Specification of Synaptic Connections between Sensory and Motor Neurons in the Developing Spinal Cord Eric Frank* and Bruce Mendelson Department of Neurobiology, Anatomy and Cell Science, Center for Neuroscience, University of Pittsburgh School of Medicine, 3550 Terrace Street, Pittsburgh, Pennsylvania 15261

SUMMARY Experimental studies of mechanisms underlying the specification of synaptic connections in the monosynaptic stretch reflex of frogs and chicks are described. Sensory neurons innervating the triceps brachii muscles of bullfrogs are born throughout the period of sensory neurogenesis and do not appear to be related clonally. Instead, the peripheral targets of these sensory neurons play a major role in determining their central connections with motoneurons. Developing thoracic sensory neurons made to project to novel targets in the forelimb project into the brachial spinal cord, which they normally never do. Moreover, these foreign sensory neurons make monosynaptic excitatory connections with the now functionally appropriate brachial motoneurons. Normal patterns of neuronal activity are not necessary for the formation of specific central connections. Neuromuscu-

lar blockade of developing chick embryos with curare during the period of synaptogenesis still results in the formation of correct sensory-motor connections. Competitive interactions among the afferent fibers also do not seem to be important in this process. When the number of sensory neurons projecting to the forelimb is drastically reduced during development, each afferent still makes central connections of the same strength and specificity as normal. These results are discussed with reference to the development of retinal ganglion cells and their projections to t h e brain. Although many aspects of the two systems are similar, patterned neural activity appears to play a much more important role in the development of the visual pathway than in the spinal reflex pathway described here.

INTRODUCTION

tact with only a subgroup of the potential target cells. And finally, in many parts of the nervous system the set of synaptic connections made initially is further refined during later development, often using correlated patterns of neural activity within the population of presynaptic and postsynaptic neurons. To understand how all this comes about, it is useful to examine a number of different neural systems in detail. Certain underlying mechanisms may be common to a large number of different systems, but it is likely that each system will also be unique. By relating the similarities and differences among the different systems to each one’s particular requirements, one may be able to gain a better understanding of why a particular combination of mechanisms is used. An attractive feature of the

A central issue in the study of the nervous system

is how neurons establish their distinctive phenotypes. One important feature of a cell’s phenotype, and the one we will focus on herc, is the set of synaptic connections it makes with other cells. Shortly after a neuron differentiates, it sprouts an axon which begins to grow along a predictable pathway. When the growing tip has reached the appropriate target area it must make synaptic conReceived June 1. 1989, accepted July 1 1, 1989 Journal ofNeurohiology, Vol 21, No I , pp 33-50 1990) 0 1990 John Wilev & Sons. Inc CCC 0022-1034/90/010033-18$0400 * To whom coriespondence should be addressed

33

34

Frunk und Mendelson

Normal Stretch Reflex muscle

triceps muscle

Reads non-triceps muscle

Figure 1 Schematic diagram of the monosynaptic stretch reflex. Stretch-sensitivesensory afferents projecting peri,pherally to muscle spindles make monosynaptic excitatoni synaptic connections with motoneurons innervating the same (homonymous)and synergistic muscles (both muscles are shaded). Much weaker connections (none are shown) are made with motoneurons supply-ingfunctionally unrelated muscles (unshaded),even when these motoneurons are located in the same region of the spinal cord.

collection of papers in this issue of the .iouriznl of Nezlrobiokgy is that it provides a way of looking at a number of such systems in juxtaposition, thereby facilitating comparisons among them. The particular system described here is the group of sensory and motor neurons whose interconnections give rise to the simple stretch reflex, found in the spinal cord of most vertebrates. As shown in the schematic diagram in Fig. 1, a particular type of sensory neuron (called a muscle spindle afferent) pro-jects to muscles and responds to changes in muscle length. These agerents, whose somata are located in dorsal root ganglia (DRGs), also project into the spinal cord, where they make monosynaptic excitatory connections with motoneurons. These peripheral and central connections result in a negative feedback system, helping the animal resist unexpected changes in muscle length. For example, if a muscle is stretched it activates the spindle afferents and thereby increases the excitatory input to the motoneurons. This extra drive increases the firing frequency of the motoneurons. increasing the tension in the muscle and thus rcsisting the initial stretch. In order for this feedback loop to operate correctly, however, it is critical that the spindle afferents from a particular muscle innervate only the appropriate subpopulation of motoneurons. They make the strongest connections with motoneurons supplying their own muscle (homonymous connections) or a synergistic muscle (for example, another head of the same musclc) and in general much weaker connections with motoneurons supplying functionally unrelated muscles (refer to Fig.

1). The pattern of these connections has been most extensively studied in cats (Eccles, Eccles, and Lundberg, 1957; for review see Burke and Rudomin, 1977) but is essentially the same in frogs (Frank and Westerfield. 1982b: Tamarova. 1977) and birds (Eide, Jansen, and Ribchester, 1982; Lee. Koebbe, and O’llonovan, 1988). Our own studies have focused on the connections made by afferents supplying the triceps brachii muscles in thc forelimb of the bullfrog. Intracellular recordings from brachial motoneurons, as illustrated in Fig. 2. show this pattern. Activity in medial triceps afferents evokes large monosynaptic excitatory postsynaptic potentials (EPSPs) in homonymous medial triceps motoneurons, fairly large EPSPs in synergistic motoneurons innervating the internal and external triceps heads, but quite small (and frequently nonexistent) EPSPs in two species of motoneurons innervating other muscles. On average, triceps sensory input is 5- 10 times larger in triceps motoneurons than in these nontriceps motoneurons (Frank et al., 1982b) despite the fact that all 4 types of motoneurons shown in Fig. 2 are located within the same region of the spinal cord (Lichtman, Jhaveri, and Frank, 1984b). The high degree of specificity in this system makes it an attractive one for developmental studies. Motoncurons are large. making it possible to make intracellular recordings of their synaptic inputs throughout development. The axons of both the presynaptic (muscle afferent) and postsynaptic (motoneuron) cells are located in muscle nerves even before the central synaptic connec-

Deve.lopmmt of Sensor)?-MotorConnecctiom

tions are made (Smith and Frank, 1988a), so they can be identified unambiguously during the recordings, something that is difficult in many other systems in the CNS. Moreover, in frogs and chicks, experimental manipulations can be made at early developmental stages so that the influence of novel peripheral targets or neural activity on the development of. these connections can be studied.

SPECIFICATION OF THE TRICEPS SENSORY PHENOTYPE What determines which particular peripheral and central targets an individual sensory afferent will innervate? For motoneurons, the equivalent decision appears to be made either before or very shortly after a cell has its terminal mitotic division (i.e., is born). Motoneurons at a particular location within the spinal cord send their axons along specific peripheral pathways to innervate a particular muscle (Landmesser, 1980). Even when bits of spinal cord are transplanted to novel locations along the neuraxis. motoneurons within the transplant still tend to project to their normal targets (LanceJones and Landmesser, I98 1 ; Lance-Jones and Landmesser, 19801, showing that the axons must Normal Projections of Medial Triceps Afferents

J-L)-/Medial

Triceps

External Triceps

+-----Subscapularis

Pectoralis

Figure 2 Intracellular recordings from four brachial motoneurons illustrating the synaptic inputs they receive from medial triceps sensory afferents, as diagramed in Fig. l . The strongest projections are made with triceps motoneurons (upper two traces); motoneurons supplying the functionally unrelated subscapular and pectoral muscles (lower-two traces) receive much less triceps input. Calibration pulses at the beginning of each trace in this and subsequent figures are 0.5 mV and 2 ms.

35

already be specified for their targets at the time they exit from the spinal cord. Are sensory afferents similarly specified at early times? Intrinsic Factors

One feature of motoneuronal development that may be important in this process is the time at which a motoneuron is born. Motoneurons in the medial part of the motor column are born before more lateral ones, but motoneurons innervating the same muscle tend to be born at the same time (Whitelaw and Hollyday, 1983). There is also evidence suggesting indirectly that sensory neurons projecting to muscle spindles might be born after those that innervate the skin (Jacobson, 1978). This prompted us to determine when triceps sensory neurons are born in relation to other brachial sensory neurons. To accomplish this, we injected 3H-th ym idi ne (3H-TdR) beginning at specific stages of development and continuing until all sensory neurons were born, about the time of metamorphosis. This procedure labeled all cells born aJev the stage at which the injections were begun. The frogs were then kept for an additional month so that cells within the brachial DRG could be clearly identified as neurons or non-neuronal cells. Finally, triceps sensory neurons were retrogradely labeled with HRP. The basic result was that triceps sensory neurons are born throughout the entire period of sensory neurogenesis; their birthdays do not occur within a restricted window. This is apparcnt from the data shown in Fig. 3 , where the percentage of 3H-labeled neurons (both triceps and other) is plotted against the developmental stage at which ‘H-TdR labeling was begun. The fraction of tt-iceps sensory neurons labeled with 3H at each stage closely parallels that of all other sensory neurons. Thus the time at which a triceps neuron is born does not play a major role in determining its phenotype. An unexpected outcome of these experiments was seen in cases where 3H-TdR injections were begun at later stages when relatively few neuroblasts were still dividing. Although only a few neurons were labeled, they tended to occur in clusters. Figure 4(A) shows 2 examples of the pattern of labeling from a frog in which only 7% of the sensory neurons were labeled. A simple interpretation is that each clustcr represents a “clone,” that is, a group of neurons all derived from a single neuroblast. If this interpretation is correct, it would mean that we have a way of examining the

36

Frank and Menddson 100 Other Brachial Neurons T

0

V

X

xv

xx

Beginning Stage of Thyrnidine Injections

Figure 3 rime course of sensory neuron production in the brachial sensory ganglion of the frog. 3H-thymidineinjections were begun at the indicated stage and continued through Stage XVIII ( n 2 5 for each point) except the 2 right-hand points ( n = 3), where injections were made between Stages XVTII and XXI. Medial triceps sensory neurons, identified by retrograde labeling with HRP, are born throughout the same developmental period as all other brachial sensory neurons. Error bars indicate 1 SD of the mean.

fate of late-stage clones of sensory cells in frogs, much as can be done with retroviral markers in chickens and rats (Sanes, Rubenstein, and Nicolas, 1986; Turner and Cepko, 1987). The interesting observation is that members of such a “clone” can have different fates. Triceps neurons (labeled with HRP) do not occur in clusters, as illustrated in Fig. 4(B). When, on occasion, one member of a “clone” is a triceps neuron, the other members are not. This suggests that even at late stages, neuroblasts are not committed to making a single type of sensory neuron. A likely possibility is that triceps sensory neurons are not clonally related, as has been shown for ganglion cells in the amphibian retina (Holt, Rertsch, Ellis, and Hams, 1988). An alternative idea which our experiments do not rule out is that intrinsically determined lineages of sensory neurons give rise to a variety of ccll types. In fact, for most examples of strict lineage relationships in which neuronal phenotype is intrinsically specified, clonal sisters do assume several different fates (Doe and Goodman, 1985; Sulston, Schierenberg, White, and Thomson, 1983; Weisblat, 1987). More direct experiments are needed to decide between these two alternatives. Extrinsic Factors There is a reason why the possibility of extrinsic, or epigenetic, determination should be seriously considered for sensory neurons. however. As demonstrated first by Le Douarin and her colleagues (LeDouarin, 1982), the fate of neural crest cells (which of course include sensory neurons) is

strongly influenced by the local environment of the cells once they have migrated away from the neural tube rather than by their place of origin. Could the position of sensory neurons along the neuraxis also influence certain aspects of their phenotype? In analogy with the neural crest transplantation experiments, we replaced the brachial sensory ganglion (DRG2) with sensory ganglia from thoracic levels (DRGs 4 and 5 ) in tadpoles (Smith and Frank, 1987). Ifthe manipulation was made sufficiently early [before Stage X (Taylor and Kollros, 1946)], the transplanted sensory neurons survived and projected both to the forelimb and to the brdchial spinal cord, as illustrated schematically in Fig. 5. Intracellular recordings from brachial motoneurons, shown in Fig. 6, demonstrated that the “foreign” sensory neurons that happened to supply the triceps muscle also made direct, monosynaptic excitatory connections selectively with triceps motoneurons. Thus, just as chick neural crest cells transplanted to a novel location along the neuraxis develop phenotypes appropriate for their new location, so sensory neurons transplanted to novel axial levels develop novel but functionally appropriate peripheral and central connections. Many sensory neurons project to their targets in the periphery hdure they make central connections (Ramon y Cajal, 1929; Windle and Orr, 1934; Windle and Baxter, 1936; Vaughn and Gricshaber, 1973; Smith, 1983), and forelimb muscle afferents in frogs are no exception (Smith and Frank, 1988b). These peripheral targets might, therefore, play an important role in determining the central connections of sensory neurons, inde-

Dmdopmcnt of'Scwsory-Motor Connections

pendent of the location of the neuronal somata. Accordingly, we induced developing foreign scnsory neurons to innervate the forelimb without moving their cell bodies, as illustrated schematically in Fig. 7. Sensory neurons in the next adjacent ganglion (DRG3) grow into the forelimb on their own if DRG2 is removed sufficiently early [Fig. 7(A)]. However, DRG4 neurons will do this only if their peripheral nerve is surgically redirected toward the forelimb [Fig. 7(B)]. I n both kinds of experiments, whenever the foreign sensory neurons projected into the forelimb, they also made extensive projections into the gray matter of the brachial spinal cord. Figure 8 shows that foreign (DRG3) afferents supplying the triceps muscles (where HRP was applied) project centrally into the specific region within the brachial spinal cord where normal triceps afferents project. This novel projection is not simply the result of a selective pruning of exuberant larval projections, because at no time during normal development do

37

thoracic afferents ever project significantly into this region (Smith et al., 1988b). And whenever there were sufficient numbers of triceps afferents so their central connections could be checked by intracellular recordings, as in the experiment illustrated in Fig. 9, these foreign triceps afferents selectively innervated triceps motoneurons (Frank and Westerfield, 1982a; Smith et al., 1988a). The interpretation of these results is that the developmental fate of sensory neurons is determined by their peripheral targets. If they supply a particular forelimb muscle, they will innervate the appropriate brachial motoneurons, whereas if they supply muscles o r skin of the thorax, they will not. Sydney Brenner has introduced names for the different ways in which cell phenotypes are determined. In the European Plan, a cell is intrinsically determined on the basis of its lineage. In the American Plan, lineage is irrelevant: cell phenotype is determined epigenetically by environmental factors. According to this naming scheme. sensory

Figure 4 Latc-born brachial sensory neurons tend to occur in clusters, but triceps scnsory neurons do not. (A) 3H-thymidine-labeled sensory neurons (solid circles indicate labeled nuclei in autoradiographs of 5 pm plastic sections) from a frog in which thymidine labeling began at Stage XIKI: 7% ofall brachial sensory neurons were labeled. Ofthe 13 labeled neurons in these 2 sections, 9 occur in clusters. Dashed lines indicatc the edges of nerve bundles coursing through the ganglion. (B) Triceps sensory neurons, retrogradely labcled with HRP, drawn from 50 pm frozen sections from a different brachial ganglion. These neurons are not grouped in clusters. Calibration bar = 100 pm.

38

Frank and Mendelson

Transdant

Normal

Spinal Nerve 4 Figure 5 Schematic diagram (dorsal view) of expenmental preparation used to study the fate of sensory neurons in transplanted ganglia. DRGs 2 and 3 were removed, and DRGs 4 and 5 were transplanted to the brachial level at an early larval stage. The transplanted sensory neurons project peripherally into the forelimb and centrally into the brachial spinal cord. After Smith et al. ( 1 987)

neurons are American but fall under the Academic Plan: their fate seems to be determined by the connections they make. An important question raised in these experiments concerns the nature of the peripheral influence. A likely possibility is that the influence is instructive; phenotypically plastic neurons are specified to be triceps sensory neurons by environmental cues. Alternatively, each sensory ganglion Projections of Transplanted Medial Triceps Afferents

7-J Medial Triceps

/------

.?A!

External Triceps

-

-.-.----.

.-

Subscapularis

Pectoralis

Figure 6 Synaptic potentials elicited by stimulation of transplanted thoracic sensory neurons that now project peripherally to the medial triceps muscles in the forelimb. These foreign sensory afferents, which would never normally project to triceps muscles, selectively innervate triceps motoneurons (upper two traces) but not two types of nontriceps motoneurons (lower two traces), just as in normal frogs.

B

Figure 7 Schematic diagrams (dorsal view) of expenmental preparations used to study the fate of thoracic sensory neurons whose peripheral axons have been rerouted at early larval stages. (A) DRG2 was removed allowing DRG3 afferents to sprout peripherally into the forelimb. (B) DRGs 2 and 3 were removed and spinal nerve 4 was redirected towards the forelimb. In contrast to the preparation shown in Fig. 5 , the position of the sensory cell bodies was left unchanged. After Smith et al. (1988a)

might be formed with a full complement of all possible types of sensory neurons, each prespecified for a particular peripheral and central target. The effect of the periphery, then, would be permissive; a neuron would die unless it found its particular prespecified target. If each peripheral target were unique, it is difficult to imagine how such a hypothesis would work. First, there is no evidence for a large number of specific trophic substances that would selectively save many different populations of sensory neurons. Second, there is a numerical problem. Every ganglion would need to contain cells prespecified for every possible target. Although a large number of sensory neurons do die during development, the fraction is probably no larger than one-half to twothirds (Prestige, 1965), so the excess in each ganglion would be insufficient to supply every possible target. One could imagine an intermediate form of the hypothesis, however, in which certain broad

Development of Sensory-Motor Corinections

classes of sensory neuron were prespecified while instructive environmental effects determined the specific phenotype within each class. As a hypothetical example (for which there is no evidence), specific classes of sensory neurons could be prespecified to project to extensor versus flexor muscles, but the choice of which motoneurons a par-

39

Projections of Transposed Medial Triceps Afferents

Medial Triceps

External Triceps

Pectoralis

Figure 9 Synaptic potentials recorded in brachial motoneurons elicited by stimulation of DRG3 sensory afferents that projected to the medial triceps muscle from a preparation as in Fig. 7(A). Despite the normal location of the thoracic sensory somata, the novel peripheral targets of these afferents resulted in novel but functionally appropriate central connections (compare with traces from a normal frog in Fig. 2).

ticular spindle afferent should innervate would be determined by the muscle it happened to supply. It will be interesting to find out just which aspects of sensory neuronal phenotype are instructively or permissively determined.

INFLUENCE OF NEURAL ACTIVITY ON CENTRAL CONNECTIVITY

Figure 8 Central projections of thoracic afferents (labeled with HRP) that now project to triceps muscles from a preparation as in Fig. 7(A). Sensory afferents on both the control (left) and experimental (right) sides give off collaterals that arborize in the region of triceps motoneuronal dendrites (VNP, ventral neuropil) but not within more dorsal laminae (DNP, dorsal neuropil) where cutaneous afferents terminate (Jhaveri and Frmk, 1983). The three camera lucida drawings were made of transverse 50 pm sections taken 200 pm rostra1 to the triceps motor pool (top), within the triceps motor pool (middle), and caudally at the level of the third dorsal root (DR3; bottom). CC = central canal.

Although our experiments (and others) illustrate the importance of peripheral targets on the development of sensory neuron phenotype, they do not help understand how the targets produce these effects. In certain cases, patterns of neural activity have been found to be crucial in specifying synaptic connections between neurons. Several articles in this issue discuss the importance of neural activity in the development of the visual and neuromuscular systems. We first explored the potential role of activity on the development of the stretch reflex by surgically moving the insertion of a muscle so its spindles would have abnormal patterns of activity. The distal tendon of the medial triceps muscle was transplanted onto the opposite surface of the radial-ulnar bone, thus making the muscle an elbow flexor instead of an elbow extensor, during the time when sensory-motor connections

40

Frurik arid Mmdelson

Regenerated Ventral Root

triceps muscle non-triceps muscle Figure 10 Schcrnatic diagram of the experimental preparation used to study the development of muscle sensory projections to motoneurons in the absence or coordinated motor activity. Resection of the ventral root (X) shortly before the normal period of synaptogenesis resulted in paralysis of the forelimb as the central connections of muscle aKcrciits with motoneurons wcrc developing. Ewn after motor axons had regenerated, limb movements were uncoordinated because the regencration was nonspecific.

were developing centrally. In all cases, the central connectivity of these afferents was normal (Frank and Jackson, 1986), suggesting that abnormal activity patterns were not playing ;I major role. A caveat in the interpretation of these results, however, is that the operation was only technically feasiblc a few stages after sensory-motor synapses had begun to form. Perhaps the “critical period” for an influence of electrical activity had already occurred. Given the large intcrcst in the influence of neural activity on synaptic specificity, it seemed prudent to design more direct experiments. Cut Ventral Root Experiments In one approach, illustrated schematically in Fig. 10, the motor innervation of the forelimb was disrupted in tadpoles by resection of the ventral root just before the time when sensory-motor synapses begin to form [Stage XVII (Frank and Westerfield, 1983; Jackson and Frank, 1987)l. As a result, forclimb muscles had no motor innervation during much of the time these synapses were forming and were consequently often atrophic. Even after the motoncurons had regenerated, they usually did so nonspecifically (Farel and Benielmans, 1986), so the frogs could not movc their affected limb. This procedure should therefore disrupt any coordinated activity of sensory and motor neurons. Intracellular recordings from motoneurons after metamorphosis showed that the central connections of triceps muscle afferents (which had not been surgically manipulated) were functionally inappropriate (Frank, 1987). Motoneurons now innervating triceps ~muscles,which

normally have large triceps EPSPs, frequently received very little triceps input, whereas motoneurons innervating other muscles that normally receivc very little triceps input sometimes had large triceps EPSPs. Further analysis showed that triceps afferents in these frogs were not just making random connections. Rather, they contacted a specific subpopulation of brachial motoneurons. Normally, as shown in Fig. 1 I all types of triceps motoncurons receive input from muscle afferents supplying both the medial and the internal-external heads of the triceps musclc, w-hercas subscapularis and pectoralis motoncurons receive little input from either group of afferents. Therefore the specificity of triceps afferent projections to brachial motoneurons could be checked even though the original identity of each motoneuron was unknown. If these projections were specific even with disrupted motor innervation, then a motoneuron that got significant input from medial triceps aferents (because it was presumably originally a triceps motoneuron) should also have input from other triceps afferents supplying the internal-external heads. In contrast. a motoneuron that had little input from medial triceps afferents (because presumably it was not originally a triccps motoneuron) should also get little from internal-external triceps afferen ts. The data presented in Fig. I2 show this is just what we found, not just for normal frogs as one would expect, but for experimental frogs as well. Motoneurons with significant inputs from medial triceps afl’erents rcceived on average 8-10 times larger inputs from internal-external afferents than those motoneurons with little medial triceps input. ~

Development of’SensoryMotor Connections

Spindle afferents from the subscapular and pectoral muscles did not discriminate between these two populations, just as in normal frogs, showing that the effect was specific for triceps afferents. Muscle afferents can therefore discriminate among different motoneurons in the absence of coordinated sensory-motor activity, arguing instead for a precise chemical recognition between appropriate synaptic partners.

Paralysis of Chick Embryos In a parallel approach, we examined the effects of blocking neuromuscular activity with curare while muscle afferents were making their connections with motoneurons. We used chick embryos for these experiments because tadpoles need to move in order to eat and breathe, and complete neuromuscular blockade would probably be fatal. Curare solutions were applied directly onto the chorioallantoic membrane beginning at St 28 (Hamburger and Hamilton, 1951) as described by Pittman and Oppenheim (1 979). This is before the establishment of direct anatomical and functional connections between muscle sensory and motor neurons (Davis, Frank, Johnson, and Scott, 1989; Lee et al., 1988). The adequacy of the blockade was checked daily by looking for limb movements and at the end of the experiment by counting the number of motoneurons histologically [curare causes a large reduction in the amount of naturally occurring cell death of motoneurons (Pittman et al., 1979)l. Curare applied in this way also blocks

the spontaneous bursts of activity in motoneurons (Landniesser and Szente, 1986). At Sts. 38-40, synaptic potentials were recorded intracellularly from identified brachial and lumbosacral motoneurons in response to stimulation of individual muscle nerves. In both normal and experimental embryos, large EPSPs were evoked in homonymous motoneurons and were often observed in synergistic motoneurons as well. In contrast, excitatory potentials were uncommon in antagonistic motoneurons; instead inhibitory inputs were often seen. Figure 13 shows examples of synaptic potentials recorded from brachial motoneurons in a normal and a curare-treated embryo that illustrate our preliminary results (Mendelson and Frank, 1989). So far we have seen no effect of the blockade on the specificity of these connections. Thus neither normal patterns of neural activity nor motoneuronal cell death appears to play a critical role in the development of specific connections between muscle sensory and motor neurons.

COMPETITIVE INTERACTIONS AMONG AFFERENTS Relatively little is known about factors that regulate the amount of innervation a particular target receives during development. Peripherally, how many motor and sensory neurons supply a particular muscle? And centrally, what determines the strength of synaptic connections between a muscle afferent and its target motoneurons? In certain

Medial Triceps Afferents

r-4’

Internal-External Triceps Afferents

Medial Triceps

.

41

External TriceDs Motoneuron Subscapularis Motoneuron Pectoralis Motoneuron

+

Figure 11 Synaptic potentials recorded from brachial motoneurons in response to stimulation of sensory afferents projecting to the medial (left) and internal-external (right) heads of the triceps muscle. Both groups of triceps afferents innervate both homonymous and synergistic triceps motoneurons much more strongly than either group innervates nontnceps motoneurons (lower two pairs of traces). This normal pattern of connectivity allows one to test the specificity of these connections in experimental preparations like that shown in Fig. 10.

42

Frank and Mendelson

A1.4

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Normal

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9 Frogs

5 Frogs

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< 0.2 rnV D 0.2 mV M. Triceps Sensory Input (mV)

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Figure 12 Correlations of different muscle sensory inputs to brachial motoneurons in normal frogs (left) and frogs like those in Fig. 10 with regenerated ventral roots (right). Motoneurons were divided into two groups based on the amplitude of the synaptic input they received from medial triceps (M. Triceps) afferents. The average amplitude of synaptic input from another group of muscle afferents to motoneurons in each group was then calculated and is shown on the ordinate. The number of motoneurons sampled is indicated for each group; error bars show 1 SE of the mean. (A) Motoneurons in both groups of frogs with significant (>0.2 mV) input from medial triceps afferents had a much larger input from internal-external triceps afferents than those that did not. The two groups of triceps arerents thus selected the same subpopulation of motoneurons, even in frogs whose ventral roots had been cut. (B) Inputs of subscapular pectoral versus medial triceps muscle afferents to brachial motoneurons were not correlated with each other. either in normal frogs or in frogs with regenerated motor axons. Motoneurons with little input from medial triceps afferents were just as likely to receive significant input from subscapular pectoral afferents as motoneurons with large triceps input. This control experiment shows that the results presented in A are not simply because some motoneurons are innervated by all muscle affercnts while othcrs are not. Rather, triceps afferents innervated a distinct subpopulation of brachial motoneurons.

+

+

cases in the peripheral nervous system, the number of cells innervating a particular target may be regulated by competition for a trophic factor (reviewed by Purves, 1988) or for postsynaptic target space (Johnson, 1988; Maelen and Nji, 1984). Centrally, the dramatic effects of monocular deprivation on the developing visual cortex (Wiesel and Hubel, 1963) also require some form of interaction between inputs from the two eyes. In frogs, one can test directly the importance of numbers of afferent fibers on the development of both peripheral and central projections of these cells. The surgical manipulations we used to cause

foreign sensory neurons to supply the forelimb also resulted in a large reduction in the total number of sensory neurons. This gave us an opportunity to see if these reductions ( 1- 16 medial triceps afferents instead of the normal 30-40) would influence quantitative aspects of these neurons' projections. Peripherally, the medial head of the triceps muscle is normally supplied by twice as many sensory afferents as the internal-external heads combined, even though each of the three heads is of similar mass and is supplied by similar numbers of motoneurons (Lichtman et al., 1984b; Mendelson

and Frank, in press). If the number of afferents innervating each head were determined by competition for a trophic substance, then a large reduction in the total number of afferents would result in each head receiving about one-third of the total triceps sensory axons, as indicated by the shaded line in Fig. 14. Instead, we found that approximately two-thirds of these axons prqjected to the medial head alone, just as in normal frogs. even when the total number ofafferents was small. Some mechanism other than competition for a trophic substance therefore appears to regulate the proportional sensory innervation of each triceps muscle head. Ccntrally, triceps EPSPs recorded from brachial motoneurons were smaller than normal, as might be expected from the smaller number of triceps afferents. The traces on the right side of Fig. 15. for example, were recorded from motoneurons in a frog that had only eight medial triceps afferents, about one-quarter the normal number. Equivalcnt traces from a normal frog are shown to the left, at one-quarter the gain. The average pmjection from single triceps afferents to each motoneuron (callcd the unitary EPSP and estimated here by dividing the composite EPSP amplitude by the number of triceps afferents) was, surprisingly, no larger than normal. The rcsults from 14 frogs yielding 21 cxNormal Animal

amples of synergistic triceps EPSPs are presented in Fig. 16. Each unitary amplitude is plotted against the number of afferents in that triceps muscle nerve. In normal frogs, synergistic unitary triceps EPSPs are about 50-100 pV (indicated for comparison with a dashed horizontal line in F-ig. 16; Lichtman and Frank, 1984a). In 12 ofthe examples. these unitary potentials were within the normal range even when there were only 2 or 3 triceps atferents. The estimates were larger than normal in the other nine cases, although the uncertainty in these estimates was large because a rather small number of motoneurons were sampled. Our tentative conclusion is that large reductions in the number of afferents need not lead to compensatory increases in the number and/or strength of connections made by each afferent despite a presumably large incrcase in ccntral target space. Rather. the number of central contacts made by individual fibers may be an intrinsic property of muscle sensory neurons. Finally, the reduction in number of afferents did not impair the specificity of their central conncctions, as was already shown implicitly for two single cases in Figures 6 and 9. As shown in Fig. 17, triceps sensory afferents consistently made stronger connections with triceps than with nontriceps (subscapularis and pectoralis) motoneuAnimal Treated with Curare

Triceps -+ Triceps

4

f'bIceps+T&ps

/

Slceps-rTriceps

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Triceps + Ulnarls

Biceps -+ Ulnarls

L. Figure 13 Intracellular recordings from three brachial motoneurons in a normal (left) and curare-treated (middle and right) chick embryo. Stimulation of' triceps muscle afferents elicited short latency (presumably monosynaptic) EPSPs in both normal and curare-treated triceps motoneurons. Stimulation of atferents in the antagonistic biceps muscle. however,

elicited a longer latency (presumably disynaptic) inhibitory potential (IPSP). The biceps IPSP in the middle traces was initially hyperpolarizing, but its polarity quickly reversed as is common for lPSPs in this preparation. Recordings from a nearby motoneuron projecting out the ulnar nerve (right) showed that it received no triceps input but did receive short latency excitatory biceps input. Muscle atferents thus make highly- selective connections with motoneurons even when limb movcmcnts are blocked with curare.

44

Frank and Mendelson

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8

10

12

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Total Number of Triceps Sensory Neurons

Figure 14 Ratios of sensory projections to the different heads of the triccps muscle in frogs with reduced numbers of afferents. The numbers of sensory neurons projecting in each muscle nerve were determined by recording from the cut peripheral end of the dorsal root with a suction electrode while gradually increasing the stimulus strength to individual triceps ncrves (Lichtman et al., 1984a: Smith et al., 1988a). The proportion of triceps neurons that innervated the medial head is plotted against the total number of triceps sensory afferents in each frog. The line at 0.33 shows the proportion that would have projected to the medial head if

each head had received equal numbers of afferents. Although the ratio for individual frogs is unreliable for small numbers of afferents (because of sampling error), the average ratio for all frogs at each number is greater than 0.5.

rons even when the number of afferents was small. In these cases, comparably few afferents innervated other forelimb muscles as well (data not shown; refer to Smith et al., 1988a). A single tri-

Normal Animal (33 Afferents)

M Triceps

DRG2 Removed

(8 Afferents)

M Triceps

M Triceps --+ iJE Triceps ?.

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.;r“““----. M Triceps -+ Subscapularis

Figure 15 The strength of medial triceps sensory input to brachial motoneurons is proportional to the number of triceps afferents. Stimulation of the approximately 33 afferents in a normal frog (left) evoked EPSPs that were approximately 4 times larger than in a frog with only 8 triceps afferents (right). Note that the normal traces are shown at one-quarter the gain. The strength of the synaptic connections made by each spindle afferent was therefore approximately normal, despite the fact that there was presumably more dendritic target space for each afferent.

ceps sensory fiber not only innervates triceps motoneurons but apparently avoids contact with, for example, pectoralis motoneurons despite the absence of “competition” from the normal number of pectoralis sensory afferents. These data, taken together with the absence of effects of neural activity, suggest a direct chemical affinity between a muscle afferent fiber and its appropriate synaptic partners that is largely independent of interactions with other inputs.

COMPARISONS WITH PROJECTIONS OF RETINAL NEURONS The picture that emerges from these studies is that at early times the determination of a muscle afferent’s phenotype is highly dependent on the environment encountered by its growing axon. Later, however, the neuron appears to be rather rigidly determined, making specific central connections independently of its electrical activity or of competitive interactions with other sensory neurons. As a way of focusing on the key features of the development of m usclc spindle afferents and their projections to the spinal cord, a comparison will be made with the development of retinal ganglion cells (RGCs), another group of neurons that project in a highly specific way to the CNS and which have been studied in considerable detail.

Development of Sensory-Motor Connections

F

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800

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.

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Total Number of Triceps Afferents

Figure 16 Average unitary triceps EPSPs in synergistic triceps motoneurons from 14 frogs with reduced numbers of triceps afferents. The unitary EPSP amplitude is plotted for each synergistic triceps connection (that is, m. triceps + i.e. triceps or i.e. triceps + m. triceps) against the number of afferents in each triceps muscle nerve (see text for details). The average projection in normal frogs (Lichtman et al., 1984a) is shown for comparison as a dashed line. Large reductions in the number of afferents usually resulted in no significant increase in unitary EPSP amplitude. Error bars show 1 SE of the mean.

Lineage

1988). A similar conclusion probably applics to the mammalian retina. Clones identified with a retroviral marker contain many different cell types (Turner and Cepko, 1987), although ganglion cells were not labeled because the virus was injected postnatally, after all ganglion cells are born. Our results are compatible with these findings, suggest-

In the amphibian retina, individual stem cells do n o t give rise to pure populations of RGCs. Injection of lineage dyes into single blast cells shows instead that a single clone can contain all types of retinal cells (Holt et al., 1988; Wetts and Fraser.

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Number of Triceps Sensory Fibers

Figure 17 Specificity of projections of triceps sensory afferents to triceps versus nontriceps motoneurons in 10 normal frogs and 19 frogs with reduced numbers of afferent fibers. For each frog, the preference of triceps afferents for triceps over nontriceps (subscapularis pectoralis) motoneurons was calculated as a specificity index:

+

x- Y SI G Y

x+

where X = average triceps EPSP amplitude in triceps motoneurons, and Y = average triceps EPSP amplitude in nontriceps motoneurons. The corresponding values of X / Y , which gives a more intuitive measure of synaptic preference, are shown on the axis to the left. Triceps afferents show an obvious preference for triceps motoneurons even when their total number is very small.

46

Frank and Mendelson

ing that triceps muscle afferents are not clonally related. This is based on the interpretation that clusters of late-born neurons represent clones, however, an assumption that remains to be proven. We are currently exploring this question more directly in the chick embryo where retroviral markers are available (Frank and Sanes, 1989). But it appears likely that in neither system is a cell’s fate specified uniquely by its lineage. Importance of Cell Position The retinotectal system is probably best known to developmental neurobiologists for its central role in thc classic experiments by Sperry (Sperry, 1943a,b) showing by behavioral testing that the RGCs in an inverted retina reestablished connections with the tectum that were appropriate for the original orientation of the eye. On the basis of these experiments, Sperry proposed that synaptic connections between the retina and brain were specified chemically (Sperry, 1963), and that the RGCs retained these chemical labels even when transplanted to novel locations. Later, Jacobson (Jacobson, 1968) found the same result when eyes were rotated in Xenopus tadpoles, before a significant number of retinal fibers had made contact with the tectum. Recently Fraser (Fraser, 1987) has shown that even when a single postmitotic retinal cell is placed in an ectopic location, it still projects to the tectum according to its original position despite the fact that all of its neighbors are projecting elsewhere. These findings make a strong case for the idea that the positional (i.e., dorsal, ventral, nasal, or temporal) phenotype of RGCs is determined at the time of transplantation. In contrast, the central projections of spinal sensory neurons are highly dependent on their choice of peripheral targets. Sensory neurons in thoracic ganglia made to supply muscles in the limb make novel but functionally appropriate projections to limb motoneurons. If the results from surgically manipulated tadpoles can be extrapolated to normal development, then the precise central projections are a consequence of the particular peripheral target a sensory neuron happens to contact. At first, this would seem to be a real difference between the two systems. The manipulations were made, however, at different times during development. Retinal ganglion cells in Xenopus are born between Stages 29 and 41 (Holt et al., 198&),and the manipulations demonstrating a fixed neuronal phenotype were performed after Stage 31 (Jacobson, 1968) or after the single transplanted cell had

had its terminaI division (Fraser, 1987). Spinal sensory neurons in bullfrogs are born much later, so that approximately 50% are born after Stage V (refer to Fig. 3). Thoracic sensory neurons grow into the forelimb most successfully if the manipulations are done around this stage, and 3H-TdR labeling begun at the time of DRG transplantation shows that a large fraction of the transplanted neurons are born after that time (Smith and Frank, 1988~).In the retinotectal system, when eye rotations are made before the majority of retinal cells have been born, RGCs project to the tectum according to their new position (Jacobson, I968), in analogy with the plasticity of transplanted DRG neurons. It will be interesting to find ways of forcing DRG neurons to supply foreign targets at later times to see if their phenotype, too, eventually becomes fixed. Anatomical Precision of Initial Central

Projections In both systems, the neurons project directly to the appropriate region of the CNS. Axons of RGCs in lower vertebrates project through the optic tract and arborize in the correct portion of the tectum from the outset (€jolt and Harris, 1983). Nor does this projection depend on competitive interactions among axons from different parts of the retina. When the temporal retina is ablated before axons begin to grow, RGCs in the nasal half bypass the vacant rostra1 tectum to arborize correctly in the caudal half (Stuermer, 1988). The retinal projection to the lateral geniculate nucleus (LGN) in mammals is somewhat more complicated; in addition to being topographically arranged, afferents from the two eyes project to different layers. Axons of single RGCs initially have very simple arbors in multiple layers and then add extensively to the arbor in the correct layer (Sretavan and Shatz, 1984; see also Shatz, 1990). In the brachial region of the frog’s spinal cord, which is only one segment long, there is no equivalent topography representing the external world. and individual muscle afferents arborize over nearly the full rostrocaudal extent of the brachial motor column (Lichtman et al., 1984b).Nevertheless, these afferents terminate in the appropriate dorsoventral laminae well before they establish synaptic contacts with motoneurons (Jackson et al., 1987; Liuzzi, Beattie, and Bresnahan, 1985). Moreover, as shown in Fig. 8, muscle afferents supplying the forelimb arborize in the appropriate location even with the large reduction in afferent input to the spinal cord caused by removal of the brachial DRG.

D t w h p m e n t of Sensory-Motor Connections

Role of Neural Activity in the Formation of Synaptic Connections The initial topographic projections in the retinotectal system do not depend on normal patterns or level of electrical activity (Harris, 1980). Ventral retina still projects normally to dorsal tectum and vice versa when action potentials In RGCs are blocked pharmacologically. However, activily appears to be important in refining this projection. The most complete physiological evidence conies from studies of optic nerve regeneration in fish. where intraocular injections of tetrodotoxin (TTX, which blocks Na-dependent action potmtials) lead to a lack of precision in the regenerated map (Meyer, 1983; Schmidt and Edwards, 1983). One of the more dramatic anatomical demonstrations of the importance of impulse activity during development of the retinotectal system comes from sludies of artificial “ocular dominance columns” produced by forcing two eyes to innervate a normally monocular tectum (Constantine-Paton and Law, 1978; see also Debski and ConstantinePaton, 1990). Chronic application of TTX prevents the formation of thcsc columns (Reh and Constantine-Paton, 1985) by a mechanism thought to involve some aspect of synaptic transmission between the presynaptic RGCs and the postsynaptic tectal cells (Cline, Dcbski. and Constantine-Paton, 1987). Neural activity has also been found to be important for the development of normal synaptic relationships in the mammalian LGN. The segregation of retinal afferents into distinct layers is prevented by chronic administration of TTX during late fetal life in kittens (Shatz and Stryker, 1988). Postnatal impulse blockade of RGC leads to dramatic changes in the response properties of LGN neurons (Dubin, Stark, and Archer, 1986). Normally, these cells are monocularly driven and respond only to the presentation or withdrawal (but not both) of a visual stimulus (ON or OFF cells). After 5 or more weeks of intraocular TTX injections, however, many cells respond to input from both eyes and have both ON and OFF response$. It is unclear whether this represents the disruption of synaptic specificity that was present from the outset or the blockade of a normal process that shapes an initially imprecise pattern of connectivity into the normal adult pattern. Dubin et al. (1986) report that LGN cells in normal kittens (at or shortly after the time when TTX injections were begun) were monocular and either O N or OFF (not both), arguing for a disruption of specificity already present by birth. Retinal affer-

47

ents first begin to contact LGN neurons during fetal life, however, and Shatj. and Kirkwood (1984) found that many LGN neurons could be driven by both eyes just after birth. It seems likely that the initial pattern, although topographically correct. undergoes substantial refinement during subsequent development. The synaptic connections mcdiating the stretch reflex, in contrast, show a high level of specificity from the earliest times that monosynaptic inputs from muscle afferents to motoneurons can be recorded (Frank et al., 1983). Triceps afferents project selectively to triceps motoneurons and not to subscapularis or pectoralis motoneurons. And, as described in this report, major disruptions of pattcrned activity in muscle afferents lead to no obvious changes in this central connectivity. In this system there appears instead to be a strong chemical afinity between appropnatc presynaptic and postsynaptic partners that operates right from the beginning when these synapses first begin to form.

CONCLUSIONS This comparison shows that RGCs and muscle affercnts have many developmental features in common. Both appear to arise in a lineage-independent manner. Their phenotypes are probably determined shortly after they are born by the environment near their inputs, which for RGCs is near their cell bodies and for muscle afferents at or near their peripheral target muscle. The central anatomical projections of both ncuronal types are largely appropriate from the outset. RGCs form a reasonably ordered topographic map on the tectum, and muscle afferents arborize within the appropriate laminae of the spinal cord. At that point, however, the two systems apparently diverge. The initial pattern of retinal projections is further refined, both in terms of topography and receptive field properties of the postsynaptic neurons. Moreover, this refinement occurs in an activity-dependen1 manner. But no such rearrangements are obvious in the connections underlying the stretch reflex, and abolishing patterned activity does not disrupt these connections. Why might synaptic rearrangement be important in one system but not in another? A possible explanation comes from a consideration of the nature of the postsynaptic neurons at the time they are receiving their synaptic inputs. There is no reason to suspect that LGN neurons that will eventually be driven by one or the other eye or will be ON or OFF type are initially differ-

48

Frank and Mendelson

ent from one another. As an extreme example, in the case of a tectum made artificially binocular, the tectal neurons now driven by the extra eye are unlikely to have come from a special, predetermined population. Instead. functionally appropriate synaptic connections can only begin to evolve once the initial projections (and perhaps connections) have been made. At that point, competitive interactions among RGCs carrying different information (left versus right eye, O N versus OFF, or similarity of receptive field position) can begin so that individual postsynaptic cells are innervated by functionally similar inputs. For many such systems, these competitive interactions are apparently mediated via neuronal activity. The postsynaptic neurons in the stretch reflex are quite different, however. Distinct populations of motoneurons are already specified to project to particular muscles shortly after the motoneurons are born, well before they begin to receive direct inputs from muscle afferent fibers. Muscle afferents, too, have already grown to their peripheral target muscles before synaptogenesis begins. It is therefore possible to predict in advance the specific subpopulation of motoneurons to which a muscle afferent should project, just as one can predict which muscle will be innervated by a particular group of motoneurons. In such a system, synaptic patterns can be rigidly prespecified, and competitive mechanisms for subsequent refinement of these patterns may not be necessary. It is a pleasure to acknowledge the valuable contributions of Drs. Patrick Jackson, Sonal Jhaveri, Jeff Lichtman, Carolyn Smith, and Monte Westerfield to the experiments described here. Dr. Lichtman also provided useful comments on the manuscript. This work was supported by grants from the National Institutes of Health and the National Science Foundation.

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sacral sensory neurons in the chick. J. Cbmp. Neurol. 279:5 56-566. DEBSKI,E., and CONSTANTINE-PATON, M. (in press). Activity-dependent tuning and the NMDA receptor. J. iVeurobiol. 21:18-32. DOE, C., and GOODMAN,C. ( I 985). Early events in insect neurogenesis. 11. The role of cell interactions and cell lineage in the determination of neuronal precursor cells. Dev. Bid. 111:206-2 19. DUBIN, M. W., STARK,L. A., and ARCHER,S. M. (1986). A role for action-potential activity in the development of neuronal connections in the kitten retinogeniculate pathway. J. Neurosci. 6: 1021- 1036. ECCLES,J . C., ECCLES,R. M.. and LUNDBERG,A. (1957). The convergence of monosynaptic excitatory afterents onto many different species of alpha motoneurones. J . Physiol. (London) 137:22-50. EIDE, A,-L., JANSEN? J. K. S., and RIBCHESTER, R. R. (1982). The effect of lesions in the neural crest on the formation of synaptic connexions in the embryonic chick spinal cord. J. Phj~sio/.(London) 324:453-478. FAREL, P., and BEMELMANS, S. (1 986). Restoration of neuromuscular specificity following ventral rhizotomy in the bullfrog tadpole, Ranu catesbeianu. J . Comp. Neurd. 254:125-132. FRANK,E. (1987). Specific synaptic connections between muscle sensory and motor neurons form in the absence of coordinated patterns of muscle activity. Neurosci. Abstr. 13:1456. FRANK, E., and JACKSON,P. C. (1986). Normal electrical activity is not required for the formation of specific sensory-motor synapses. Brain Res. 378: 147151. FRANK, E.. and SANES,J. (1989). Cell lineage in chick sensory ganglia studied with a retroviral marker. Neurosci. Absfr. 15:600. FRANK, E., and WESTERFIELD. M. (1982a). The formation of appropriate central and peripheral connexions by foreign sensory neurones of the bullfrog. J. Physiol. (London) 324:495-505. FRANK, E., and WESTERFIELD, M. ( 1 982b). Synaptic organization of sensory and motor neurones innervating triceps hrachii muscles in the bullfrog. J. Phy,siol. (London) 324:479-494. FRANK, E., and WESTERFIELD, M. (1983). Development of sensory-motor synapses in the spinal cord of the frog. J . Physiol. (London) 343:593-6 10. FRASER,S. ( 1987). Intrinsic positional information guides the early formation of the retinotectal projection of Xenopus. Neurosci. Ab.str. 133368. HAMBURGER, V., and HAMILTON,H. C. (1951). A series of normal stages in the development of the chick embryo. J . Moryhol. 88:49-92. HARRIS,W. A. (1980). The effects of eliminating impulse activity on the development of the retinotectal projection in salamanders. J. Comp. Neurol. 194:303-317. HOLT, C., BERTSCH,T., ELLIS,H., and HARRIS,W. (1988). Cellular determination in the Xenopus retina

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