The Development of the Lamprey Pattern Generator for Locomotion Avis H. Cohen,*t Tamara A. Dobrov, Guan Li,t Timothy Kiernel,* and Margaret T. Baker Section of Neurobiology and Behavior, Cornell University, Ithaca, New York 14853, USA

SUMMARY The life cycle of the lamprey includes a larval stage that can last for several years. The motor behavior of the larval lamprey, the ammocoete, has been only minimally studied and little is known of the neural correlates of that behavior. Comparison of known larval behavior to that of adults leaves unclear whether there are large or small changes in t h e spinal nervous systcm during transformation. The motor output of isolated larval and transforming spinal cords when stimulated to “swim” with D-glutamate has some differences from that of

comparable adult preparations, but shares many important features with adults. Primarily, the fictive swimming is less well regulated and less stable than adults of the same species. We propose that a major difference in the structure and organization of the central pattern generator for locomotion between adults and ammocoetes is a relative lack or immaturity of some cell types that participate in the coordination of the segments and the generation of the rhythm of the periodic bursting.

INTRODUCTION

species of our study), probably spends 5-6 years as an ammocoete (Hardisty and Potter, 1971). In contrast. I . micuspis spends an estimated 3 larval years followed by an extended juvenile and feeding adult stage (H. A. Purvis. personal communication in Hardisty and Potter, 197 1). A developmental strategy in which ammocoetes remain protected in the silt may well afford them a selective advantage as the only limitations on their growth are those imposed by the mechanics of their microphagous feeding processes rather than the availability of food resources. Within 1-3 weeks following hatching (Potter, 1980) the so-called proammocoetes emerge from the nest, move downstream, and after quickly finding an acceptable habitat, burrow themselves into fine mud where they remain concealed, exiting to swim only briefly at night. At this stage and for some time thereafter larvae are vulnerable to predators and have been found in large numbers in the gut of several fish species (Potter, 1980). Field observations on ammocoetes of different sizes suggest that smaller animals are also quite vulnerable to the effects of flooding (Manion and Smith, 1978). In any case, larvae tend to disperse in a predominately downstream direction. Although ammocoetes appear capable of swimming quite

Ammocoetes remain at their stage of development for such a long time and their metamorphosis is so prolonged and gradual that it was not until the nineteenth century that they were established as the larval stage of lampreys (Muller, 1856 in Hardisty and Potter, 1971). The fact that lampreys have survived so well through geologic time and are presently so widely distributed (Hardisty and Potter, 197 1 ) owes much to the length of the ammocoete stage. On the basis of a comparison of closely related “paired species” Hardisty ( 1979; Hardisty and Potter, 197 I ) has even suggested that the larval life span lengthens during phylogenetic development (Youson, 1988). Ichthyornyzon J;?ssar, for example, a nonparasitic form thought to derive from the ancestral parasite, 1.unicuspis (the Received June 23, 1990: accepted June 26, 1990 Journal of Neurobiology, Vol. 21, No. 7, pp 958-969 (1990) C 1990 John Wiley & Sons, Inc. CCC 0022-3034/90/0709S8- 1 2W4.00 * To whom correspondence should be addressed. Present address: Department of Zoology, Ilniversity of Maryland, College Park, Maryland 20742. i:Presenl address: Center for Complex Systems, Florida Atlantic University, Boca Raton, Florida 3343 I .

958

Lamprey Puttern Generator

rapidly, it is unlikely that thcy can sustain such speed for long (Potter, 1980). This relative weakness stands in distinct contrast to the endurance of adult lampreys which swim with a very high efficiency ratio U/V ((Williams, 1986: McClellan and Grillner, 1983) where U = forward velocity of the animal and V = the backward velocity of the wave with respect to the head). Thus the adult has evolved, above all. to be efficient (Williams et al., 1989), a quality which is no doubt valuable during the latter phase of life when lampreys must swim upstream against fast currents, spawn, and build nests-all after they’ve stopped feeding! As larval lampreys grow in size, they exhibit behavioral and morphological changes suggestive of an increase in swimming efficiency. Tagging experiments show that the breadth of distribution away from the nest is proportional to the lampreys’ G7c (Potter. 1980). Larger animals, moreover, exhibit a preference for a coarser substratum (Beamish and Medland, 1988), with land-locked species preferring slightly deeper waters. Although the size and age at which an ammocoete begins to transform depends on its species and condition (Purvis, 1979, 1980), larval growth, in general. is characterized by an overall increase in length and certain changes in body proportions. Following their departure from the nest, young ammocoetes of 3-7 m m will often grow steadily to reach a total length in excess of 100 mm . The prebranchial, branchial, and middle trunk regions, on the other hand, decrease in relative length (Beamish, 1982; Beamish a nd Austin, 1985). Other physical changes that have been described include: (1) an abrupt increase in relative tail length in the first year of larval life followed by steady growth for its remainder; (2) relative body depth increasing in the dorso-ventral plane at the cloaca and dorsal fin and gradually decreasing at the third branchiopore; (3) progressive rearrangement in muscle fiber orientation within myotomes (Youson, 1980); and (4)an increase in dorsal fin height in some advanced ammocoetes. Overall, these changes lead to improved hydrodynamic efficiency. Thcre have been no investigations to date of the alterations in the nervous system in response to these physical changes during the larval phase. When considering the precise matching process in adults between neural activation and mechanical trav e I i ng waves (determ i n ed by the natural frequency ofthe body as an oscillator: Williams et al., 1989), it seems possible that larval motor activity reflects ongoing adjustments required by an evolving physical form. As new motoneurons differen-

959

tiate, for example, they must form connections with parts of the spinal nervous system. Other changes may alter the relative roles of movementrelated feedback and descending control systems which affect pattern-generating circuitry. These changes may aid in the tuning of spinal neural circuitry. Metamorphosis enables lampreys to withstand a move into salt water (anadromous spp.), to prey on fish (parasitic forms), and to swim upstream to build nests and spawn. This prolonged transformation introduces external morphological changes that further decrease hydrodynamic drag and enhance propulsive thrust: for example, height increase in dorsal fin (Beamish and Austin, 1985) and associated muscle volume increase (Rovainen and Birnberger, 1971), increase in general body depth and decrease in girth in the region of the branchial basket. We might expect remarkable internal changes to attend the transformation of an ammocoete, burrowing. blind, and suspensionfeeding in a freshwater stream, to a juvenile with suctoral mouth, rasping tongue, salivary anticoagulant, and well-developed eyes as well as a new form of ventilation and innumerable changes to the digestive. excretory, and respiratory systems. From the onset of larval life through metamorphosis, however, changes in motor control, especially at the level of the spinal pattern generator. are difficult to predict and until this time virtually uninvestigated. In contrast to the prediction of dramatic changes, it could equally well be argued that little change attends transformation. One report has suggested that thc swimming movements of ammocoetes a n d adults are essentially identical (Ayers, Carpenter, Currie, and Kinch, 1983). In general, our limited knowledge suggests that the adults primarily swim through a uniform medium and rarely if ever burrow, whereas ammocoetes primarily burrow through non-uniform media and swim only briefly (Hardisty and Potter, 1971). However, both adults and ammocoetes appear able to perform the movements ofthe other. Given the similarity in the animals’ shape and motor patterns. it is possible that during transformation the spinal cord simply grows in size with the only changes being in the brain’s organization and responses to sensory and hormonal stimuli. This is especially plausible given Rovainen’s observation that the total numbers of cells in the spinal cord is unchanged during transformation (Rovainen, 1979). The possibility of simple expansion can be ruled out by our studies of the isolated spinal cords of

960

Cohen et al.

adults and ammocoetes in a single species, Zchthyomyzon unicuspis. As we will demonstrate, the motor outputs of the isolated spinal cords of lampreys at different life stages are very different when they are activated by bath application of D-glutamate. The major difference is in the stability of the motor output, but the temporal pattern of the bursting is also considerably changed. The differences in the motor pattern cannot be accounted for by a simple expansion of the spinal cord; some qualitative changes are also suggested. For purposes of the present discussion, we will deduce the necessary developmental changes in the spinal cord by a close comparison of the motor outputs of the spinal cords of ammocoetes, transformers, and adults. To date our research has centered around the locomotor output of the isolated spinal cord. Consequently, we will focus our discussion on the characteristics of “fictive locomotion.” Therefore, for now we will only address the intrinsic organization of the neuronal circuitry of the spinal cord and will not discuss the interactions of this circuitry with either the brain or the sensorium. This does not reflect the relative unirnportance of these interactions, quite the contrary. As will become apparent, our results demonstrate the necessity of sensory and/or descending inputs in shaping the animal’s adaptive behavioral repertoire. The limitation of the discussion is more a reflection of our present ignorance.

ADULT MOTOR PATTERN We will begin with the adult, the best characterized of the life stages. In normal intact lampreys, swimming consists of a sequence of traveling waves that progress down the animal in such a way that the body retains approximately one wavelength regardless of the speed of swimming or frequency of the traveling waves (Williams, 1986). The underlying muscle activity observable during normal swimming in lampreys is similar to that for other anguilliform fish as was first described by Grillner and Kashin (1 976) in dogfish. The motor pattern consists of a wave of periodic muscle contractions that progresses down the animal’s body. During regular swimming, the body always assumes a single Wavelength and this results from the close relationship between the neural and the mechanical waves (Williams et al., 1989). In the neural wave, the single wavelength is reflected in the temporal pattern of activity among the segments. The ventral root output in one segment is followed by activity in the next segment by a delay equal to 1% of

the cycle period regardless of the speed of swimming. The accumulated delays among the roughly 100 segments thus produce one wavelength per cycle. The capacity to produce such temporal constancy exists in isolated lamprey spinal cords even though there is a complete absence of sensory and descending input (Cohen and W a l k 1980: Wallkn and Williams, 1984). In such a reduced preparation, the temporal pattern of the so-called “fictive locomotion” can be well regulated shortly after drug application [Fig. l(a)] and will remain so until the drug is removed. (For the present discussion, we define cycle period as the time from one burst onset to the subsequent burst onset in the same segment and the phase as the delay from the rostral to the more caudal segment divided by the period of the rostral segment.) The stability of the fictive motor pattern is evidenced by two analyses of the activity, the cycle periods when plotted over time and the histograms of the relative phases between two segments [cf. Fig. 2(a)]. In adult bursting, the periods 10 min after drug application are generally relatively stable (cf. Table 1 for the range of means and coefficients of variation) and the phases are tightly clustered about some preferred value. Thus the adult spinal cord has a strong intersegmental coordinating system capable of rapid and reliable entrainment of the segments even in the complete absence of sensory and descending inputs (Cohen, 1987). The adult locomotor pattern generator is organized and distributed in such a way that all portions of the spinal cord are equally capable of producing a stable fictive motor pattern. Isolated spinal cord pieces as long as 60 segments or as short as 10 segments all share this characteristic. The minimum number of segments found to generate a reasonable pattern is four (Cohen and Wallkn, 1980); fewer than this have not yet been shown to suffice. Cord pieces of 10-12 segments cut from the same spinal cord will differ insofar as their periods are concerned. All their periods will typically be similar in magnitude to those of much longer pieces [Figs. l(a), 3(b)] although it is not uncommon for any one of the pieces to be less stable than the others even when all are subjected to identical conditions. The organization of the central pattern generator (CPG), which is believed responsible for the adult fictive locomotor pattern, is basically that of a chain of segmental oscillators coupled along the cord by coordinating fibers in the medial regions and the lateral tracts. There are relatively short coordinating fibers in both lateral and medial por-

Lumpre)>Puttern Generator 12.0289 Adult Control

961

4r-----7

25.01.90 Early Transformer Control

ic

c 0

ZOO

I00

Time

0

I00

200

Is)

300

400

Time (s)

12.01.89 Ammocaele Control

24.12.89 Late Transformer Conlrol

1

& *

-. 0

loo

100

Time 1s)

loo

200

3CO

Time 1s)

Figure 1 Pattern of variation of pcriod over time for diffcrent life stagcs for thc most stable bursting for that life stage. Period is plotted over time to give a visual impression of the variability. In each such plot, note the scale. For these analyses, the bursting for the ammocoete was allowed to stabilize for 60 min. The adults required only 10 min or less. (a) Adult. (b) Ammocoete. (c) Early transformer. (d) Late transformer.

tions of the spinal cord and long fibers only in the lateral tracts (Fig. 4) (Cohen. 1987; 1988). Each subset of the coordinating system. that is, the medial short, lateral short, and the lateral long fibers are each capable of sustaining stable coordination in the absence of the others (Cohen, 1987). Several pieces of indirect evidence suggest that there are both ascending a n d descending coordinating neurons (Cohen, 1987; Rovainen, 1985; Williams, Sigvardt, Kopell, Errnentrout, and Remler. 1990) and that the ascending may be more effective than the descending coordinating fibers (Williams et al., 1990).

AMMOCOETE AND TRANSFORMER MOTOR PATTERN The ammocoete motor pattern induced by D-glutamale was first described for the sea lamprey, Petromyzon marinus (Cohen, Mackler, and Selzer, 1986). The onset of activity was often marked by long, slow bursting with cyclc periods in excess of

5 s. The phase delays were long and not well regulated compared to the motor output of adult 1. zinicuspis At that time we were unsure whether this slow pattern was seen only in the ammocoetes or was also produced by adult P. marinus. We have since found that adult spinal cords of P. marinus also exhibit this slow rhythm, but adult I. ziniciispis rarely do. Larval I . unicuspis begin with slow activity, but it is often less regular than in P. marznus. The observation of the slow rhythm in adult P rnurinziS demonstrates that the slow rhythm is not unique to the immature nervous system; however, because of its transitional character, it has not yet been well studied. What follows relates primarily to I. unzcuspzs for which considerably more is known for all life stages; P. marinus will not be discussed further. For some time after the application of D-glutamate to the isolated ammocoete spinal cord, the evoked segmental motor nerve discharge is irregular and not well patterned [Fig. 5(a) compared to Fig. 2(b)]. This can continue for 50 min or more. With time, the ammocoete burst pattern does be-

962

Cohen et al.

come more regular and a fast, more adult-like bursting is seen [Fig. l(b),2(b)]. However, the stability is only relative. as the bursting remains far

Table 1 Coefficients of Variation for the Period of Best Performances in Each Life Stage

12112.89 Adult Control

Life Stage

n

Ammocoete Early transformer Late transformer Adult

4 4 6

13

Coefficient of Variation' for Averages of Cycle Periods

Range

0.22' 0.122 0.06 0.09

0.1 1-0.27 0.07-0.20 0.05-0.08 0.03-0.22

Note: The values are taken from 200 or more cyclcs of bursting when the spinal cord was most stable. The adults' data were taken from past recordings from intact control animals chosen at random. The adults were from a diverse range of sizes and populations. S.D.

Phase

1201.89 AmmoCL4m Contrnl

I__

X

Significantly different from adults with p < 0.01.

Phase

14113.8P Esrly Trmaformer Control

Phase

24.12.119 Lam Tnndormr Control

1 PCa3e

Figure 2 Phase histograms for the bursting of animals of different life stages. (a) Adult. (b) Ammocoete. (c) Early transformer. (d) Late transformer. The segment numbers are given on the right of the figures. Note the scale for the ordinate differs among the graphs.

less regular than typical adult bursting [compare Figs. l(a,b) and 2(a,b): Table I]. This relative instability is also seen in 10-12 segment pieces of ammocoete spinal cord [Fig. 3(a)]. Perhaps most striking is the variability in the pattern of phase delays. Within a single swimming bout. the phases among rostra1 segments 10-12 segments apart can vary 0.0 around +/- 0.2 [Fig. 61, that is, the bursting can be synchronous ($ = 0.0), or it can exhibit typical forward ( 4 = 0.2) or backward ($ = -0.2) phase delays. The range of variability itself is atypical. The usual adult behavior is for the cord to settle at some preferred phasc relations among the segments. In ammocoete cords, it is often seen especially during the slow rhythm that the phases can be highly variable and can even be as great as 0.5 among nearby segments, but this extreme value, although it can occur, is less common in the fast rhythm. We have just begun our analysis of transformers, so our conclusions must be viewed as preliminary. The transformers that have been fully examined include only some from early (2) and late stages (8). To date, we have been staging the transformers on the basis of maturing external characters described for the head and gill region. These staging criteria, however, have only been developed for other species of lampreys (Potter, Wright, and Youson, 1978 and Youson and Potter, 1979 in Potter, Hilliard, and Bird, 1982). Detailed descriptions of metamorphosis in I. uniczispis with more appropriate criteria could modify our present conclusions either qualitatively or quantitatively.

Lamprey Pattern Generator

963

Figure 3 Cycle periods for four pieces of spinal cord cut from a single animal and subjected to identical conditions. The legends in the right corner of the figure refer to the pieces, with ROI, the most roslral. and R04, the most caudal of the pieces. (a) Pieces cut from an ammocoetc and given timc to stabilizc. Note the scale is larger than in Figure la. This was neither the best nor the worst of the group tested ( n = 4). (b) Pieces cut from an adult. Note that the periods are roughly equal to those of the singlc long piece in Figure la. One ofthe four pieces was somewhat less stable than the others. This was not unusual. The regional origin of the piece that was less stable could vary from animal to animal.

tween ammocoetes and adults in the development of their pattern generator for locomotion. At the other extreme of metamorphosis arc the stage 8 or late transformers. Late transformers look virtually identical to young adults; the only evidence for incomplete transformation is the absence of suctoral feeding and the presence of fat surrounding thc internal organs (indicating continuing lipogenesis). These animals can exhibit burst patterns identical to those of adults. The frequency can be well regulated along with the phase delays among the rostra1 and caudal segments [Figs. l(d), 2(d); Table I]. To date, we have found only two differences between the late transformers and typical I. unzcuspis adult cords, that is ( I ) the concentration of excitatory amino acid necessary for evoking stable fictive swimming and (2) the regions of the cord that were sufficient to sustain coordination among the segments. The concentrations of excitatory amino acid most often used for evoking fictive swimming in ammocoetes were 0.125-0.25 mM. Lower concentrations were not tested frequently, but when they werc used thcy rarcly produced good bursting, whereas higher concentrations mainly evoked intense highly irregular activity. In young adults, 0.25 mM is usually the concentration of choice, but 0.125 m M or 0.5 m M can produce stable bursting. By contrast, in the late transformers. the concentration that was most often effective was 0.06 mM or less. In our experiments, this concentration of D-glutamate in adults has never been effective in inducing fictive locomotion. Higher concentrations wcrc completely ineffective i n these late transformers and generally only pro-

One immediate conclusion from our experiments is that despite relativcly few morphological differences, individual early transformers can vary considerably in their fictive motor patterns. Most of the early transformers shared the characteristic that their bursting stabilized more rapidly and more fully than the arnmocoetes [Figs. l(c), 2(c); Table I]. However, in some early stage 2 transformers, the phases could vary in the same fashion as the ammocoetcs, that is. around 0.0 +/- 0.2, whereas in others that were also apparently stage 2, almost adult-like patterns were seen. The latter group of early transformers differed from adults in that their bursting was somewhat less stable and the phases were somewhat shorter [Fig. l(c)]. One conclusion can be safely drawn from these data. namely, that transformers appear transitional be-

Figure 4 An illustration of the distribution of coordinating fibcrs across the adult lamprey spinal cord. The stippled areas represent the cellular gray flanked by the lateral tracts. The dots in the middle represent the midline ofthe spinal cord. Note that there are long fibers in the lateral tracts, short fibers in the lateral tracts, and short fibers in the medial region just adjacent to thc medial tracts. (From Cohen. 1988.)

Cohen et al.

964

duced intense and irregular activity, which did not stabilize even after some time. The sample for these experiments is from a single population and is still small ( n = 6). Conceivably, this observation is not a regular phenomenon. However, regardless of future results, this finding remains interesting, because it implies that the excitability of the late transformer CPG can be very high. The other difference between the late transformers and typical I. unicuspis adults was found by lesioning either the lateral or medial fiber tracts 12.01.89 Ammocoele 1 S I Onset

to test each region's capacity for sustaining coordination in the absence of the other. In adults, lesions of neither lateral tracts nor medial regions badly disrupt coordination (Cohen. 1987) (Fig. 7). Similarly, in late transformers, medial lesions did not disrupt coordination [Fig. 8(a,b)], but lateral lesions could be completely disruptive [Fig. 8(c,d)]. In two of the three spinal cords tested, the coordination was seriously disrupted; in the third. coordination was preserved. This implies that the lateral tracts have their full complement of coordinating fibers in these animals, but the medial regions may not. The differences in the results of lateral lesions seems to suggest some variation in the degree of development of the coordinating system among these similar staged animals.

CONCEPTUAL FRAMEWORK FOR THE COORDINATING SYSTEM

Phase

11.03.89 Tnmfwmr onvl 01 iSavnV

Phase

13.05.87 Adult 1s1onset

3c

c

n 21

z c

c

Figure 5 Phase delays 10 min after drug application. (a) Ammocoete spinal cord illustrated in Figures l b and 2b, but after only 10 min of exposure to drug. Notice that the phases are completely unregulated. (b) Phases from an early transformer after 10 min exposure to drug. (c) Adult phases after 10 min of exposure to drug.

To gain some insights into what could be responsible for the diverse results we observed, we have examined mathematical models that reflect some of the more important aspects of the system. The modeling has been an integral part of our research on the CPG for some years (Cohen, Holmes, and Rand, 1982; Rand, Cohen. and Holmes, 1988) and has evolved as ideas have emerged and new results have forced change in the basic assumptions. The basic structure of the models reflects two key properties of the locomotor CPG in the lamprey: (1) individual parts of the spinal cord (pieces of at least four segments) are capable of producing oscillations (Cohen and Wallin. 1980), and (2) different parts of the spinal cord are functionally connected. Thus from a mathematical perspective, the spinal generator of the lamprey can be thought of as a chain of coupled oscillators. For a complete description of the modeling and the conceptual framework see Cohen et al. (1982) and Kopell (1988b). The modeling makes two additional and central assumptions: ( 1) the coupling between oscillators depends only on the phase difference bctween the oscillators and not on their absolute phase; and (2) the effects of multiple connections onto the same oscillator are additive.' These assumptions are made for a variety of reasons. First, they are I The model is 4 = w t ZLl;~-, Hk(0, k d,), i = I , . . . , n, where 0, is the phase of the i-th oscillator and w is the common natural frequency of all n oscillators. The functions Hk, which describe coupling of length k ( k > 0 descending, k < 0 ascending). are characterized by their phases $A.and strengths elk, where Hk($k) = 0 and ff'($k) = o(k > 0.

Lampre?)Pattern Generator

965

12.01.89 Ammocoete 1 .o

0.8

.

.

-

.

&

m l -

2

0.6

..

0

K al

.

c .

v)

m c

n

04

. ..

02

00 200

400

600

time (s)

Figure 6 Variability of phase over time for an ammococtc spinal cord. The activity began with a relatively slow rhythm with long phase delays as evidenced by the clustering in the region between 0.4-0.7. At the arrow. the rhythm jumped to a faster. more stable rhythm with shorter phase delays. The values for this part of the activity began to distribute themselves around 0.0-0.3. With time, the predominant value drifted through 0.0 and began to show up on the top of the graph with values predominating at about 0.9 and getting as low as 0.8. Thus because 0.8 is also -0.2. the drifting values can be viewed as passing from f0.2through 0 to -0.2, but the values clearly are not strongly regulated at some fixed value, but rather flip around within these values.

known to be valid in a wide range of conditions. In particular they will hold if the coupling between oscillators is sufficiently weak (Kopell, 1988a). In the lamprey spinal cord what is known is consistent with this condition. Second, the assumptions are consistent with the experimental evidence available at present, and third, the assumptions make the model tractable and thus useful as a tool to gain intuition about the sometimes counter-intuitive behavior of coupled biological oscillators. Besides the central assumption of additive phase difference coupling, additional assumptions are made on a case by case basis in order to test the consequences of certain hypotheses. The case we consider here is the time-varying phase relationships found between segments in the ammocoete. Whereas in the adult, the phase between segments is relatively stable over time, in the ammocoete

this is not typically true. Instead, one can find the following situation, as dcscribed above (cf. Fig. 6). The phase value between two segments will not remain at one stable value, but may drift from a positive value of about 1% of the cycle per segment, through zero, to a value equivalent to the positive phase, but opposite in sign. Throughout this wandering, the values will not be tightly regulated, but will form a cloud around the appropriate average value. The model could produce such behavior if we start with simple bidirectional short-distance coupling that produces synchronous activity among the segmental oscillators. Electrically coupled cells would serve this function (Cohen et al., 1982; Kopell, 1988b) and are known to be fairly common in the lamprey spinal cord (Rovainen, 1979). If we add long-distance coupling in an appropriate

966

Cbhrn ct al.

manner to produce phasc delays equivalent to those observed (cf. Kiemel, 1990 for method) then we see the small average phase differences indicative of a traveling wave. In the model, traveling waves in both the forward and backward directions can be stable for the same parameter values, so the combination of the positive and negative phase delays requires no additional assumptions. This bistability is particularly robust if there is long-distance coupling that is roughly antiphase (cf. Cohen et al., 1982), a condition that may be met by a population of cells [thc lateral interneurons (LINs)] thought to be coordinating neurons (cf. below). If the long-distance coupling is weak relative to the short-distance coupling, then shifts between synchronous activity and traveling waves can occur. This means that if the strength of the long-distance coupling is small and variablc, then we can see shifts between synchrony and positive and negative phase differences. Presumably, with development, the long-distance coupling increases and stabilizes the proper phase

-

Coordination preserved Coordination dismpted

Figure 7 Data from adult spinal cords in which acute lesions wcre made and the coordination was measured across the lesioned segmcnt. The solid bars indicate those lesions that preserved coordination indicating that the remaining cord contained sufficient fibers to maintain the regulation. Open bars indicate those lesions that disrupted coordination indicating that the remaining cord had insufficient fibers to preserve coordination. (From Cohen, 1987.)

value. Interestingly, although the intact adult can produce backward-swimming movements (McClellan and Gi-illner, 1983), the isolated adult spinal cord only rarely produces rostral-directed traveling waves (A. H. Cohen, unpublished). Somehow, the system must prebent their occurrence by some mechanism not yet present in the ammocoete cord, perhaps through the addition of medium-range connections whose action lies somewhere between inphase and antiphase.

CONCLUSIONS Two features of the ammocoete motor pattern seem to be most reflective of the immaturity of the CPG. These are the instability of the fictive motor pattern a n d the abnormal variability of phase delays. The model presented above makes some straightfonvard suggestions regarding the origin of the shifts among 0.0 versus negative and positive phase delays seen in the ammocoete and early transformer motor patterns. We will primarily relate the model to what is already known about the circuitry in trying to understand the source of the phenomenon, but it is likely that other neurons still unidentified will be part of the final picture. There is a class of long. descending inhibitory neurons known that could be the source of the behavior. The lateral interneurons appear only in the rostra1 region of the cord and send their axons down to the caudal half of the cord (Rovainen, 1983). These cells make inhibitory connections upon several classes of neurons and are believed to be part of the network that gives rise to the segmental oscillations (Grillner. Buchanan. a nd Lansner, 1988). What does seem plausible is that if some LlNs or some other class(es) of long, descending coordinating neurons are beginning to be present but have not yet become fully established, then they would cause the variability described above. It is now believed that the ascending coordinating neurons are actually stronger than the descending ones (Williams et al.. 1990). There are as yet no good candidates for ascending coordinating neurons yet identified, but some neurons form diverse classes that potentially include neurons with ascending axons. These are the crossed inhibitory neuron: (CCINs) and the excitatory interneurons (EINs). both described by Buclianan. The former include crossed and descending inhibitory neurons (CCINs) of at least three subtypes, one of which has both ascending and descending branches (Buchanan, 1982). The latter are small local inter-

Cutnprq PatfPrn Generator

967

18.12.89 Conlrol 21.12.89 control

Phase L19-LZ9

Phase L21-L30

18.12 8s medial lesion 63

,

3c

,1,_ 05

,

05

21.12.89 lateral lesion 50 segs

0

0 Phase L19-LZ9

Phase L21-L30

Figure 8 Phase histograms for late transformers in which lateral and medial lesions were made. The histograms are the phases for segments separated by the lesion site. (a) Control phases for (b) the medial lesion. Kotc that after the medial lesion the phases remain well regulated indicating that thc lateral tracts contain sufficient fibers to preserve coordination. (c) Control for (d) the lateral lesion. Note that after the lateral lesion the phases become totally disrupted indicating that the medial regions contain insufficient fibcrs to maintain coordination despite the fact that the adults are quite capable of coordination following such an acute lesion.

neurons (EINs) (Buchanan, Grillner, Cullheim, and Risling, 1989). The potential diversity of the EIN class of cells has not been investigated. and may include neurons with ascending fibers. Results from the late transformers suggest there are some fibers that are not yet fully developed in the medial region of the cord. The fibers of these undeveloped neurons could well be the absent ascending coordinating neurons. Another possible source in the transformers for developing coordinating fibers could be the caudal cord. It should be noted that the ammocoete undergoes considerable caudal growth (Beamish, 1982: Beamish and Austin, 1985). and newly differentiated coordinating cells may arise in the tail sending axons rostrally to complete the interscgmental coordinating system. Because the late transformers had stablc well-coordinated patterns

with the lateral tracts intact. it can be assumed that whether ascending or descending, the long, tract fibers known to traverse in the lateral tracts are fully developed along with the short fibers that are also known to pass in this region. The overall instability of the ammocoete activity is the other feature that seems to require some explanation. The fact that 10- 12 segment pieces of ammocoete cord are as unstable as long pieces suggcsts that the segmental oscillators themselves are unstable and are not simply being driven by eratic and inappropriate coordinating input from distant segments. Thus we should look for some source of instability that is at the local level. One source is suggested by the network model for the segmental oscillator set forth by Grillner et al. (1988). In their model, the LINs, CCINs, and EINs are all a part of the mechanism that generates the

968

Cohen et al.

oscillation. As there is good evidence that these neurons or some similar coordinating neurons are absent or still immature, it seems reasonable to suggest that the oscillator network is still not fully populated in the ammocete segments. This could well make them unstable and easily perturbed. The only assumption that must be made for this hypothesis is that the coordinating neurons are also part of the oscillator mechanism, an assumption that is most likely acceptable. The exact identity of thc missing or immature neurons need not be exactly those suggested; other similar classes of as yet unidentified neurons could equally well be lacking as essential elements of the oscillator. An alternate explanation for the instability could derive from immature membrane properties. In either case, the proof of the conjecture should be obtainable. One final caveat is in order, namely that the ammocoetes we examined were all fairly large and presumably near transformation. Because the precise onset of metamorphosis is difficult to ascertain and the first 2 stages are known to be of very short duration (Potter, 1980), it is possible that our ammocoetes had, in fact, begun transformation and were undergoing some critical modifications in their spinal circuitry. Alternatively, during the final year of larval lifc, considered an “arrested growth” phase, (Hardisty and Potter, 1971 ), there could be internal changes in the spinal cord despite the absence of external morphological changes. Before we can rule out the possibility that the pattern generator has begun to change during the final ammocoete year, we must test some very small ammococtes for the stability of their fictive motor patterns. If they are stable. then we will have to test whether the instability of the large ammocoetes is a function of early premetamorphic spinal cord changes or whether the instability emerges at some earlier time in ammocoete life. If they are unstable, then we can be more certain that the oscillators are truly not fully formed in the larval phase. Some safe conclusions are possible from a purely phenomenological perspective. One is that the large ammocoetes must be augmenting their locomotor CPG in some way to produce the adaptive movements that they are clearly capable of making. This could be accomplished by sensorimotor interactions or by descending regulation of neural activity, or some combination of both, but something must be stabilizing and adapting the spinal motor apparatus in the intact animal. This i s especially important given the prolonged period of ammocoete life. Indeed, evolutionarily speaking, the ammocoete period is, if anything, becom-

ing increasingly protracted, Whether the roles for sensory and descending systems undergo some major changes to accommodate the prolonged ammocoete development is unclear, but this is certainly suggested by these findings. We would like to thank Dr. Naomi Altman for helpful discussions on the statistics used here. This research was funded by NIH grant No. NS16803 and AFOSR contract No. F49620-89-C-0013.

REFERENCES AYERS, J. A., CARPENTER, G. A., CURRIE, S., and UNCH, J. ( 1 983). Which behavior does the lamprey central motor program mediate? Science 221:13121314. BEAMISH, F. W. H. (1982). Biology of the Southern Brook Lamprey, Ichthyomyzon gugei. Env.Bid. Fish. 7~305-320. BEAMISH, F. W. H., and AUSTIN, L. S. (1985). Growth of the Mountain Brook Lamprey Ichthyomyzon grec/eyi Hubbs and Trautman. Copeia 4:88 1-890. BEAMISH, F. W. H., and MEDLAND, T. E. (1988). Metamorphosis of the Mountain Brook Lamprey Ichthyovijlzon greeleyi. E m . Biol.Fish. 23:45-54. BUCHANAN, J. T., GRILLNER, S., CUILHEIM,S., and RISLING,M. ( 1989). Identification of excitatory interneurons contributing to generation of locomotion in lamprey: Structure. pharmacology, and function. J. Neztrophysiol. 62:59-69. BUCHANAN, J. T. (1982). Identification of interneurons with contralateral: caudal axons in the lamprey spinal cord: synaptic interactions and morphology. J. Neurophysiol. 47:96 1-975. COHEN,A. H. f 1988). Studies of the lamprey central pattern generator for locomotion: A close relationship between modeling and experimentation. In: Dynamic Patterns in Complex Systems. J. A. S. Kelso, A. J. Mandell and M. F. Shelsinger, Eds., World Scientific Pub., Singapore, pp. 143-155. COHEN,A. H. ( I 987). Intersegmental coordinating system ofthe lamprey central pattern generator for locomotion. J. Comp. Physid. 160:181-193. COHEN, A. H., MACKLER,S. A,, and SELZER,M. E. ( 1986). Functional regeneration following spinal transection demonstrated in the isolated spinal cord of the larval sea lamprey. Proc. Nut. Acud. Sci. USA 83:2763-2766. COHEN, A. H., HOLMES,P. J.. and RAND, R. H. (1982). The nature of‘the coupling between segmental oscillators of the lamprey spinal generator for locomotion: A mathematical model. J. Muth. Biol. 13:345-369. COHEN,A. H., and W A L L ~ N P., ( 1 980). The neuronal correlate of locomotion in fish: “fictive swimming” induced in an in v i m preparation of the lamprey spinal cord. Exp. Bruin Rcs. 41:ll-18. GRILLNER,S., BUCHANAN, J . T., and LANSNER,A.

Lamprey Pattern Generator (1988). Simulation of the segmental burst generating network for locomotion in lamprey. Neurosci. Lett. 89:31-35. GRILLNER, S., and KASHIN, S. ( 1 976). On the generation and performance of swimming in fish. In: Neural Control of' Locomotion. R. Herman, S. Grillner, P. Stein, and D. Stuart, Eds., Vol. 18, Plenum Press, New York, pp. 18 1-202. M. W. (1 979). Biology of the Cyclostomes, HARDISTY, Chapman and Hall, London. HARDISTY, M. W., and POTTER,1. C. (1971). The behavior, ecology and growth of larval lampreys. In: 7he Biology of'Lampreys. M. W. Hardisty and I. C. Potter, Eds., Vol. I , Academic Press, London and New York, pp. 85-127. KIEMEL, T. L. (1990). Three problems from the mathematics of neural oscillations. Ph.D. Thesis, Cornell University. KOPELL,N. (1988a). Chains of oscillators and the effects of multiple coupling. In: Dynamic Putterns in Complex Systems. J. A. S. Kelso, A. J. Mandcll, and M. F. Shelsinger, Eds., World Scientific Pub., Singapore, pp. 156-161. KOPBLL,N. (1988b). Toward a theory of modeling central pattern generators. In: Neural Control ($Rhythmic Movements in Vertebrates. A. H. Cohen, S. Rossignol, and s. Grillner, Eds., John Wiley & Sons, Inc., New York, pp. 369-413. MANION,P. J., and SMITH,B. R. (1978). Biology of larval and metamorphosing sea lampreys, Petrorny-on marinus, ofthe 1960 year class in the Big Garlic River, Michigan Part 11, 1966-72. Great Lakes Fish. Comm. Tech. Rep. 30: 1-35. MCCLELLAN, A. D., and GRILLNER, S. ( 1 983). Initiation and sensory gating of 'fictive' swimming and withdrawal responses in an in vitro preparation of the lamprey spinal cord. Brain Rev. 269:237-250. POTTER, I. C. (1980). Ecology of Larval and Metamorphosing Lampreys. Cun. J . Fish. Aquut. Sci. 37: 1641-1 657. POTTER,I. C., HILLIARU, R. W., and BIRD,D. J. (1982). Stages of Metamorphosis. In: The Biology oJ-Lurnpreys. M. W. Hardisty and I. C. Potter, Eds., Vol. 4B, Academic Press: London and New York, pp. 137-164. PURVIS,H. A. (1979). Variation in growth, age at transformation, and sex ratio of sea lampreys reestablished in chemically treated tributaries of the upper Great Lakes. Great Lakes Fish. Comm., Tech. Rep. 35: 1-36.

969

H. A. (1980). Effects of tempcrature on metaP~JRVIS, morphosis and the age and length at metamorphosis in sea lamprey (Petrornyzon murinus) in the Great Lakes. Can. J. k'ish. Aquat. Sci. 37: 1827- 1834. RAND,R. H., COHEN:A. H., and HOLMES, P. J. (1988). Systcrns of coupled oscillators as models for central pattern generators. In: Neural Control ef' Rhythmic Movements in Vertebrutes. A. Cohen, S. Rossignol, S. Grillner, Eds., pp. 333-368. ROVAINEN, C. M. (1985). Effects of groups of propnospinal interneurons on fictive swimming in the isolated spinal cord of the lamprcy. J . Neurophysiol. 54:959-977. ROVAINEN, C. M. (1983). Identified neurons in the lamprey spinal cord and their roles in fictive swimming. Svmp. Soc. Exp. Biol.37:305-330. ROVAINEN; C. M. (1979). Neurobiology of lampreys. Physiol. Rev. 59:1007-1077. C . M.. and BIRNBEKGEK, K. L. (1971). ROVAINEN, Identification and properties of motoneurons to fin muscle of the sea lamprey. J. Neurophy.sio1. 34:974982. WALL~N P.,, and WILLIAMS, T. L.. (1984). Fictive locomotion in the lamprey spinal cord in vitro compared with swimming in the intact and spinal animals. J. PhJ)Siol.347~225-239. WILLIAMS, T. L., SIGVARDT, K. A., KOPBLL,N., ERMEYTROUT, G. E., and REMLER, M. P. (1990). Forcing of coupled non-linear oscillators: Studies of intersegmental coordination in the lamprey locomotor central pattern generator. J. Neurophysiol. (in press). T. L., GRILLNER, S., SMOLJANINOV, V. V.. WILLIAMS, WAIJ.~N, P.. &SHIN, S., and ROSSIGNOL, S. (1989). Locomotion in lamprey and trout: the relative timing of activation of movement. J. Exp. Bio. 143559-566. WILLIAMS,T. L. (1986). Mechanical and neural patterns underlying swimming by lateral undulations: Review of studies on fish, amphibia, and lamprey. In: Neurohiology qf' Vertebrate Locomoiion. P. S. G. Stein, D. G. Stuart, H. Forssberg, and R. Herman, Eds., Macmillan, London, pp. 141-155. YOUSON,J. €1. (1980). Morphology and Physiology of Lamprey Metamorphosis. C a n J . Fish. dqirat. Sci. 37:1687-1710. YOUSON,J. H. (1988). First Metamorphosis. In: Fi.rk Physiolog,v, Vol. XIB. Acadcmic Prcss Inc, pp. 135- 196.