31 Bloch, R.J., Acetyfcholine receptor clustering in rat myotubes: requirement for CaZ* and effects of drugs which depolymerize microtubules, 1. Neurosci., 3 (1983) 26702680. 32 Bolsover, S.R. and Spector, I., Measurements of calcium transients in the soma, neurite, and growth cone of single cultured neurons,J. Neeurosci., 6 (1986) 1934-1940. 33 Bonner, T.I., Buckley, N.J., Young, A.C. and Brann, M.R., Identification of a family of muscarinic acetylcholine receptor genes, Science, 237 (1987) 527-532.

27x 34 Bostwick, J.K.. Appel. S.H and Perez-Polo. J.R.. DIStinct influences of nerve growth factor and a central cholinergic trophic factor on medial septal explants. Brait? Res. * 4x (1987) 92-Y% 35 Botticelli. L.J. and Wurt~Ian. R.J., Scpt(~h~pp~~c~~rnp~~l cholinergic neurons are regulated trans.synaptic~~lly by endorphin and corticotropin neuropeptides. J. :Yeuro.sci., 2fl982) 1316-1321. 36 Brenneman, D.E.. Fitzgerald, S. and Nelson. P.G.. Interaction between trophic action and electrical activity in spinal cord cultures, Dev. Brain Res.. 15 (1984) 21 I-217. 37 Brenneman, D.E.. Neale, EA., Habig. W.H.. Bowers. L.M. and Nelson. P.G., Developmental and neurochemical specificity of neuronal deficits produced by electrical imoulse blockade in dissociated spinal cord cultures. De%,. B&n Ref., 9 (1983) 13-27. . 38 Brimijoin. S., Molecular forms of acetylcholinesterase in brain. nerve and muscle: nature, localization and dynamics, Prog. Neurobiol.. 21 (1983) 291-322. 39 Brown, M.C., Sprouting of motor nerves in adult muscles: a recapitulation of ontogeny, TINS, 7 (1984) 10-14. 40 Brown. M.C.. Holland, K.L. and Hopkins, W.G., Motor nerve sprouting, Annn. Rev. Nrurosci.. 4 (1981) 17-42. 41 Brown. M.C. and Ironton. R.. Sprouting and regression of neuromuscular synapses in partially denervated mammalian muscles, .I. Physiof. (Lund.), 278 (1978) 325-348. 42 Brown. M.C.. Jansen. J.K.S. and Van Essen. D., Polyneuronal innervation in new-born rats and its elimination during maturation. .I. Physivl. (Land.), 261 (1976) 387-422. 43 Buck. C.R., Martinez. H.J.. Black. I.B. and Chao, M.V., Developmentally regulated expression of the nerve growth factor receptor gene in the periphery and brain, Proc. Narl. Acud. Sci. U.S.A., 84 (1987) 3060-3063. 44 Burden. S., Development of the neuromuscular junction in the chick embryo: the number, distribution, and stability of acetylcholine receptors. Dev. Biol., 57 (19771 317-329. 45 Burden, S.J.. The extracellular matrix and subsynaptic sarcoplasm at nerve-muscle synapses. In M.M. Salpeter (Ed.), The Vertebrate Neuromuscular Junction. Liss. New York. 1987, pp. 163-186. 46 Bursztajn. S.. McManaman. J.L. and Appel, S.H., Organization of acetylcholine receptor clusters in cultured rat myotubes is calcium-dependent. I. Cell Biol., 98 (1984) 507-s 17. 41 Busis, N. A.. Daniels. M.P., Bauer, H.C., Pudimat, P.A., Sonderegger, P., Schaffner. A.E. and Nirenberg. M., Three cholinergic neuroblastoma hybrid cell lines that form few synapses on myotubes are deficient in acetylcholine receptor aggregation molecules and large dense core vesicles. Brain Res., 324 (1984) 201-210. 48 Carbonetto, S. and Muller. K.J., Nerve fiber growth and the cellular response to axotomy, Curr. Topics Dev. Biol., 17 (1982) 33-76. 49 Chiappinelli, V., Giacobini, E., Pilar, G. and Uchimura, H., Induction of cholinergic enzymes in chick ciliary ganglion and iris muscle cells during synapse formation, J. Physiol. (Land.), 257 (1976) 749-766. 50 Chiu, A.Y. and Sanes, J.R., Development of basal lamina in synaptic and extrasynaptic portions of embryonic rat muscie, Dev. Biof., 103 (1986) 456-467. 51 Chu-Wang, L-W. and Oppenheim, R.W., Cell death of motoneurons in the chick embryo spinal cord. I. A light

and electron microscopic study of naturally occurring and induced cell loss during development, I. CoruIl. ,Nrur~/., 177 (1978) 33-58. s2 Chu-Wang. I.-W. and Oppenheim, R.W.. C‘ell death of motoneurons in the chick embryo spinal cord. Il. A quantitative and qualitative analysis of degeneration in the ven:raI root. including evidence for axon outgrowth and limb innervation prior to cell death. .I. C(lmp. Neural.. 177 :1978) so-86. 53 Cohen. M.W.. The development of neuromuscular connexions in the presence of D-tubocurarine. Brain Res., 41 :1973) 451-463. 54 Cohen, M.W. and Weldon, P.R., Localization of acetyl:holine receptors and synaptic ultrastructure at nervemuscle contacts in culture: dependence on nerve type, .I. CeII Biuf., 86( 1980) 388-401. 55 Cohen, S.A., Early nerve-muscle synapses in vitro release transmitter over postsynaptic membrane having low acetylcholine sensitivity, Proc. Nail. Acad. Sci. U.S.A., 77 (1980) 644-648. 56 Connold, A.L., Evers, J.V. and Vrbova, G.. Effect of low calcium and protease inhibitors on synapse elimination during postnatal development in the rat soleus muscle. Dev. Brain Res., 28 ( 1986) 99- 107. 57 Connor, J.A., Digital imaging of free calcium changes and of spatial gradients in growing processes in single, mammalian central nervous system cells, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 6179-6183. 58 Cotman, C.W, Nieto-Sampedro, M. and Harris, E.W.. Synapse replacement in the nervous system of adult vertebrates, Physiol. Rev., 61(1981) 684-784. 59 Cowan, W.M., Fawcett, J.W., O’Leary, D.D.M. and Stanfield, B.B.. Regressive events in neurogenesis. Science, 225 (1984) 1258- 1265. 60 Coyle, J.T.. Price, D.L. and DeLong, M.R., Alzheimer’s disease: a disorder of cortical cholinergic innervation. Science, 219 (1983) 1184-l 189. 61 Crain, SM. and Peterson. E.R., Development of paired explants of fetal spinal cord and adult skeletal muscle during chronic exposure to curare and hemicholinium. In Vi&o. 6 (1971) 373. 62 Crawford, G.D., Correa, L. and Salvaterra, P.M., Interaction of monoclonal antibodies with mammalian choline acetyltransfe~se. Proc. Natl. Acud. Sci. U.S.A., 79 (1982) 7031-7035. 63 Creazzo, T.L. and Sohal. G.S.. Effects of chronic injections of a-bungarotoxin on embryonic cell death, Exp. Neural., 66 (19X1) 135-145. 64 Cunningham, T.J., Naturally occurring neuron death and its regulation by developing neuron pathways. Inf. Rev. Cytol., 74 (1982) 163-186. 65 Czeh. G., Gallego. R.. Kudo, N. and Rune, M.. Evidence for the maintenance of motoneurone properties by muscle activity, .I. I’hysiof. (Lond.j, 281 (1978) 239-252. 66 Davey, D.F. and Cohen, M.W., Localization of acetylcholine receptors and cholinesterase on nerve-contacted and non-contacted muscle cells grown in the presence of agents that block action potentials, .I. Neurosci., 6 (1986) 673-680. 67 Dennis. M.J., Development of the neuromuscular junction: inductive interactions between cells. Annu. Ret,+. Neurosri., 4 (1981) 43-68. 68 Dennis, M.J. and Harris, A.J., Transient inability of neonatal rat motoneurons to reinnervate muscle. Dev. Biol.,

279 74 (1980) 173- 183. 69 Dennis, M.J., Z~skind-~onhaim, L. and Harris, A.J., Development of neurom~ular junctions in rat embryos, Dev. &of., 81(1981) 266-279. 70 Eckenstein, F. and Thoenen, H., Cholinergic neurons in the rat cerebral cortex demonstrated by immunohistochemica1 localization of choline acetyltransferase, Neurosci. Lett., 36 (1983) 211-215. 71 Eusebi, F., Pasetto, N. and Siracusa, G., Acetylcholine receptors in human oocytes, J. Physiol. (Land.), 346 (1984) 321-330. 72 Fambrough, D.M., Development of cholinergic innervation of skeletal, cardiac, and smooth muscle. In A.M. Goldberg and I. Hanin (Eds.), Riofogy of Chofinergic Function, Raven, New York, 1976, pp. 101-160. 73 Fambrough, D.M., Control of acetylcholine receptors in skeletal muscle, Physiol. Rev., 59 (1979) 165-227. 74 Farel, P.B. and Bemelmans, SE., Specificity of motoneuron projection patterns during development of the bullfrog tadpole (Rana catesbeiana), J. Cornp. Neural., 238 (1985) 128-134. 75 Fauquet, M., Smith, .I., Ziller, C. and Le Douarin, N.M., Differentiation of autonomic precursors in vitro: cholinergic and adrenergic traits in cultured neural crest cells, 1. Neurosci., l(1981) 478-492. 76 Festoff, B.W. and Hantai, D., Plasminogen activators and inhibitors: roles in muscle and neuromuscular regeneration, Prog. Brain Res., 71 (1987) 423-431. 77 Fields, K., Neuronal and glial surface antigens on cells in culture. In J.E. Battenstein and G. Sato (Eds.), Cell Cuiture in the Neurosciences, Plenum, New York, 1985, pp. 45-93. 78 Filogamo, G. and Marchisio, P.C., Acetylcholine system and neural development, Neurosci. Res., 4 (1971) 29-63. 79 Fischbach, G.D., Frank, E., Jessell, T.M., Rubin, L.L. and Schuetze, SM., Accumulation of acetylcholine receptors and acetylcholinesterase at newly formed nerve-muscle synapses, Pharmacoi. Rev., 30 (1979) 411-428. 80 Fishman, M.C. and Nelson, P.G., Depolarization-induced synaptic plasticity at cholinergic synapses in tissue culture, J. Neurosci., 1 (1981) 1043-1051. 81 Frank, E. and Fischbach, G.D., Early events in neuromuscular junction formation in vitro. Induction of acetylcholine receptor clusters in the postsynaptic membrane and morphology of newly formed synapses, J. Cell Biol., 83 (1979) 143-158. 82 Freund, H.-J., Motor unit and muscle activity in voluntary motor control, Physiol. Rev., 63 (1983) 387-436. 83 Fukada, K., Hormonal control of transmitter choice in sympathetic neurone cultures, Nature (Lund.), 287 (1980) 553-555.

84 Fukada, K., Purification and partial characterization of a cholinergic neuronal differentiation factor, Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 8795-8799. 85 Furber, S., Oppenheim, R.W. and Prevette, D., Naturally-occurring neuron death in the ciliary ganglion of the chick embryo following removal of preganglionic input: evidence for the role of afferents in ganglion cell survival, f. Neurosci., 7 (1987) 1816-1832. 86 Furshpan, E.J., MacLeish, P.R., O’Lague, P.H. and Potter, D.D., Chemical transmission between rat sympathetic neurons and cardiac myocytes developing in microcultures: evidence for cholinergic, adrenergic, and dual-function neurons, Proc. Natl. Acad. Sci. U.S.A., 73 (1976)

4225-4229. 87 Galper, J.B., Klein, W. and Catterall, W.A., Muscarinic acetylcholine receptors in developing chick heart, J. Biol. Chem., 252 (1977) 8692-8699. 88 Gehring, W.J., Homeo boxes in the study of development, Science, 236 (1987) 1245-1252. 89 Giacobini, E., Pilar, G., Suszkiw, J. and Uchimura, H.,

Normal distribution and denervation changes of neurotransmitter related enzymes in cholinergic neurones, J. Physiof. (Lond.), 286 (1979) 233-253. 90 Giacobini, G., Embryonic and postnatal development

of choline acetyltransferase activity in muscles and sciatic nerve of the chick.J. Neurochem., 19 (1972) 1401-1403. 91 Giacobini, G., Filogamo, G., Weber, M., Boquet, M. and Changeux, J.P., Effects of a snake a-neurotoxin on the development of innervated skeletal muscles in chick embryo, Proc. Natl. Acad. Sci. U.S.A., 70 (1973) 1708-1712. 92 Giess, M.-C. and Weber, M.J., Acetylcholine metabolism in rat spinal cord cultures: regulation by a factor involved in the determination of the neurotransmitter phenotype of sympathetic neurons, J. Neurosci., 4 (1984) 1442-1452. 93 Giller, E.L., Neale, J.H., Bullock, P.N.. Schrier, B.K. and Nelson, P.G., Choline acetyltransferase activity of spinal cord cell cultures increased by co-culture with muscle and by muscle-conditioned medium, J. Cell Biol., 74 (1977) 16-29. 94 Glicksman, M.A. and Sanes, J.R., Differentiation of motor nerve terminals formed in the absence of muscle fibres, J. Neurocytol., 12 (1983) 661-671. 95 Gnahn, H., Hefti, F., Heumann, R., Schwab, M.E. and Thoenen, H., NGF-mediated increase of choline acetyltransferase in the neonatal rat forebrain: evidence for a physiological role of NGF in the brain?, Dev. Bruin Res., 9 (1983) 45-52. 96 Godfrey, E. W., Schrier, B.K. and Nelson, P.G.. Source and target cell specificities of a conditioned medium factor that increases choline acetyltransferase activity in cultured spinal cord cells, Dev. Biol., 77 (1980) 403-418. 97 Gordon, T., Dependence of periphera1 nerves on their target organs. In G. Burnstock et al. (Eds.), Somaric and Autonomic Nerve-M~cle Interactions, Elsevier Science Publishers, New York, 1983, pp. 289-325. 98 Gordon, T., Perry, R., Tuffery, A.R. and Vrbova, G., Possible mechanisms determining synapse formation in . . . ctevetopmg skeletal muscles of the chick, Cell Tissue Res., 155 (1974) 13-25. 99 Gordon, T. and Stein, R.B., Time course and extent of recovery in reinnervated motor units of cat triceps surae muscles, J. Physiol. (Land.), 323 (1982) 307-323. 100 Gray, D.B. and Tuttle, J.B., [3H]Acetyicholine synthesis in cultured cihary ganglion neurons: effects of myotube membranes, Dev. Biol., 119 (1987) 290-298. * 101 Greenberg, J.H. and Schrier, B.K., Development of choline acetyltransferase activity in chick cranial neural crest cells in culture, Dev. Riot., 61 (1977) 86-93. 102 Greenberg, M.E., Greene, L.A. and Ziff, E.B., Nerve growth factor and epidermal growth factor induced rapid transient changes in proto-oncogene transcription in PC12 cells, J. Riot. Chem., 260 (1985) 14101-14110. 103 Greene, L.A. and Shooter, E.M., The nerve growth factor: biochemistry, synthesis and mechanism of action, Annu. Rev. Neurosci., 3 (1980) 353-402.

104 Grinnell, A.D. and Herrera, A.A., Specificity and plasticity of neuromuscular connections: long-term regulation of

2x0 motoneuron function. Pros. Neurobioi.. 17 (19x1) 203-282. 105 Grinvald, A. and Farber, I.C., Optical recording of calciurn action potentials from growth cones of cultured neurons with a laser microheam, Srience. 212 (lY81) 1164-l 167. 106 Guroff, G., PC12 cells as a model of neuronal differentiation. In J.E. Bottenstein and G. Sato (Eds.), Cell Culture in rhe Neurusciences, Plenum, New York. 1985, pp. 245-272. 107 Habgood, M.D., Hopkins, W.G. and Slack. J.R., Muscle size and motor unit survival in mice, /. Physiol. (Lo&.). 356 (1984) 303-314. 108 Hamburger. V., Regression versus peripheral control of differentiation in motor hypoplasia. Am. J. Anar., 102 (1958) 365-410. 109 Hamburger. V.. The developmental history of the motor neuron. Neurosci. Res. Prog. Bull., Suppl. 15 (1977) l-37. 110 Hamburger, V., Brunso-Bechtold, J.K. and Yip, J.W., Neuronal death in the spinal ganglia of the chick embryo and its reduction by nerve growth factor. J. Neurosci., I (1981)60-71. I i 1 Hamburger, V. and Oppenheim, R. W., Naturally occurring ncuronal death in vertebrates, Neurosci. Comment., 1 (1982) 39-55. 112 Hammond, D.N., Wainer. B.H.. Tonsgard, J.H. and Heller, A., Neuronal properties of clonal hybrid cell lines derived from central cholinergic neurons. Science, 234 (1986) 1237-1240. 113 Harris. A.J.. Embryonic growth and innervation of rat skeletal muscles. I. Neural regulation of muscle fibre numbers, Phil. Trans. R. Sot. Lund., 293 (1981) 257-277. 114 Harris. A.J.. Embryonic growth and innervation of rat skeletal muscles. III. Neural regulation of junctional and extra-junctional acetylcholine receptor clusters, Phil. Trans. R. Sot. Land., 293 (1981) 287-314. 115 Harris, A.J. and McCaig. C.D., Motoneuron death and motor unit size during embryonic development of the rat, J. Neurosci., 4 (1984) 13-24. 116 Harris, W.A.. Neural activity and development, Annu. Rev. Physiof., 43 (1981) 689-710. 117 Haverkamp, L.J., Anatomical and physiological development of the Xenopus embryonic motor system in the absence of neural activity, J. Neurosci., 6 (1986) 13381348. 118 Hawrot, E.. Cultured sympathetic neurons: effects of cellderived and synthetic substrata on survival and development, Dev. Biof., 74 (1980) 136-151. 119 Hefti, F.. Nerve growth factor promotes survival of septal cholinergic neurons after fimbrial transections, J. Neurosci., 6 (1986) 2155-2162. 120 Hefti, F., Hartikka, J., Eckenstein, F., Gnahn, H., Heumann, R. and Schwab, M., Nerve growth factor increases choline acetyltransferase but not survival or fiber outgrowth of cultured fetal septal cholinergic neurons, Neuroscience, 14 (1985) 55-68. 121 Hefti, F., Hartikka, J. and Frick, W., Gangliosides alter morphology and growth of astrocytes and increase the activity of choline acetyltransferase in cultures of dissociated septal cells, J. Neurosci., 5 (1985) 2086-2094. 122 Hefti. F. and Weiner. W.J., Nerve growth factor and AIzheimer’s disease, Ann. Neural., 20 (1986) 275-281. 123 Henderson, L.P., Smith, M.A. and Spitzer, N.C., The absence of calcium blocks impulse-evoked release of acetyl-

choline but not de novo formation of functional neuromuscular synaptic contacts in culture, J. Neurosci., 4 (1984) 3140-3150. 124 Henderson. L.P. and Spitzer. N.C.. Autonomous early differentiation of neurons and muscle cells in single cell cultures, Dev. &of., 1I3 (1986) 381-387. 125 Hersh, L.B., Coe. B. and Casey. L.. A fluorometric assay for choline acetyltransferase and its use in the pu~fication of the enzyme from human placenta, .1. Neurochem., 30 (1978) 1077-108s. 126 Hcumann, R., Korsching, S., Bandtlow, C. and Thoenen. H., Changes of nerve growth factor synthesis in nonneuronal cells in response to sciatic nerve section. J. Cell &of., IO4 (1987) 1623-i631. 127 Heumann, R., Schwab, M., Merkl, R. and Thoenen, H., Nerve growth factor-mediated induction of choline acetyltransferase in PC12 cells: evaluation of the site of action of nerve growth factor and the involvement of lysosomal degradation products of nerve growth factor, J. Neurosci.. 4 (1984) 3039-3050. 128 Hollyday. M.. Motoneuron histogenesis and the development of limb innervation, C&r. Topics Dets. &of.. IS (1980) 181-215. 129 Hollyday, M., Rules of motor innervation in chick embryos with supernumerary limbs, J. Camp. Neural., 202 (1981) 439-465. 130 Hollyday, M. and Hamburger, V.. Reduction of the naturally occurring motor neuron loss by enlargement of the periphery, J. Comp. Neural., 170 (1976) 31 t-320. 131 Hollyday, M. and Hamburger. V., An autoradiographic study of the formation of the lateral motor column in the chick embryo, Brain Res., 132 (1977) 197-308. 132 Honegger, P.. DuPasquier, P. and Tenot. M.. Choiinergic neurons of fetal rat telencephalon in aggregating cell culture respond to NGF as well as to protein kinase C-activating tumor promoters. Dev. Brain Res., 29 (1986) 217-223. 133 Hume, RI., Role. L.W. and Fischbach, G.D.. Acetylcholine release from growth cones detected with patches of acetylcholine receptor-rich membranes, Nature (Lund.). 305 (1983) 632-634. 134 Iacovitti. L., Job, T.H., Albert. V.R., Park, D.H., Reis, D.J. and Teitelman, G., Partial expression of carecholaminergic traits in cholinergic chick ciliary ganglia: studies in vivo and in vitro, Dev. Biol., 110 (1985) 402-412. 135 Ishida, I. and Deguchi, T., Effect of depolarizing agents on choline acetyltransferase and acetylcholinesterase activities in primary cell cultures of spinal cord, J. Neurosci.. 3 (1983) 1818-1823. 136 Itoh. N., Slemmon, J.R., Hawke, D.H., Williamson. R.. Morita. E., Itakura, K., Roberts, E., Shively. J.E., Crawford, G.D. and Satvaterra. P.M., Cloning of ~rosphfZ~ choline acetyltransferase cDNA, Proc. Narl. Acad. Sci. U.S.A., 83 (1986) 4081-4085. 137 Jacobson. M., Clonal analysis and cell lineages of the vertebrate nervous system, Annu. Rev. Neurosci.. 8 (1985) 71-102. 138 Jacobson,

M. and Moody, S.A., Quantitative lineage analysis of the frog’s nervous system. I. Lineages of ROhon-Beard neurons and primary motoneurons, J. Neuro-

xi., 4 (1984) 1361-1369. 139 Johnson, EM.. Caserta, M.T. and Ross, L.L., Effect of

destruction of the postganglionic sympathetic neurons in neonatal rats on development of choline acetyltransferase and survival of preganglionic cholinergic neurons. Brain

281 Res., 136 (1977) 455-464. 140 Jope, R.S., High affinity choline transport and acetyl CoA production in brain and their roles in the regulation of acetylcholine synthesis, Brain Res. Rev., 1(1979) 313-344. 141 Kahn, C.R., Coyle, J.T. and Cohen, A.M., Head and trunk neural crest in vitro: autonomic neuron differentiation, Dev. Biol., 77 (1980) 340-348. 142 Kashihara, Y., Kuno, M. and Miyata, Y., Cell death of axotomized motoneurones in neonatal rats, and its prevention by peripheral reinnervation, J. Physiol. (Land.), 386 (1987) 135-148. 143 Katz, M.J. and Lasek, R.J., Evolution of the nervous system: role of ontogenetic mechanisms in the evolution of matching populations, Proc. Nad. Acad. Sci. U.S.A., 75 (1978) 1349-1352. 144 Kawamura, Y. and Dyck, P.J., Permanent axotomy by amputation results in loss of motor neurons in man, J. Neuroparhol. Exp. Neurol., 40 (1981) 658-666. 145 Kelly, A.M. and Zacks, S.I., The fine structure of motor endplate morphogenesis, J. Cell Biol., 42 (1969) 154-169. 146 Kelly, R.B., Carlson, S.S. and Caroni, P., Extracellular matrix components of the synapse. In T.N. Wight and R.P. Mecham (Eds.), Biology of Proreoglycans, Academic, New York. 1987, pp. 247-265. 147 Kessler, J.A., Parasympathetic, sympathetic, and sensory interactions in the iris: nerve growth factor regulates cholinergic ciliary ganglion innervation in vivo, .I. Neurosci., 5 (1985) 2719-2725. 148 Kessler, J.A., Differential regulation of cholinergic and peptidergic development in the rat striatum in culture, Dev. Biol., 113 (1986) 77-89. 149 Kessler, J.A., Conn, G. and Hatcher, V.B., Isolated plasma membranes regulate transmitter expression and facilitate effects of a soluble brain cholinergic factor, Proc. Nad. Acad. Sci. U.S.A., 83 (1986) 3528-3532. 150 Keynes, R.J., Schwann cells during neural development and regeneration: leaders or followers?, TINS, 10 (1987) 137-139. 151 Kidokoro, Y., Anderson, M.J..and Gruener, R., Changes in synaptic potential properties during acetylcholine receptor accumulation and neurospecific interactions in Xenopus nerve-muscle cell culture, Dev. Biol., 78 (1980) 464-483. 152 Kidokoro, Y. and Brass, B., Redistribution of acetylcholine receptors during neuromuscular junction formation in Xenopus cultures, J. Physiol. (Paris), 80 (1985) 212-220. 153 Kidokoro, Y., Heinemann, S., Schubert, D., Brandt, B.L. and Klier, F.G., Synapse formation and neurotrophic effects on muscle cell lines, Cold. Spring Harbor Symp., 40 (1975) 373-388. 154 Kidokoro, Y. and Yeh, E., Synaptic contacts between embryonic Xenopus neurons and myotubes formed from a rat skeletal muscle cell line, Dev. Biol., 86 (1981) 12-18. 155 Kidokoro, Y. and Yeh, E., Initial synaptic transmission at the growth cone in Xenopus nerve-muscle cultures, Proc. Natl. Acad. Sci. U.S.A., 79 (1982) 6727-6731. 156 Kinutani, M. and Le Douarin, N.M., Avian spinal cord chimeras. I. Hatching ability and post-hatching survival in homo- and hetero-specific chimeras, Dev. Biol., 111 (1985) 243-255. 157 Ko, C.-P., Formation of the active zone at developing neuromuscular junctions in larval and adult bullfrogs, J. Neurocylol., 14 (1985) 487-512. 158 Koenig, J., Oren, M. and Melone, M.A.B., Establish-

ment of neuromuscular contacts in cultures of rat embryonic cells: effect of tetrodotoxin on maturation of muscle fibers and on formation and maintenance of acetylcholinesterase and acetylcholine receptor clusters, Dev. Neurosci., 5 (1982) 314-325. 159 Korshing, S., Auburger, G., Heumann, R., Scott, J. and Thoenen, H., Levels of nerve growth factor and its mRNA in the central nervous system of the rat correlate with cholinergic innervation, EMBO J., 4 (1985) 1389-1393. 160 Krishnan, S., Lowrie, M.B. and Vrbova, G., The effect of reducing the peripheral field on motoneurone development in the rat, Dev. Brain Rex, 19 (1985) 11-20. 161 Krystosek, A. and Seeds, N.W., Plasminogen activator release at the neuronal growth cone, Science, 213 (1981) 1532-1534. 162 Krystosek, A. and Seeds, N.W., Peripheral neurons and Schwann cells secrete plasminogen activator, J. Cell Biol., 98 (1984) 773-776. 163 Kubo, T. et al., Cloning, sequencing and expression of complementary DNA encoding the muscarinic acetylcholine receptor, Nature (Land.), 323 (1986) 411-416. 164 Kullberg, R.W., Lentz, T.L. and Cohen, M.W., Development of the myotomal neuromuscular junction in Xenopus laevis: an electrophysiological and fine-structural study, Dev. Biol., 60 (1977) 101-129. 165 Kuromi, H. and Kidokoro, Y., Nerve disperses preexisting acetylcholine receptor clusters prior to induction of receptor accumulation in Xenopus muscle cultures, Dev. Biol., 103 (1984) 53-61. 166 Kushner, P.D., A library of monoclonal antibodies to Torpedo cholinergic synaptosomes, J. Neurochem., 43 (1984) 775-786. 167 Laing, N.G., Motor projection patterns to the hind limb of normal and paralysed chick embryos, J. Embryol. Exp. Morphol., 72 (1982) 269-286. 168 Lamborghini, J.E., Rohon-Beard cells and other large neurons in Xenopus embryos originate during gastrulation, J. Comp. Neurol., 189 (1980) 323-333. 169 Lamborghini, J.E., Revenaugh, M. and Spitzer, N.C., Ultrastructural development of Rohon-Beard neurons: loss of intramitochondrial granules parallels loss of calcium action potentials, J. Comp. Neural.. 183 (1979) 741-752. 170 Lance-Jones, C.. Motoneuron cell death in the develooine lumbar spinal cord of the mouse, Dev. Brain Res., 4 (1982) 473-479.

171 Lance-Jones, C., The somitic origin of limb muscles in the chick embryo: a correlation with motor innervation, Sot. Neurosci. Abstr., 11 (1985) 975. 172 Lance-Jones, C. and Landmesser, L., Motoneurone projection patterns in embryonic chick limbs following partial deletions of the spinal cord, J. Physiol. (Land.), 302 (1980) 559-580.

173 Lance-Jones, C. and Landmesser, L., Motoneurone projection patterns in the chick hind limb following early partial reversals of the spinal cord, J. Physiol. (Land.), 302 (1980) 581-602.

174 Landis, S.C. and Keefe, D., Evidence for neurotransmitter plasticity in vivo: developmental changes in properties of cholinergic sympathetic neurons, Dev. Biol., 98 (1983) 349-372.

175 Landmesser, L., The development of motor projection patterns in the chick hind limb, J. Physiol. (Land.), 284 (1978) 391-414.

282 176 Landmesser, L.T.. The generation of neuromuscular specificity, Annu. Rev. Neurosci., 3 (1980) 279-302. 177 Landmesser. L., Peripheral Guidance Cues and the Pormation of Specific Motor Projections in the Chick, 1987. 178 Landmesser,

L. and Morris. D.G., The development of functional innervation in the hind limb of the chick cmbryo, J. Physiol. (Land.), 249 (1975) 301-326. 179 Landmesser, L. and Pilar, G., The onset and development of transmission in the chick ciliary ganglion, 1. Phy.rio/. (Land.), 222 (1972) 691-713. 180 Landmesser. L. and Pilar, G., Synaptic transmission and cell death during normal ganglionic development, J. Physiol. (Land.). 241 (1974) 737-749.

181 Landmesser, L. and Pilar, G., Fate of ganglionic synapses and ganglion cell axons during normal and induced cell death, J. Cell Biol., 68 (1976) 357-374. 182 Lanser, M.E.. Carrington, J. L. and Fallon, J.F., Survival of motoneurons in the brachial lateral motor column of limbless mutant chick embryos depends on the periphery. I. Neurosci., 6 (1986) 2551-2557. 183 Lanser, M.E. and Fallon, J.F., Development of the lateral motor column in the limbless mutant chick embryo, J. Neurosci., 4 (1984) 2043-2050.

184 Lauder, J.M. and Krebs, H., Do neurotransmitters, neurohumors, and hormones specify critical periods? In W.T. Greenough and J.M. Juraska (Eds.), Developmental Neuropsychobiology, Academic, 1986, pp. 119-174. 185 Leblanc, G. and Landis, S., Development of choline acetyltransferase in the sympathetic innervation of rat sweat glands, J. Neurosci., 6 (1986) 260-265. 186 Le Douarin, N., The Neural Crest, Cambridge Univ. Press, Cambridge, 1982. 187 Le Douarin, N.M., Renaud, D., Teillet, M.-A. and Le Douarin, G.H., Cholinergic differentiation of presumptive adrenergic neuroblasts in interspecific chimaeras after heterotopic transplantations, Proc. Natf. Acad. Sci. U.S.A.,

72 (1975) 728-732.

188 Le Douarin, N.M., Teillet, M.A., Ziller, C. and Smith, J., Adrenergic differentiation of the cells of the cholinergic ciliary and Remak ganglia in avian embryo after in vivo transplantation, Proc. Natl. Acad. Sci. I.I.S.A.. 75 (1978) 2030-2034.

189 Le Lievre, C.S., Schweizer, G.G., Ziller, C.M. and Le Douarin, N.M., Restrictions of developmental capabilities in neural crest cell derivatives as tested by in vivo transplantation experiments, Dev. Biol., 77 (1980) 362-318. 190 Letinsky, M.S., Physiological properties of developing frog tadpole nerve-muscle junctions during repetitive stimulation, Dev. Biol., 40 (1974) 154-161. 191 Levey, A.I., Armstrong, D.M., Atweh, S.F.. Terry, R.D. and Wainer, B.H., Monoclonal antibodies to choline acetyltransferase: production, specificity, and immunohistochemistry, J. Neurosci., 3 (1983) l-9. 192 Lewis, J., Chevallier, A., Kieny, M. and Wolpert, L., Muscle nerve branches do not develop in wings devoid of muscle, J. Embryol. Exp. Morphol., 44 (1981) 211-232. 193 Lieberman, A.R., The axon reaction, Int. Rev. Neurobiol., 14 (1971) 49-124. 194 Lomo, T. and Waerhaug, 0.) Motor endplates in fast and slow muscles of the rat: what determines their differences?, J. Physiol. (Paris), 80 (1985) 290-297. 195 Lowrie, M.B., O’Brien, R.A.D. and Vrbova, G., The effect of altered peripheral field on motoneurone function in

developing rat soleus muscles. J. Physiol. (Land.).

368

(1985) 513-524.

196 Maehlen, J. and Nja, A.. The effects of electrical stimulation on sprouting after partial denervation of guinea-pig sympathetic ganglion cells. J. Physioi. (Land.). 322 (1982) 151-166.

197 Magchielse, T. and Meeter. E., The effect of neuronal activity on the competitive elimination of neuromuscular junctions in tissue culture. Dev. Brain Res., 25 (1986) 21 l-220.

I98 Mark, R.F.. Synaptic repression at neuromuscular junctions, Physioi. Rev., 60 (1980) 355-395. 199 Marshall. L.M.. Sanes, J.R. and McMahan, U.J., Reinnervation of original synaptic sites on muscle fiber basement membrane after disruption of the muscle cells, Proc. Natl. Acad. Sci. U.S.A.,

74 (1977) 3073-3077.

206 Martinez, H.J., Dreyfus, C.F., Jonakait, G.M. and Black, I.B., Nerve growth factor promotes cholinergic development in brain striatal cultures. Proc. Nan. Acad. Sci. U.S.A., 82(1985)7777-7781. 201 Masuko, S. and Shimada, Y.. Neuronal cell-surface specific antigen(s) is expressed during the terminal mitosis of cells destined to become neuroblasts, Dev. Biol.. 96 (1983) 396-404.

202 Maxwell, G.D., Sietz, P.D. and Rafford, C.E., Synthesis and accumulation of putative neurotransmitters by cultured neural crest cells. J. Neurosci., 2 (1982) 879-888. 203 McLennan, I.S., Hill, C.E. and Hendry, I.A., Glucocorticosteroids modulate transmitter choice in developing superior cervical ganglion, Nature (Land.), 283 (1980) 206-207.

204 McLennan, I.S., Size of motoneuron pool may be related to number of myotubes in developing muscle, Dev. BioI., 92 (1982) 263-265.

205 McLennan, IS., Neural dependence and independence of myotube production in chicken hindlimb muscles, Dev. Bioi., 98 (1983) 287-294.

206 McMahon, D., Chemical messengers in development: a hypothesis, Science, 185 (1974) 1012-1021. 207 Mendell, L.M., Modifiability of spinal synapses, Physiol. Rev.,

64(1984) 260-324.

208 Mishina, M., Takai, T., Imoto, K., Noda, M., Takahashi, T., Numa, S., Methfessel, C. and Sakmann, B., Molecular distinction between fetal and adult forms of muscle acetylcholine receptor, Nature (Land.), 321 (1986) 406-411. 209 Mobley, W.C., Rutkowski, J.L., Tennekoon, G.I., Buchanan, K. and Johnston, M.V., Choline acetyltransferase activity in striatum of rats increased by nerve growth factor, Science, 229 (1985) 284-287. 210 Mobley, W.C.. Rutkowski, J.L., Tennekoon, G.I., Gemski, J., Buchanan, K. and Johnson, M.V., Nerve growth factor increases choline acetyltransferase activity in developing basal forebrain neurons, Mol. Brain ‘Res., 1 (1986) 53-62. 211 Moody, S.A. and Jacobson, M., Compartmental relationships between anuran primary spinal motoneurons and somitic muscle fibers that they first innervate, J. Neurosci., 3 (1983) 1670-1682.

212 Moody-Corbett,

F. and Cohen, M.W., Increased adhe-

siveness at sites of high acetylcholine receptor density on embryonic amphibian muscle cells cultured without nerve, J. Embryol. Exp. Morphol., 72 (1982) 53-69. 213 Nadler, J.V., Matthews, D.A., Cotman, C. W. and Lynch, G.S., Development of cholinergic innervation in

283

214

215

216

217 218

219

220

221

222

223

224

225

the hippocampal formation of the rat. II. Quantitative changes in choline acetyitransferase and acetylcholinesterase activities, Dev. Biol., 36 (1974) 142- 154. Nakajima, Y., Kidokoro, Y. and Klier, F.G., The development of functional neuromuscular junctions in vitro: an ultrastructural and physiological study, Dev. Biol., 77 (1980) 52-72. Nakajima, Y., Nakajima, S., Obata, K., Carlson, C.G. and Yamaguchi, Dissociated cell cultures of cholinergic neurons from nucleus basalis of Meynert and other basal forebrain nuclei, hoc. Natl. Acad. Sci. U.S.A., 82 (1985) 6325-6329. Narayanan, C.H. and Narayanan, Y., On the origin of the ciliary ganglion in birds studied by the method of interspecific transplantation of embryonic brain regions between quail and chick, J. Embryol. Exp. Morphol., 47 (1978) 137-148. Newgreen, D.F. and Erickson, C.A., The migration of neural crest cells, hat. Rev. Cytol., 103 (1986) 89-145. Nishi, R. and Berg, D.K., Two components from eye tissue that differentially stimulate the growth and development of ciliary ganglion neurons in cell culture, J. Neurosci., l(1981) 505-513. Nishi, R. and Berg, D.K., Effects of high KS concentrations on the growth and development of ciliary ganglion neurons in cell culture, Dev. Biol., 87 (1981) 301-307. Nitkin, R.M., Smith, M.A., Magill, C., Fallon, J.C., Yao, Y.-M.M., Wallace, B.G. and McMahan, U.J., Identification of agrin, a synaptic organizing protein from Torpedo electric organ, J. Cell Biol., 105 (1987) 2471-2478. Noakes, P.G. and Bennett, M.R., Growth of axons into developing muscles is preceded by cells that stain with Schwann cell antibodies, f. Comp. Neurol., 259 (1987) 330-347. Obata, K., Development of neuromuscular transmission in culture with a variety of neurons and in the presence of cholinergic substances and tetrodotoxin, Bruin Res., 119 (1977) 141-153. O’Brien, R.A.D., Postnatal development of the innervation of mammahan skeletal muscle. In G. Burnstock et al. (Ed?..), Somatic and Autonomic Nerve-M~cle Interactions, Elsevier Science Publishers, 1983, pp. 153-184. O’Brien, R.A.D., Ostberg, A.J.C. and Vrbova, G., Protease inhibitors reduce the loss of nerve terminals induced by activity and calcium in developing rat soleus muscles in vitro, Neuroscience, 12 (1984) 637-646. O’Brien, R.A.D. and Vrbova, G., Acetylcholine synthesis in nerve endings to slow and fast muscles of developing chicks: effects of muscle activity, Neuroscience, 3 (1978) 1227-1230.

226 O’Donovan, M.J., Caldwell, R.T., Morrison, J.A. and Denburg, J.L., Monoclonal antibodies that distinguish among neuronal subsets in the spinal cord of the chick embryo, Sot. Neurosci. Abstr., ll(l985) 1064. 227 Okado, N. and Oppenheim, R.W., Cell death of motoneurons in the chick embryo spinal cord. IX. The loss of motoneurons following removal of afferent inputs, J. Neurosci., 4 (1984) 1639-1652. 228 Olek, A.J., Effects of alpha and beta bungarotoxin on motor neuron loss in Xenopus larvae, Neuroscience~ 5 (1980) 1557-1563.

229 Oppenheim, R.W., Neuronal cell death and some related regressive phenomena during neurogenesis: a selective historical review and progress report. In Studies in Devel-

opmental Neurobiology. Essays in Honor of Victor Hamburger, Oxford Univ. Press, New York, 1981, pp. 74-133.

230 Qooenheim, R.W., Cell death of motone~o~ in the chick embryo spinal cord. VIII. Motoneurons prevented from dying in the embryo persist after hatching, Dev. Eiol., lOl(l984) 35-39. 231 Oppenheim, R.W. and Chu-Wang, I.-W., Aspects of naturally-occurring motoneuron death in the chick spinal cord during embryonic development. In Somatic and Autonomic Nerve-Muscle Interactions, Elsevier Science Pubiishers, New York, 1983, pp. 57-107. 232 Oppenheim, R.W., Chu-Wang, I.-W. and Maderdrut, J.L., Celi death of motoneurons in the chick embryo spinal cord. III. The differentiation of motoneurons prior to their induced degeneration following limb-bud removal, J. Comp. Neurol., 177(1978) 87-112. 233 Oppenheim, R.W. and Nunez, R., Electrical stimulation

of hindlimb increases neuronal cell death in chick embryo, Nature (Land.), 295 (1982) 57-59.

234 Patrick, J., Ballivet, M., Boas, L., Claudio, T., Forrest, J., Ingraham, H., Mason, P., Stengelin, S., Ueno, S. and Heinemann, S., Molecular cloning of the a~etylcholine receptor. In Molecular Neurobiology, Cold Spring Harbor Symp. Quant. Biol., 48 (1983) 71-78. 235 Patterson, P.H., Environmental determination of autonomic neurotransmitter functions, Annu. Rev. Neurosci.,

1(1978) l-17. 236 Patterson, P.H., On the role of proteases, their inhibitors and the extracellular matrix in promoting neurite outgrowth, J. Physiol. (Paris], 80 (1985) 207-211. 237 Peng, H.B., Bridgman, P.C., Nakajima, S., Greenberg, A. and Nakajima, Y., A fast development of presynaptic function and structure of the neuromuscular junction in Xenopus tissue culture, Brain Res., 167 (1979) 379-384. 238 Perez-Polo, J.R., Neuronotrophic factors. In J.E. Bottenstein and G. Sato (Eds.), Cell Culture in the Neurosciences, Plenum, New York, 1985, pp. 95-123. 239 Peterson, E.R. and Crain, SM., Maturation of human muscle after innervation by fetal mouse spinal cord explants in long-term cultures. In A. Mauro et al. (Eds.), Muscle Regenera~on, Raven, New York, 1979, pp. 429-441. 240 Pette, D. and Vrbova, G., Neural control of phenotypic expression in mammalian muscle fibers, Muscle and Nerve, 8 (1985) 676-689. 241 Pfeiffer, S.E., Jacob, H., Mikoshiba,

K., Dubois, P., Guenet, J.L., Nicolas, J.-F., Gaillard, J., Chevance, G. and Jacob, F., Differentiation of a teratocarcinoma line: preferential devetopment of cholinergic neurons, J. CeZi

BioZ., 88 (1981) 57-66.

242 Phelps, P.E., Barber, R.P., Houser, CR., Crawford, G.D., Saivaterra, P.M. and Vaughn, J.E., Postnatal development of neurons containing choline acetyltransferase in rat spinal cord: an immunocytochemical study, .I. Comp. Neural., 229 (1984) 347-361. 243 Pilar, G., Jenden, D.J. and Campbell, B., Distribution of acetylcholine in the normal and denervated pigeon ciiiary ganglion, Brain Res., 49 (1973) 245-256. 244 Pilar, G. and Landmesser, L., Axotomy mimicked by localized colchicine application, Science, 177 (1972) 11161118, 245 Pilar, G. and Landmesser, L., Ultrastructural differences during embryonic cell death in normal and peripherally deprived ciliary ganglia, J. Cell Biol., 68 (1976) 339-356.

284 246 Pilar, G., Landmesser. L. and Burstein. L., Competition for survival among developing ciliary ganglion cells. J. Neurophysiol., 43 (1980) 233-254. 247 Pilar. G., Nunez, R.. McLennan. IS. and Meriney, SD.. Muscarinic and nicotinic synaptic activation of the developing chicken iris. J. Neurosci., 7 (1987) 3813-3826. 248 Pilar, G.. Tuttle, J. and Vaca, K., Functional maturation of motor nerve terminals in the avian iris: ultrastructure, transmitter metabolism and synaptic reliability, J. Physiol. (Land.). 321 (1981) 175-193. 249 Pittman. R.N.. Release of piasminogen activator and a calcium-dependent metalloprotease from cultured sympathetic and sensory neurons. Dev. Biol., 110 (1985) 91-101. 250 Pittman. R. and Oppenheim, R.W., Cell death of motoneurons in the chick embryo spinal cord. IV. Evidence that a functional neuromuscular interaction is involved in the regulation of naturally occurring cell death and the stabilization of synapses. J. Camp. Neurol., 187 (1979) 425-446. 2.51 Price, J., Turner, D. and Cepko, C., Lineage analysis in the vertebrate nervous sytem by retrovirus-mediated gene transfer, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 156-160. 252 Puro, D.G.. Glucocorticoid regulation of synaptic development. Dev. Brain Res., 8 (1983) 283-290. 2.53 Pure, D.G., DeMeIlo, F.G. and Nirenberg, M., Synapse turnover: the formation and termination of transient synapses, Prof. Nail. Acad. Ski. U.S.A., 74 (1977) 4977-4981. 254 Puro, D.G., Battelle, B.-B. and Hansmann,

opment of cholinergic

K.E., Develneurons of the rat retina. Dev.

Biol., 91 (1982) 138-148. 255 Purves, D. and Lichtman,

J.W., Formation and maintenance of synaptic connections in autonomic ganglia, Physiol. Rev., 58 (1978) 821-862. 256 Purves, D. and Lichtman, J.W., Elimination of synapses in the developing nervous system, Science, 210 (1980) X53-157. . 257 Redfern, P.A., ~euromuscuIar transmission in new-born rats,.l. Physic& (Land.), 209 (1970) 701-709. 2.58 Reist, N.E., Magill, C. and McMahan, U.J., Agrin-like molecules at synaptic sites in normal, denervated, and damaged skeletal muscles, 1. Cell Biol., 105 (1987) 2457-2469. 259 Ribchester,

R.R. and Taxt, T., Motor unit size and synaptic competition in rat lumbrical muscles reinnervated by active and inactive motor axons, J. Physiol. (Land.), 344

(1983)89-111. 260 Ribchester, R.R. and Taxt, T., Repression of inactive mo-

tor nerve terminals in partially denervated rat muscle after regeneration of active motor axons, J. Physiol. (Lond.J, 347 (1984) 497-511. 261 Richardson, P.J., Walker, J.H., Jones, R.T. and Whittak-

er, V.P., Identification of a cholinergic-specific antigen Chol-1 as a gang~ioside, J. Neurochem., 38 (1982) 1605-1614. 262 Rickman, M., Fawcett, J.W. and Keynes, R.J., The mi-

gration of neural crest cells and the growth of motor axons through the rostra1 half of the chick somite, J. Embryof. Exp. Morphol., 90 (1985) 437-455. 263 Ridge, R.M.A.P. and Betz, W.J., The effect of selective,

chronic stimulation on motor unit size in developing rat muscle, J. Neurosci., 4 (1984) 2614-2620.

264 Riley. D.A..

265

266

267

268

269

Ultrastructural evidence for axon retraction during the spontaneous elimination of the rat coleus murcle, J. Neurocytol., 10 (1981) 425-440. Rimvall. K., Keller, F. and Waser, P.G., Development of cholinergic projections in organotypic cultures of rat scpturn, hippocampus and cerebellum, Dev. Brain Res., lY (1985) 267-278. Role, L.W.. Matossian, V.R., O’Brien, R.J. and Fischbath, G.D., On the mechanism of acetylcholine receptor accumulation at newly formed synapses on chick myotubes, J. Neurosci., 5 (1985) 2197-2204. Romanes, G.J.. Motor localization and the effects of nerve injury on the ventral horn ceils of the spinal cord, J. Anar.. 80(1946) 117-131. Rotundo, R.L., Biogenesis and regulation of acetylcholinesterase. In M.M. Salpeter (Ed.), The Vertebrate Neurumuscular Junction, Liss. New York, 1987, pp. 247-284. Rubin, E., Development of the rat superior cervical ganglion: ingrowth of preganglionic axons, J. Neurosci.. 5

(1985) 685-696 270 Rubin. E., Development

of the rat superior cervical ganglion: initial stages of synapse formation, J. Neurosci., 5 (1985) 697-704. 271 Rubin, L.L.. Increases in muscle Ca’+ mediate changes in acetylcholinesterase and acetylcholine receptors caused by muscie contraction, Prac. Nafl. Acad. Sci. U.S.A., 82 (1985) 7121-7125. 272 Rubin, L.L., Schuetze, SM., WeiH, CL. and Fischbach, G.D., Regutation of acetylcholinesterase appearance at neuromuscular junctions in vitro, Nature (Lund./, 283 (1980) 264-267.

273 Salpeter, M.M., Development and neural control of the neuromuscular junction and of the junctional acetylcholine receptor. In The Vertebrate Neuromuscular Junction, Liss, New York, 1987, pp. .55- 115. 274 Sanes, J.R., Roles of extracellular matrix in neural development, Annu. Rev. Physiol., 45 (1983) 581-600. 275 Sanes, J.R.. Feldman. D.H., Cheney, J.M. and Lawrence, J.C., Brain extract induces synaptic characteristics in the basal iamina of cultured myotubes, J. Neurosci., 4 (1984) 464-473. 276 Sanes, J.R. and Lawrence, J.C., Activity-dependent accumulation of basal lamina by cultured rat myotubes, Dev. Biol., 97 (1983) 123- 136. 277 Sanes, J.R., Rubenstein, J.L.R. and Nicolas, J.-F., Use of a recombinant retrovirus to study post-implantation cell lineage in mouse embryos, EMBO J., 5 (1986) 3133-3142. 278 Sastry, B.V.R. and Sadavongvivad, C., Cholinergic systems in non-nervous tissues, Pharmacol. Rev., 30 (1979) 65-132.

279 Schafer, T., Schwab, M.E. and Thoenen, H., Increased formation of preganglionic synapses and axons due to a retrograde trans-synaptic action of nerve growth factor in the rat sympathetic nervous system, J. Neurosci., 3 (1983) 1501-1510. 280 Schaffner, A.E., Nelson, P.G. and Fishman, M.C., Synapse repression in cell culture, Dev. Bruin Res., 12 (1984) 159-165. 281 Schlaepfer, W.W. and Zimmerman, U.-J.P., Calcium-activated protease and the regulation of the axonal skeleton. In J.S. Elam and P. Cancalon (Eds.), Axonal Transport in Neuronal Growth and Regeneration, Plenum, New York, 1984, pp. 261-273.

285 282 Schmalbruch, H., Motoneuron death after sciatic nerve section in newborn rats, J. Comp. Neurol., 224 (1984) 252-258. 283 Schuetze, S.M. and Role, L.W., Developmental regulation of nicotinic acetylcholine receptors, Annu. Rev. Neurosci., 10 (1987) 403-457. 284 Seiler, M. and Schwab, M.E., Specific retrograde transport of nerve growth factor from neocortex to nucleus basalis in the rat, Bruin Res., 300 (1984) 33-39. 285 Sheard, P., McCaig, CD. and Harris, A.J., Critical periods in rat motoneuron development, Dev. Biol., 102 (1984) 21-31. 286 Shelton, D.L., Nadler, J.V. and Cotman, C.W., Development of high affinity choline uptake and associated acetylcholine synthesis in the rat fascia dentata, Bruin Res., 163 (1979) 263-275. 287 Sieber-Blum, M. and Cohen, A.M., Clonal analysis of quail neural crest cells: they are pluripotent and differentiate in vitro in the absence of noncrest cells, Dev. Biol., 80 (1980) 96-106. 288 Sieber-Blum, M. and Kahn, CR., Suppression of catecholamine and melanin synthesis and promotion of cholinergic differentiation of quail neural crest cells by heart cell conditioned medium, Stem Cells, 2 (1982) 344-353. 289 Slack, J.R., Hopkins, W.G. and Pocket& S., Evidence for a motor nerve growth factor, Muscle and Nerve, 6 (1983) 243-252. 290 Smith, J., Cochatd, P. and Le Douarin, N.M., Development of choline acetyltransferase and cholinesterase activities in enteric ganglia derived from presumptive adrenergic and cholinergic levels of the neural crest, Cell Differ., 6 (1977) 199-216. 291 Smith, J., Fauquet, M., Ziller, C. and Le Douarin, N.M., Acetylcholine synthesis by me~ncephalic neural crest cells in the process of migration in vivo, Nature ftond.], 282 (1979) 8.53855. 292 Smith, R.G. and Appel, S.H., Extracts of skeletal muscle promote increased neuritic outgrowth and cholinergic activity of rat spinal motor neurons, Science, 219 (1983) 1079-1081. 293 Smith, R.G., McManaman, J. and Appel, S.H., Trophic effects of skeletal muscle extracts on ventral spinal cord neurons in vitro: separation of a protein with morphologic activity from proteins with cholinergic activity, J. Cell Biol., lOl(l985) 1608-1621. 294 Smith, R.G., Vaca, K., McManaman, J. and Appel, S.H., Selective effects of skeletal muscle extract fractions on motoneuron development in vitro, J. Neurosci., 6 (1986) 439-447. 295 Sohal, G.S., Stoney, S.D., Arumugam, T., Yamashita, T.

and Knox, T.S., Influence of reduced neuron pool on the magnitude of naturally occurring motor neuron death, J. Camp. Neurol., 247 (1986) 516-528. 296 Soreq, H., Zevin-Sonkin, D., Avni, A., Hall, L.M.C. and Spierer, P., A human acetylcholinesterase gene identified by homology to the Drosophila gene, Proc. Natl. Acad. Sci. U.S.A., 82(1985) 1827-1831. 297 Sperry, D.G. and Grobstein, P., Regulation of neuron numbers in Xenopus luevis: effects of hormonal manipulation altering size at metamorphosis, J. Comp. Neurol., 232 (1985) 287-298. 298 Srihari, T. and Vrbova, G., The role of muscle activity in

the differentiation of neuromuscular junctions in slow and fast chick muscles, J. Neurocyiol., 7 (1978) 529-540.

299 Steinbach, J.H., Developmental changes in acetylcholine receptor aggregates at rat skeletal neuromuscular junctions, Dev. Biol., 84 (1981) 267-276. 3oa Steinbach, J.H. and Bloch, R.J., The distribution of acetylcholine receptors on vertebrate skeletal muscle cells. In R.M. Gorczynski (Ed.), Receptors in Cellulur Recognit~~ and Developmentul Processes, Academic, New York, 1986, pp. 183-213. 301 Steinbach, J.H., Harris, A.J., Patrick, J., Schubert, D. and Heinemann, S., Nerve-muscle interaction in vitro. Role of acetylcholine, J. Gen. Physiol., 62 (1973) 255-270.

302 Stern, C.D., Sisodiya, S.M. and Keynes, R.J., Interactions between neurites and somite cells: inhibition and stimulation of nerve growth in the chick embryo, J. Embryol. Exp. ~orphol.,

91(1986)

209-226.

303 Sun, Y.-A. and Poo, M.-M., Evoked release of acetylcholine from the growing embryonic neuron, Proc. Natl. Acad. Sci. D.S.A.,

84 (1987) 2540-2544.

304 Takahashi, T., Nakajima, Y., Hirosawa, K., Nakajima, S. and Onodera, K., Structure and physiology of developing neuromuscular synapses in culture, J. Neurosci., 7 (1987) 473-481. 305 Tanaka, H. and Landmesser, L.T., Interspecies selective motoneuron projection patterns in chick-qu~l chimeras, J. Neurosci., 6 (1986) 2880-2888. 306 Tanaka, H. and Landmesser, L.T., Cell death of lumbosacral motoneurons in chick, quail, and chick-quail chimera embryos: a test of the quantitative matching hypothesis of neuronal cell death, J. Neurosci., 6 (1986) 2889-2899. 307 Tanaka, H. and Obata, K., Developmental changes in unique cell surface antigens of chick embryo spinal motoneurons and ganglion cells, Dev. Biol., 106 (1984) 26-37. 308 Taniuchi, M., Clark, H.B. and Johnson, E.M., Induction of nerve growth factor receptor in Schwann cells after axotomy, Proc. Natl. Acud. Sci. U.S.A., 83 (1986) 4094-4098. 309 Tapscott, S.J., Bennett, G.S. and Holtzer, H., Neuronal precursor cells in the chick neural tube express neurofilament proteins, Nature (Land.), 292 (1981) 836-838. 310 Teitelman, G., Joh, T.H., Grayson, L., Park, D.H., Reis, D.J, and Iacovitti, L., Cholinergic neurons of the chick ciliary ganglia express adrenergic traits in vivo and in vitro, J. Neurosci., 5 (1985) 29-39. 311 Thiriet, G., Kempf, J. and Ebel, A., Expression of cholinergic markers in the developing chick embryo spinal cord, ht. J. Dev. Neurosci., 4 (1986) 451-463. 312 Thoenen, H. and Barde, Y.-A., Physiology of nerve growth factor, Physiol. Rev., 60 (1980) 1284-1335. 313 Thompson, J.M., Increase in acetylcholine release from . . . chrck embryo retina during development, Dev. Bruin Res., 4 (1982) 259-264. 314 Thompson, W., Synapse elimination in neonatal rat muscle is sensitive to pattern of muscle use, Nature (Land.),

302 (1983) 614-616. 315 Torda, C. and Wolff, H.G., Effect of pituitary hormones, cortisone and adrenalectomy on some aspects of neuromuscular function and acetylcholine synthesis, Am. J. Physiol., 169 (1952) 140-149. 316 Tosney, K.W. and Landmesser, L.T., Pattern and specificity of axonal outgrowth following varying degrees of chick limb and ablation, f. Neurosci., 4 (1984) 25182527. 317 Tosney, K.W. and Landmesser, L.T., Specificity of earIy motoneuron growth cone outgrowth in the chick embryo,

286 J. Neurosci., 5 (1985) 2336-2344. 318 Tosney, K. W. and Landmesser, L.T., Growth cone mor-

319

320

321

322

323

phology and trajectory in the lumbosacral region of the chick embryo, J. Neurosci., 5 (1985) 2345-2358. Tuttle, J.B., Interaction with membrane remnants of target myotubes maintains transmitter sensitivity of cultured neurons. Science, 220 (1983) 977-979. Tuttle, J.B., Vaca, K. and Pilar, G., Target influences on (“H]ACh synthesis and release by ciliary ganglion neurons in vitro, Dev. Bioi., 97 (1983) 255-263. Tweedle, C.D. and Stephens, K.E., Development of complexity in motor nerve endings at the rat neuromuscu!ar junction, Neuroscience, 6 (1981) 1657-1662. Usdin, T.B. and F~schbach, G.D., Purification and characterization of a polypeptide from chick brain that promotes the accumulation of acetylcholine receptors in chick myotubes, J. Cell Biol., 103 (1986) 493-507. Vaca, K., Basic FGF in muscle and its effects on cultured motoneurons and Schwann cells, Sot. Neurosci. Absrr., 14 (1988).

tent at the neuromuscuiar junction of fast muscle after cross-union with the nerve of slow muscle in the chick. BrainRes., 26(1971)443-445. 334 Walicke, P.A., Campenot. R.B. and Patterson, PH..

335 336

337

338

339

324 Vaca, K., Johnson, D. and Pilar, G., Modulation of transmitter synthesis and release in choiinergic terminals, J. Physiol. (Paris), 78 (1982) 385-391.

32.5Vaca, K., Nunez, R. and Pilar, G., Neural control of acetylcholine receptor and contraction in the iris, Sot. Neuruxi. Absrr., 6 (1980) 99. 326 Vaca, K. and Pilaf, G., Mechanisms controlling choline transport and acetylcholine synthesis in motor nerve terminals during electrical stimulation, J. Gen. Physiol., 73 (1979) 605-628. 327 Vaca, I(., Stewart, S.S. and Appel, S.H., Identification of basic FGF as a cholinergic growth factor from muscle. submitted. 328 Valinsky, J.E. and Loomis, C., The cholinergic system of the primitive streak chick embryo, Cell Differ., 14 (1984) 287-294. 329 Van Essen, D.C., Neuromuscular synapse elimination: structural, functional, and mechanistic aspects. In N.C. Spitzer (Ed.), Neuronal Development, Plenum, New York, 1982, pp. 333-376. 330 Varmus, H., Cellular and viral oncogenes. In G. Stamatoyannopoulos et al. (Ed%), The Molecular Bask of Blood Diseases, Saunders, Philadelphia, PA, 1987, pp. 271-346. 331 Varon, S., Manthorpe, M. and Longo, F.M., Growth factors and motor neurons. In L.P. Rowland (Ed.), Human Motor Neuron Diseases, Raven, New York, 1982, pp. 453-471. 332 Vrbova, G., Neuromuscular diseases viewed as a disturbance of nerve-muscle interactions. In G. Burnstock et al. (Eds.). Somatic and Aufonom~c Nerve-Muscle Interactions, Elsevier Science Publishers, New York, 1983, pp. 359-383. 333 Vyskocil, F., Vyklicky, L. and Huston, R., Quantum con-

340

341

342

Determination of transmitter function by neuronal activity, Proc. Nat/. Acad. Sri. U.S.A., 74 (1977) 5767-5771. Watson, W.E.. Cell Biology of Brain, Chapman and Hall, London, 1976. Weber, M.J.. Raynaud, B. and Deheii, C., Molecular properties of a cholinergic differentiation factor from muscle-conditioned medium, J. Neurochem., 45 (1985) 1541-1547. Weldon, P.R. and Cohen, M.W., Development of synaptic ultrast~cture at neuromuscular contacts in an amphibian cell culture system, J. Neurocytol., 8 (1979) 239-259. Whitelaw, V. and Hollyday, M., Thigh and calf discrimination in the motor innervation of the chick hindlimb following deletion of limb segments, J. Neurosci., 3 (1983) 1199-1215. Whitelaw, V. and Hollyday, M., Position-dependent motor innervation of the chick hindlimb following serial and parallel duplications of limb segments, J. Neurosci.. 3 (1983) 1216-1225. Whitelaw, V. and Hollyday, M., Neural pathway constraints in the motor innervation of the chick hindlimb following dorsoventral rotations of distal limb segments, 1. Neurosci., 3 (1983) 1226-1233. Whitemore, S.R., Larkfors, L., Ebendal, T., Holets, V.R., Ericsson, A. and Persson, H., Increased p-nerve growth factor messenger RNA and protein levels in neonatal rat hippocampus following specific cholinergic lesions, J. Neurosci., 7 (1987) 244-25 1. Williams, L.R., Varon, S., Peterson, G.M.. Wictorin, K., Fischer, W., Bjorklund, A. and Gage, F.H., Continuous infusion of nerve growth factor prevents basal forebrain neuronal death after fimbria fornix transection, Proc.

Natl. Acad. Sci. U.S.A., 83 (1986) 9231-9235. 343 Wolinsky, E.J. and Patterson, P.H., Rat serum contains a

developmentaIly

regulated cholinergic inducing activity,

J. Neurosci.. 5 (1985) 1509-1512.

344 Wright, L., Cell survival in chick embryo ciliary ganglion is reduced by chronic ganglionic blockade, Dev. Brain Res., l(l981) 283-286. 345 Yntema. C.L., Deficient efferent innervation of the extremities following removal of neural crest in Amblystoma, J. Exp. Zooi., 94 (1943) 319-349. 346 Young, S.H. and Poo. M., Spontaneous release of transmitter from growth cones of embryonic neurones, Nature (Land.), 305 (1983) 634-637. 347 Zitler, C., Dupin, E., Brazeau,

P., Paulin, D. and Le Douarin, N.M., Early segregation of a neuronal precursor cell line in the neural crest as revealed by culture in a chemically defined medium, Cell, 32 (1983) 627-638.