Changing Concepts in Developmental Neurobiology Viktor Hamburger Perspectives in Biology and Medicine, Volume 18, Number 2, Winter 1975, pp. 162-178 (Article) Published by Johns Hopkins University Press DOI: https://doi.org/10.1353/pbm.1975.0002

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CHANGING CONCEPTS IN DEVELOPMENTAL NEUROBIOLOGY* VIKTOR HAMBURGER^

I should like to take this opportunity to meditate on half a century of explorations in the field of experimental neurogenesis which I have witnessed or participated in. I shall do this in a rather personal and informal way. This, I am sure, would have the blessing of George Bishop, who had little use for formalities. To me it is still a miracle to watch the nervous system transform itself

within a few weeks from a simple tube, composed of a few hundred seemingly undifferentiated embryonic cells, into the most complex organ system that has evolved in nature. So many interlocking production lines must operate with the highest precision, so many tightly programmed schedules must be met, so much could go wrong that one marvels that we all function as well as we do. But unraveling and under-

standing these intricacies is another matter. The neuroembryologist is overwhelmed by problems on the cellular, supercellular, and ultrastruc-

tural level. How do the literally hundreds of neuron strains, each with its own structural and biochemical identity, originate? How do they organize themselves into the supercellular units, the strata, columns, and nuclei in their precise topographic relationships? How is the circuitry established? How does an axon know on which dendritic spine to settle

down? As if all this were not enough, we have to cope with the propen-

sity of the neurons to establish intimate relations with any number of

peripheral structures. This confronts us with problems of directional axon outgrowth and specific sensory and motor connections. Moreover, these relationships involve mutual dependencies of such stringency that they often decide on the life or death of the partners.

By posing the problems the way I do, I reveal my bias, which is that of the experimental neuroembryologist brought up in a school where the

dynamics of developmental relations and mutual interactions between

*Nineteenth George H. Bishop Lecture in Experimental Neurology, given on April 19, 1974, at the Washington University School of Medicine, Saint Louis. tDepartment of Biology, Washington University, Saint Louis 63130. Research has been generously supported by the NINDS, NIH, Bethesda, Maryland.

162 I Viktor Hamburger · Developmental Neurobiology

Fig. 1.—George H. Bishop (1889-1973)

embryonic primordia were the primary concern. My bias was acquired in the laboratory of H. Spemann, which in the twenties and thirties (while I

was there first as a Ph.D. candidate and later as a Privatdozent) was at the

zenith of its activity. The "organizer" story was unraveling, and although

I did not participate directly in this adventure, I became imbued with the

spirit and the canons of experimental embryology, which in essence is the inquiry into the immediate causes or factors that determine the fate

of cells and organ primordia. Today one hardly speaks of causality; we

analyze mechanisms. And the term "determination," which was then the

key concept, is now translated into computer language as "programPerspectives in Biology and Medicine · Winter 1975 I 163

Fig. 2.—Viktor Hamburger

ming," with little net gain in basic insight. The art of microsurgery on amphibian embryos was brought to high perfection by the masters of experimental embryology, H. Spemann and R. Harrison. The aesthetic appeal added to the satisfaction of being engaged, as Spemann put it, in a direct dialogue with the living embryo, a pleasure which most modern molecular embryologists have to forgo.

The focus of interest was on embryonic induction, which is a special

category of developmental interactions. As it happens, the primordium

of the nervous system, the neural plate, owes its existence to such an interaction, which occurs during a very early stage, the gastrulation 164 I Viktor Hamburger · Developmental Neurobiology

phase. During that process, the mesoderm invaginates and its median portion applies itself closely to the overlying outer layer, the ectoderm. The mesoderm mantle then induces the formation of the neural plate in the overlying ectoderm; that is, it initiates neural differentiation by a chemical interaction. The induction was demonstrated by H. Spemann and Hilde Mangold in the classical organizer experiment on salamander embryos [1], which earned Spemann the Nobel Prize. If by appropriate transplantation a piece of mesoderm is brought into contact with a part of ectoderm that would normally form body wall, a secondary neural plate is induced in that region. Transplant and induced structure could be distinguished by choosing a salamander species with unpigmented eggs as donor and one with pigmented eggs as host. If the induced structure is allowed to continue its differentiation, the secondary nervous system, together with the transplant and additionally induced organs, forms a whole secondary embryo on the flank of the host embryo; hence, the transplanted mesoderm was designated the "organizer." By the way, the inductive act creates notjust neuralization in general but the regional patterning into three brain divisions and the spinal cord. This seems to be accomplished by two macromolecules distributed in the

mesoderm mantle along two concentration gradients in opposite directions along the main axis [2]. I have always considered it a particularly friendly gesture of the embryo toward the neurologist to single out the creation of the nervous system as. the most prominent and celebrated, Nobel Prize-winning event in all embryonic development. At the suggestion of Spemann, I began to work on developmental relationships in later phases of neurogenesis, and thus moved into the orbit of R. Harrison, the founder of experimental neuroembryology, who at that time was "the Chief " (as everybody called him) at Yale. His historic contribution was the definitive demonstration that the axon is a

pseudopodial outgrowth of the neuroblast, which he observed under the microscope in fragments of explanted neural tube of frog embryos. The experiment settled a highly controversial issue and at the same time gave us the tissue culture method. He also confirmed an old observation of

Ramón y Cajal, dating back to 1890, who had described in silver preparations the formation of a club-shaped "growth cone" at the end of the advancing tip of the axon. Harrison states in his 1907 paper: "These observations show beyond question that the nerve fiber develops by the outflowing axoplasm from the central cells. This protoplasm retains its ameboid activity at the distal end, the result being that it is drawn out

into a long thread which becomes the axis cylinder" [3]. As you see, axoplasmic flow is an old story. Its present popularity dates from the discovery of P. Weiss that it is not limited to embryonic and regenerating fibers but is a continuous process [4]. Both Cajal and Harrison recognized an essential corollary to the nerve outgrowth theory—the posPerspectives in Biology and Medicine · Winter 1975 ! 165

tulate that the growth cone must be endowed with some kind of sensitivity to perceive cues and signals that guide it to its destination. The Detwiler Experiment One of the earliest experiments in this field, done in the early twenties

at the suggestion of Harrison by his oldest and most active student and co-worker, S. Detwiler [5], illustrates the way in which the experimental

embryologist tackles problems such as directional fiber outgrowth. To answer the simple question of how outgrowing axons would behave in a foreign environment, he transplanted forelimb primordia in the tail bud stage of the salamander embryo one to five segments caudad. The gen-

eral result was that trunk nerves alone or in combination with limb

nerves would form a reasonably typical limb nerve pattern. Two conclusions are warranted: (1) that the limb tissues make a major contribution to nerve pattern formation by providing tracks and (2) that, at least in this instance, the responsiveness of growth cones to environmental cues is rather broad and unspecific. The ingenuity of this particular experimental design becomes apparent when one realizes that it led to two other major discoveries. Detwiler found that a limb transplanted to the

flank would participate in coordinated walking movements only if at least one of its nerves originated in the brachial segments of the cord. If innervated exclusively by trunk nerves, it would perform irregular

twitches at best. This implies a basic difference between the limbinnervating and the thoracic segments of the cord; only the former have the capacity to build the circuitry for coordinated locomotion. This dif-

ference exists already in very early prefunctional stages, when neuronal

differentiation has hardly begun. Detwiler made another observation. He found that those ganglia

which were deprived of their peripheral field of innervation became hypoplastic and thoracic ganglia that were overloaded by the implanted limb became hyperplastic. Thus, the "trophic" dependency of developing neural structures on nonnervous structures was revealed. This de-

ceptively simple experiment, which required only a pair of iridectomy scissors, some glassware, and a few embryos from a nearby pond, thus identified three of the major issues in neurogenesis which I shall deal

with.

Trophic Effects I shall take up first the trophic effects. I indicated at the beginning that trophic relationships between neuroblasts and their target organs may become obligatory to the point that the absence of one partner may spell death to the other. The fate of denervated muscle in the adult is 1 66 I Viktor Hamburger · Developmental Neurobiology

well known. In amphibian and chick embryos, one can obtain nerveless limbs by preventing nerve ingrowth from the start. The general morphogenesis is relatively normal, but the limbs are smaller than normal and atrophic. Their musculature differentiates to the point of crossstriation but then breaks down [6]. In insects, lack of innervation has more severe effects. In moths, the larval musculature is broken down

during pupation and the adult musculature is built anew from remnants of larval muscle fibers. If a thoracic ganglion of the pupa is extirpated, adult muscle differentiation is completely inhibited; indeed, it does not even get started [7]. A spectacular illustration of trophic nerve influence

was found in amphibian limb regeneration. A denervated amputation stump in a salamander larva not only fails to regenerate but actually regresses rapidly down to the base of the arm [8]. We still refer to this remarkable extracurricular activity of the axon by the century-old term "trophic," which indicates that we have made little advance in the clarification of the mechanism involved. But it is obvious that impulse propagation is only one aspect of neuron function. To quote George Bishop, What is a nerve fiber for, anyway? and what, in fact, is a nerve cell? The intrigu-

ing mechanism, by means of which it generates and conducts an impulse, has chiefly preoccupied three generations of neurophysiologists with the performance of too many and ingenious experiments. . . . Conduction of an impulse is in fact somewhat incidental to another essential functioning of a neuron, however useful as a sign that the neuron has functioned. Where does one come out,

if he looks at the neuron as a secretory organ? This proposal is not new, but nobody has done much lobbying for it. To wit, the prime function of a neuron is to produce and apply to other tissues a chemical activator. [9, p. 14]

The reverse relationship has been analyzed in considerable detail. If a limb bud of the chick embryo is extirpated at 21A days, this is not noticed by the primary motor and sensory centers for a while. Differentiation goes on until the time when the axons would normally have contacted muscle fibers and sense organs in the growing limb. But then, at around 6-7 days, a sudden dramatic disintegration of the motor column sets in which wipes out the entire population of 20,000 cells within 3 days [10]. The ganglia are also reduced greatly. Obviously, unspecified conditions at the periphery control the maintenance of the centers. There exists an interesting parallel between this breakdown process and a similar phenomenon that occurs in normal development. The motor column, like other neuron populations, normally engages in overproduction of neuroblasts, which is followed by a depletion that amounts to 40 percent in the case of the chick motor column [H]. The fact that it occurs at

approximately the same time as the breakdown after limb ablation suggests a similar mechanism in both instances. Probably only those neuroPerspectives in Biology and Medicine · Winter 1975 | 167

blasts survive whose axons manage to make contacts at the periphery. This would explain selective survival in the normal situation and total loss in the experimental situation. The hypoplasia story has had a much happier ending for me than for

the motor neuroblasts. I confirmed in the chick the old observation of

Detwiler that an increase of the peripheral field by implantation of a supernumerary limb results in a distinct hyperplasia of the spinal gan-

glia and a less conspicuous effect on the motor column. I proposed a feedback hypothesis which I thought was quite clever in that it would explain both hyper- and hypoplasia by the same mechanism. I suggested that the demand for nerve fibers at the periphery might regulate the production of neuroblasts at the center. The hypothesis turned out to be essentially wrong; but my error was a blessing in disguise. It resulted in two related events: the appearance of Dr. Rita Levi-Montalcini on the scene, in 1948; and the discovery of the nerve growth factor (NGF) a few

years later. Rita had done the same limb extirpation experiments in Italy during the war. She had obtained the same results, but her interpreta-

tion was different and, as usual, the correct one: absence of the limb

results in the retrograde degeneration of the frustrated neuroblasts. I

invited her to Saint Louis, and we settled the argument. We then tried to get to the heart of the matter of hyperplasia. We repeated a rather bold

experiment of one of my former students, Bueker, who had implanted a piece of mouse tumor in chick embryo in the hope that this fast-growing tissue would incite a more impressive hyperplasia than the supernumerary limbs. He did find invasion of the tumor by nerves but was not

encouraged by the results. At this point, Rita's flair for detecting subtle clues which the embryo manages to hide from the eyes of other observers asserted itself, and in rapid sequence the NGF yielded its secrets. This story has been told by her [12], but I would like to reminisce on two milestones in the early days. One day she showed me a chick embryo with a large intraembryonic tumor. The tumor had produced the typical conspicuous hyperplasia of spinal and sympathetic ganglia, and it had

been invaded massively by nerve fibers from these ganglia. But she had made another, novel observation. Some prevertebral sympathetic ganglia, quite remote from the tumor, were also hyperplastic; their axons ended in adjacent viscera rather than in the tumor. This was the first

hint that a diffusible agent was involved. It did not take long to prove the point. Tumor tissue was transplanted to the chorioallantoic membrane, far from the embryo, where it grows well. It exerted the familiar effect: hyperplasia of ganglia, by remote control via the circulation [13].

The second memorable event was the arrival of a sketch from Rita

from Rio de Janeiro, where she was working with a tissue-culturing friend. The sketch showed the first ganglion in tissue culture with a halo of fibers induced by a piece of tumor which had been placed at some 168 I Viktor Hamburger · Developmental Neurobiology

distance from it and had released NGF. To this day, the halo provides

the indispensable bioassay for NGF—and honors for Rita. To conclude this "trophic" chapter: I am not sure of the actual role of NGF in the normal development of adrenergic neurons, but I am sure that this discovery is a major breakthrough in developmental neurobiology. It is the one instance in which a highly target-specific nerve growth regulator has been tracked down to its molecular structure. With the

sequencing of the active NGF protein by Ruth Angeletti and R. Bradshaw in 1971 [14], the road is open for an understanding of its mechanism of action. But the fact that this discovery, which grew out of

a seemingly peripheral problem (peripheral in every sense of the word),

has blazed so many new trails is its greatest contribution to neuroem-

bryology.

Central Circuitry Of the other two problems raised by the Detwiler experiment, that of the construction of central circuitry has been advanced most successfully. In the more precise formulation as the problem of formation of specific synapses, it has moved to the center of the stage. Before taking up this topic, I shall mention briefly two experiments which support the contention of Detwiler that the central circuitry for locomotion is built

into the system prior to function and without benefit from it. Narayanan and I found very early specification of the brachial and lumbosacral segments of the spinal cord for their respective functional assignments. We used a different experimental design, which, incidentally, also goes back to Detwiler. In 1 Mi-day chick embryos, we switched the brachial to the lumbosacral level, and vice versa. A few embryos with legs inner-

vated by a brachial plexus were hatched. The legs moved only synchronously, as in wing flapping, but they never showed alternating stepping movements. Obviously, the program for the construction of the central circuitry is built into the limb segments of the spinal cord even before the onset of neuroblast differentiation, and it cannot be modulated by the

appendages [15]. Straznicky [16] had reported similar findings. The same point was demonstrated by Bentley and Hoy in a more sophisticated way. They studied the embryonic origin of the neural circuitry which commands the motor output to wing muscles in the flight pattern of the cricket [17]. Crickets undergo approximately 10 molts before emergence of the adult. Individual flight muscles are innervated by only one to three neurons. Motor output was monitored by electromyograms taken simultaneously from four muscles: hind-wing depressor, hind-wing elevator, fore-wing depressor, and fore-wing elevator. The tests were made by suspending the larvae in a wind tunnel. In the last instar before final molt, the complete flight pattern is estabPerspectives in Biology and Medicine · Winter 1975 | 169

lished; long trains of spikes are performed, equivalent to dozens of wing strokes, although the wing primordia are immobile and tightly packed in

the larval envelope. The gradual emergence of flight muscle coordination, which reflects the gradual establishment of neuronal circuitry, was

traced back to the fourth to the last instar, when the first irregular spikes

occur in the hind-wing depressors, which are the muscles that eventually lead the flight pattern. There could be no better demonstration of the

nonparticipation of functional activity in the buildup of circuitry for complex, stereotyped motor patterns.

Synaptogenesis Before I turn to the modern aspects of synapse specificity, I would like to indicate the conceptual metamorphosis which the problem has un-

dergone. In the twenties and thirties there prevailed among develop-

mental psychologists a belief in the powerful role of learning, experi-

ence, and sensory input in the molding of behavior and a downgrading oí instincts and built-in action systems. These views carried over into neurogenesis. It was seriously proposed that neuronal connections were

at first indiscriminate and random and that functionally adaptive path-

ways and synapses would be reinforced by practice and would survive

while all others would vanish. This extreme view of the creative role of

function was founded on shaky evidence and challenged by neuroembryologists such as Coghill, P. Weiss [18], and others. Their observational and experimental data supported the diametrically opposed view

that the maturation of neural connectivities precedes and conditions

functional activity and behavior, not vice versa. The neuroembryologists have won the argument, but, as we shall see, the final, or rather the

present-day, verdict is by no means quite that simplistic. It was in this context, as a challenge to the viewpoint of functional adaptation, that Roger Sperry initiated his analysis of retinotectal rela-

tions [19]. The choice of the retinotectal system was fortunate and was one major reason for the rapid advances in our insight into the mechanism by which neuronal connections are formed in synap-

togenesis. It is of interest that his major discoveries were based on purely behavioral observations on lower vertebrates, frogs and salamanders, which he subjected to minor surgery. His basic experimental design capitalizes on the capacity of the optic and other nerves in the frog to

regenerate to the point of full functional recovery. In the first crucial experiment, optic nerve transection was combined with the rotation of

the eyeball by 180°. The optic fibers were scrambled at the cut surface to

reduce the chance of their return to their old channels. After functional

regeneration, the visual experience of the frog was found to be abnormal: objects were seen upside-down and backward instead of forward, 170 I Viktor Hamburger · Developmental Neurobiology

and this maladaptation was never corrected. If only the dorsoventral axis of the eye was rotated (by shifting the left eye to the right orbit, over the head), then an object presented above was seen as if it were located below; if only the anteroposterior axis was inverted (by shifting the left eye to the right orbit around the nose), then the forward-backward direction only was misjudged. In other words, the errors were not ran-

dom, but systematic. The explanation for abnormal responses was based on the well-known fact that there is a precise topographic projection of the different quadrants of the retina onto the optic tectum. Sperry then postulated that the retinal ganglion cell axons had reestablished synapses at exactly the same tectal region with which they had been connected before. We speak of "position specificity." Numerous experiments on other sensory systems supported this conclusion. Sperry then

made the bold and ingenious inference from the behavioral data to a biochemical mechanism of synapse formation. He proposed the chemoaffinity hypothesis, according to which synapse formation is based on matching or complementary biochemical affinities between the axonal growth cone and the neuron which it contacts. In the course of time, the wide gap between data and inference was almost filled by him and others.

The first step was taken by M. Gaze in England [20]. He confirmed the precise retinotectal projection in the frog by electrophysiological recording from the tectum. Then he applied the mapping technique to inverted eyes. He showed that, indeed, retinal ganglion axons returned to their specific locations on the tectum. Attardi and Sperry [21] came a

step closer to histological verification of the synaptic site in an experiment in which optic nerve transection was combined with ablation of half-retinas. The residual fibers returned to their specific sites, leaving other tectal areas vacant, or even bypassing the inappropriate areas. But

none of these approaches could trace the axon terminals to the actual synaptic sites; and they all dealt with the reestablishment of previously existing connections. In both respects, the recent experiments of Cowan and his co-workers on the retinotectal projection in chick embryos [22] provide the finishing touch. The half-retina experiments were repeated on 2 M¡-day chick embryos, and the ingrowth of the fibers from the residual retina to their correct positions was ascertained histologically and by autoradiography. The crucial point is that the actual synaptic sites could be identified by two different methods. The first was based on rapid axoplasmic flow: labeled protein precursors were injected into the miniature eyes at prehatching stages. The sites of projection of the ganglion cells were determined from the grain condensations found in those strata of the optic tectum which are known to be the sites of optic fiber termination. In the other method, which is more precise, eyes with reduced retinas were extirpated some time after hatching, and a few days Perspectives in Biology and Medicine · Winter 1975 I 171

later the sites of degenerating synapses were identified by the appearance of ringlike boutons. Since we are dealing here with initial embryonic synaptogenesis, the "position specificity" of optic fiber connections is established beyond doubt.

But the final step, from position specificity to chemoaffinity, remains to be taken. So far, chemoaffinity is an abstract concept devoid of substantive content, not an explanation. It is a challenge to the molecular neuroembryologist to give it a concrete meaning. Synapse formation is a special case of cell recognition. The latter topic is now under intense investigation in cell dissociation and reaggregation studies. It is obvious that the solution to the problem of synapse specificity will be in terms of the biophysical and biochemical properties of membranes. The beginnings that have been made with regard to retinotectal adhesive recognition are promising. Preferential adhesion of dissociated retina cells to appropriate tectal sites has been reported [23]. In another experimental design, isolated plasma membranes from chick embryo retina cells were

found to inhibit differentially the reaggregation of retina cells and, to a lesser degree, that of tectal cells [24]. Of particular interest is the demonstration that cell membrane recognition changes profoundly with stage of development.

This is not the only unresolved problem. The pursuit of the question concerning the precision of neuronal specification has led to difficulties which present a serious challenge to the chemoaffinity hypothesis. No-

body thinks in terms of a rigid one-to-one relationship between individual retinal and tectal cells. But what are the limits of "recognition"? Sperry built into his hypothesis a certain degree of plasticity in the form of two overlapping gradient fields, one perpendicular to the other, and each representing a major axis in the retina and tectum, respectively. A neuron group would be identified by its relative position in this coordinate system [25]. Gradient fields have been invoked in experimental

embryology whenever the problem of regulation becomes acute, because

the relative position of the subunits remains unchanged when the fields are experimentally reduced or expanded or even distorted. An unex-

pected degree of regulation in the retinotectal projection was revealed

when the size of the tectum rather than that of the retina was reduced.

For instance, if half of the tectum or a median strip is removed, the entire retina is projected onto the residual tectum surface in a compressed, orderly fashion [26, 27]. Further difficulties arise in mammals,

in which the retina projects onto several different centers [28, 29]. It remains to be seen whether these new data can be accommodated in

Sperry's double-gradient model, or whether the chemoaffinity hypothesis will have to undergo a major revision. Another series of recent investigations makes it appear as if we were on the way to returning full circle to the viewpoint of functional adapta172 I Viktor Hamburger · Developmental Neurobiology

tion. Recent experiments from different laboratories give clear evidence that cells in the visual cortex of newborn mammals can be specified as to their functional role by exclusive exposure to particular visual patterns, such as vertical bars [30-32]. I do not believe that these findings are in conflict with those on prefunctional specification. It would seem that built-in fixity and functional plasticity both have their place in synaptogenesis, the former as the basis of stereotyped behavior and funda-

mental organization and the latter in higher centers, where the capacity for postnatal adjustments and refinements would be of adaptive value.

Finally, one has to ask, To what extent are the findings on the visual system paradigmatic for other systems? A clear answer cannot be given at this moment. Extensive work on neuromuscular specificity, which my limited time does not permit me to review, has not given unequivocal

results, and the issue is highly controversial. We are not much better off in the matter of the central organization of localized skin reflexes. In both instances, two alternative possibilities have been debated: that the peripheral structures specify the nerves which contact them, or that

prespecified nerves selectively establish contact with the proper end organ. Recently, skepticism has been expressed concerning both viewpoints [33]. Indeed, we are far from any generalization in this field. However, this should not minimize the great heuristic value of the chemoaffinity concept, which has served as a guide and frame of reference for all subsequent experimental analysis.

Peripheral Nerve Patterns In contrast to synaptogenesis, the problem of how nerves reach their goals has not advanced much. I state this with regret, because this problem has interested me for many years and I worked on it in my earlier days. It is not even generally realized that pathfinding is a separate

problem, distinct from synapse formation. Fibers must arrive before they can make connections. There is one way to combine both aspects in a unified theory, namely, by assuming that fibers are attracted at a distance by their targets. This was indeed the prevailing view when tropisms were fashionable, around the turn of the century. Ramón y

Cajal was one of the first to suggest chemotropism as the guiding force; others postulated galvanotropism. Yet there is an inherent improbability in the tropism theories: How can overlapping local gradients in an embryo remain sufficiently stable for a long enough period to be effective? How would one account for the branching of dorsal root fibers in opposite directions? Then there is the fact that the major branching pattern,

for instance in the embryonic limb, is laid down before the individual muscles are formed. Apart from such considerations, all efforts to test Perspectives in Biology and Medicine · Winter 1975 | 173

tropism theories in vitro or in vivo have given negative results. But perhaps the last word has not been said in this matter. Harrison has always stressed the importance of the solid substrate on which the axon is spun out, and I mentioned earlier that the growing limb provides tracks for nerves. Weiss once thought that the micellar organization of the substrate might provide the necessary directional cues; this is expressed in the contact guidance concept [34]. But mechanical guidance alone cannot be the answer. Since growth cones are com-

pelled to make choices at every branching point and have to decide whether to enter a proximal or a distal muscle or a particular sense organ, one has to postulate additional cues which would provide more specific guidance than mechanical tracks can give. The obvious suggestion is that substrates are biochemically specified. Thus, we are back at chemoaffinity, this time between the growth cone and the substrate on which it advances [25, 34].

This brief survey of facts and fancies in pathfinding and synapse formation should end on an optimistic note. If the prevailing view of the inherent structural, functional, and biochemical specificity of neuron strains is correct, and if one also accepts the corollary that interneuronal and neuron and target recognition is based on matching biochemical membrane specifications, then we are in sight of one of the most formidable problems in neurogenesis: How does neuronal strain specificity originate in the embryo? What constellation of intrinsic and extrinsic agencies is instrumental in conferring identity on a neuron? Is this done in one step or in a sequence of steps? Do the partners acquire their matching specifications independently, or does one consult with the other? Here is a great challenge for a new generation to invent the

concepts and tools necessary for the pursuit of this goal—indeed an optimistic outlook.

Embryonic Motility Near the start of my career, I wrote an article for Naturwissenschaften in

which I outlined a research program for the rest of my life [35]. I have followed it—to some degree. But I had omitted one point—the embryology of behavior—for reasons that are now not clear to me. Perhaps, as an embryologist, I looked at the developing nervous system primarily as a playground for collecting developmental correlations, and I was apparently not fully aware oí the raison d'etre of the nervous system. This

omission has been corrected in the last decade with the help of a number of able collaborators, M. Balaban, J. Decker, C. H. Narayanan, R. Oppenheim, R. Provine, S. Sharma, and E. Wenger [36-38]. In the course of this work, again, a fundamental shift in conceptualization was forced on us by the phenomena. It will not surprise you to learn that the change 174 I Viktor Hamburger · Developmental Neurobiology

of outlook is in the same direction as for previously discussed areas, namely, from a behavioristic, stimulus-response notion to the primacy of

built-in, autonomously generated spontaneous motor activity. For de-

velopmental psychologists of the thirties and forties, like Kuo and Schneirla, who dominated the field, the role of sensory input, learning, and similar experience in the emergence of behavior was self-evident, and the extensive observations of Kuo on chick embryo were interpreted in this way, without experimental validation. A competing theory ofthat period, that is, Coghill's notion that behavior is integrated from the first head movement of the embryo to the adult stage [39], may be valid for his material, the salamander, but it is definitely not applicable to birds and mammals.

My contention that embryonic behavior results from autonomous,

nonreflexogenic activity of the central nervous system is based on three lines of evidence: observation of some peculiar features of overt motility on chick and rat embryos, deafferentation experiments, and electrophysiological recordings. The peculiar features I refer to are (1) the spontaneity of embryonic movements, not caused by any obvious stimulation; (2) the periodicity of motility, activity phases alternating with inactivity phases, which is evident in the chick embryo from the beginning of motility at 3 1A. to 13 days; and (3) the lack of coordination of parts, such as right and left leg or head movements. During an activity

phase, different parts move in seemingly random and unpredictable combinations (I do not speak of muscle coordination) and in a jerky,

convulsive-like fashion. These features suggest that an integrating influence of sensory information is not yet present. This peculiar motility pattern is in contrast to a highly integrated, quite different behavior type which starts at 17 days and is instrumental in hatching. The fetal motility of the rat between 16 days and parturition shows the same

characteristics, that is, spontaneity, periodicity and lack of coordination. I believe that this type of motility is characteristic of amniote embryos in general. Experimental evidence for the nonreflexogenic nature of the em-

bryonic motility type was obtained by deafferentation experiments in which the primordia of sensory ganglia to the leg, the primordia of the trigeminal ganglia, and those of the accousticovestibular system were extirpated in early chick embryos. I shall discuss only the leg deafferentation, which was accomplished by a double operation; the removal of a thoracic section of the cord to prevent input from rostral regions; and the extirpation of the dorsal half of the lumbosacral cord, which includes

the primordia of sensory ganglia that feed into the lumbosacral plexus. Periodicity and general characteristics of the motility were normal, up to

15-17 days, when the half-cord began to deteriorate. Normalcy in these

features was also found in the other deafferentation experiments. Perspectives in Biology and Medicine · Winter 1975 | 1 75

Since the activity is not blocked by chronic gaps in the cervical cord, we concluded that the motility results from autonomously generated discharges of the spinal cord. This assumption was substantiated by direct recordings from curarized embryo, in vivo done mostly by Provine [40].

He found, after preliminary exploration of the lumbosacral cord, a close correlation between patterns of polyneuronal bursts generated in the ventral (motor) part of the spinal cord and the overt motility patterns.

This holds for all stages, from the beginning of motility, at 4 days, to hatching. The conformity of bursts and activity phases was definitely established when floating electrodes were used to record from uncurarized, freely moving embryos. One observer recorded the motility phases, and an oscillograph recorded the bursts. There was precise synchrony throughout development. Finally, the proof that the bursts are the cause of motility, and not movement or other artifacts, came from recordings that were made before, during, and after a 15-minute curarization period. It was found that bursts continue while the embryo is immobilized.

This peculiar embryonic spontaneous motility pattern has no parallel in postnatal behavior; it persists, perhaps, in the phasic movements of REM sleep. Its adaptive significance is probably in preventing muscle atrophy and fusion ofjoints; both occur when embryos are immobilized for longer periods. The strange performance of the chick embryo and of the mammalian

fetus leaves us in a puzzling situation. It looks as if integrated activities such as walking or pecking of food which are performed with reasonable perfection soon after hatching or birth by all precocious animals have no antecedents in prenatal motility. This apparent paradox can be resolved if one assumes that the neuronal circuitry for these activities is prepared during embryonic development but does not find expression in the overt motility of the embryo. In fact, it can be shown that, at least at the level of muscle coordination within the leg, more order exists than is apparent from the jerks and twitches that the embryonic leg performs or from the burst pattern. Anne Bekoff, who is presently monitoring the motor output in embryos in situ by electromyography, finds antagonistic inhibition in leg muscles as early as 7 days of incubation, that is, shortly after onset of leg motility [41].

Concluding Remarks When I was invited to give this lecture and asked for a title, I tried to think of something timely and "relevant," and it occurred to me that "Law and Order in Neurogenesis" would be a fitting title. But, on second thought, I discarded this idea, not only because I have too much respect for the nervous system to associate it with dubious company, but also 176 I Viktor Hamburger · Developmental Neurobiology

for another reason. Although there is definitely order, I was at a loss to think of a single "law" or even a broad generalization. Apparently, experimental neuroembryology, after passing the half-century mark, is still too young to show such signs of maturity. The old-timer admires the vigor and vitality of its second youth and the rapid and imaginative progress that has occurred in neuroembryology in recent years. In the

same spirit, the younger generation that is now taking over should be grateful to us old-timers for not being smart enough to solve all the problems.

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ON HUMAN PURPOSE*

The purpose of life is what we make it, Yet it is deep within us. As an individual it is

Enjoyment through healthy function, love, growth, development, and maintenance of species; As a society, it is To provide an environment In which people of all races Can develop their individual abilities To discover, examine critically, Preserve, and transmit

The knowledge, wisdom, and values That will help ensure the survival Of the present and future generations With improvement in the quality of life And in human dignity. Van Rensselaer Potter

*An extension of a statement on the "Purpose and Function of the University" by V. R.

Potter et al., Saence 167(1970): 1590.

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