THE JOURNAL OF EXPERIMENTAL ZOOLOGY 253:20-29 (1990)
Unexpected Divergence Among Identified Interneurons in Different Abdominal Segments of the Crayfish Procambarus clarkii JAMES L. LARIMER AND CRAIG M.PEASE Department of Zoology, University of Texas, Austin, Texas 78712 ABSTRACT The command elements that initiate and coordinate the abdominal movements in crayfish show little similarity between the various abdominal segments. Our criteria for similarity among interneurons were based on both cell morphology and electrophysiology. By contrast, previously published evidence shows much greater intersegmental similarity in the skeletal, muscular, motoneuronal, and sensory components of the abdominal system in crayfish, structures that are controlled by or send information to the command elements. Therefore, unlike the command elements, these structures have retained nearly identical form and function in the various segments. We also found in different ganglia examples of interneurons involved with abdominal positioning behavior that have similar morphology but different function and vice versa. Such interneurons could represent divergent pairs of serial homologues. It is unknown why so many of the abdominal positioning interneurons have become different. The various ganglia may perform subtly different functions, requiring differences in the positioning interneurons but not in the motor neurons or muscles. Alternatively, some of the abdominal positioning interneurons underlie more than one behavior; consequently, selection acting on these multiple functions may have changed these interneurons through evolution.
Serial homology is a fundamental consequence It is known that in crayfish the skeletal and of the metameric body plan. In the primitive con- muscular structures as well as many motor and dition, for example, in the annelids, serial homol- sensory neurons involved in abdominal positionogy is apparent since the segments resemble each ing behaviors are quite similar among different other in detail. Evolutionary modification and segments. We show, however, that the command specialization of serially homologous structures elements that coordinate abdominal positioning has created much of the morphological diversity behaviors are not very similar among different found in the annelids, arthropods, and verte- ganglia. This result is of interest, because the command elements are situated between the senbrates. Before presenting the data, it is instructive to sory and motor neurons, which are similar from consider the concept of serial homology itself. Spe- segment t o segment. In the discussion, we suggest cial problems arise in defining serial homology, as how this divergence among the command eleopposed to homology of structures in different ments could have evolved. We compiled a large catalogue of identified species. Two structures can be serially homologous either because they are the products of a neurons that are involved in crayfish abdominal determinant cell lineage present more than once positioning behavior (Larimer, '88; Larimer and in the developing animal or because they were Pease, '88). Detailed cellular morphology and subjected to a developmental cue present in two physiology were gathered for each of the 79 intersegments. Unfortunately, in crayfish individual neurons in this study. The data in this catalogue adult neurons have not yet been traced back were gathered over a period of more than 5 years, through development to their origins, and we do and the catalogue was used.in a previous study not know the critical developmental cues; hence (Larimer and Pease, '881, which, like the present serial homologs cannot be recognized with either one, quantified aspects of the organization of the of these criteria. Because of this difficulty we in- crayfish abdominal positioning system. We will vestigated the percentage of cells that retain similar form and function. Received January 20, 1989; revision accepted June 9, 1989. 0 1990 WILEY-LISS, INC.
UNEXPECTED DIVERGENCE OF IDENTIFIED NEURONS
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TABLE 1 . Intersegmental similarities of abdominal structures in crayfish and related crustaceans 1.Immunohistochemical maps show that ganglia A2 to A4 have the same number of serotonergic neurons and that these neurons have similar soma and tract locations. However, the ganglia at the extremes of the series, i.e., A1 and A5, do exhibit different numbers of serotonergic neurons. Lobster Homarus americanus. (Beltz and Kravitz, '83) 2. Immunohistochemical maps of proctolin cells show that approximately the same number of such neurons are present in A2 to A5 and that these neurons have similar soma and tract locations. As indicated above, ganglia A1 and A6 are different, however. Lobster H . americanus and crayfish Procambarus clarkii. (Siwicki and Bishop, '86) 3. A segmentally repeated giant neuron is situated between the phasic flexor motor neurons and the lateral and medial giant interneurons. Crayfish Pacifastacus leniusculus and other crustaceans. (Heitler and Darrig, '86) 4.A local, nonspiking interneuron involved in swimmeret control is present in A3, A4, and A5 and shows very similar morphology and function in these ganglia. Crayfish Pa. leniusculus. (Paul and Mulloney, '85) 5 . Lateral giant fibers are formed by end to end gap junctions of segmentally repeated neurons that are similar in morphology and position. Crayfish Cambarus limosus and other crustaceans. (Johnson, '24) 6. Soma maps of the motor neurons controlling abdominal flexor and extensor muscles are similar in ganglia A2 and A3, and the GABA maps of these motoneurons are the same. Lobster H . americanus. (Otsuka et al., '67) 7. Ganglia A1 to A4 have the same number of tonic and phasic extensor motor neurons, and soma maps obtained by backfilling these motoneurons are very similar for these ganglia. However, A5 has a different number of extensor motoneurons. Four efferent neurons to the muscle receptor organs have similar structure in ganglia A1 t o A5. Crayfish Pr. clarkii. (Wine and Hagiara, '77) 8. The superficial flexor muscles of each hemisegment form a sheet of about 40 fibers. They are innervated by five excitor and one inhibitor neurons on each side in each segment. These motor neurons are readily identifiable in any segment from their impulse amplitudes, and each one innervates the sheet in a unique, gradient fashion. Crayfish Pr. clarkii (Kennedy and Takeda, '65; Velez and Wyman, '78) 9. The same number of tonic flexor motor neurons innervate the superficial flexor muscles of each segment and have similar structures in the various ganglia. Crayfish Pr. clarkii. (Wine et al. '74) 10. The soma maps, axon courses, and branching patterns of the tonic flexor motor neurons are similar in each abdominal ganglion. The phasic flexors are also somewhat similar, but vary in number from ganglion to ganglion. Ganglia A2 to A4 are more alike than A1 and A5. Crayfish Pr. clarkii. (Mittenthal and Wine, '78) 11. In spite of the fact that the terminal ganglion, A6, is derived from the fusion of two neuromeres, each abdominal ganglion, i.e., A1 through A6, contains a nearly constant number of neurons. Several counts estimate there are approximately 650 neurons per ganglion. Crayfish Pr. clarkii. (Reichert et al., '82; Kondoh and Hisada, '86) 12. Detailed anatomy of the superficial extensor muscles and the receptor muscles of the muscle receptor organs of the two most anterior abdominal segments are very similar. Crayfish Pr. clarkii (Pilgrim and Wiersma, '63) 13. Two pairs of muscle receptor organs (MRO) are present in each abdominal segment of crayfish and lobsters. The MROs in each segment consist of similar receptor muscles, receptor neurons, motor neurons, and accessory control neurons, and they have similar innervation patterns. Lobster Homarus vulgaris and Palinurus vulgaris, also found in cravfish and other crustaceans. (Alexandrowicz. '51: Evov. '76: Bastiani and Mullonev. '88)
compare the intersegmental similarity between these interneurons with data already published on segmentally repeated sensory and motor neurons, muscles, limbs, and skeletal structures in the abdomen of crayfish and their close relatives. An extensive literature shows that crayfish abdominal segments 2 through 5 have retained a great deal of their ancestral similarity (see Table 1).
At the gross anatomy level the segments of the carapace look alike, and each has a pair of swimmerets. Additionally, at the internal anatomical level, the muscles controlling the swimmerets, and particularly those that are used in tonically positioning the abdominal segments, are clearly similar. At the cellular level each ganglion contains almost exactly the same number of neurons, and the roots emerging from the surface of each
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J.L. LARIMER AND C.M. PEASE
ganglion contain the same classes, if not the exact number, of axonal processes. The motor neurons in each ganglion innervating the fast and slow flexor muscles, as well as the fast and slow extensor muscles, are quite similar in number and function among ganglia. For comparison, remarkable serial similarity also occurs among the leg motor neurons in locusts (Wilson, 1979a,b). The two pairs of large stretch receptor neurons of each segment look remarkably the same, and each is associated with very similar receptor muscles and motor and inhibitory neurons. The well-known lateral giant fibers in crayfish and lobsters are clearly segmental. Finally, at the molecular level, the immunocytochemical maps for serotonin (in the lobster CNS) and for proctolin (in the crayfish CNS) are alike. Prior t o this study, the interneurons controlling abdominal positioning behavior had not been investigated for evidence of similarity. One might expect that the interneurons occurring in different ganglia that are involved in these behaviors would be as similar as those cells and structures described above. We will show, however, that only about 25% of these command elements are similar in form and function to a cell in another ganglion and will suggest how this divergence might have come about.
MATERIALS AND METHODS The data utilized in the present study were originally gathered to survey the command elements involved in abdominal positioning behavior in the crayfish Procambarus clarkii (Larimer and Jellies, '83; Larimer and Moore, '84; Jellies and Larimer, '85, '86; Moore and Larimer, '87). A complete description of the data base is given by Larimer and Pease ('88). Briefly, an abdominal ganglion was probed with a dye-filled microelectrode, impaling a single neuron. This neuron was depolarized, and the output of tonic abdominal flexion and extension motor roots of two ganglia were recorded. Neurons that produced motor output in response to depolarization were considered command elements, and only these neurons were included in the data base. Such neurons were filled with Lucifer dye (Stewart, '78), and their morphology was traced using a Zeiss drawing tube. Each crayfish contributed only one, or at most two, neurons t o the data base. The catalogue used for the present study consists of 79 neurons, including 29 from the third abdominal ganglion (A3), 30 from the fourth (A4), and 20 from the fifth (A5).
This catalogue is known t o be incomplete, since new command elements are still encountered occasionally in all the ganglia. We presented in a previous paper a statistical procedure t o estimate the total number of command elements, both seen and unseen, in these ganglia (Larimer and Pease, '88). To accomplish this, we used both morphological and electrophysiological criteria to recognize identified neurons. We then grouped the cells from each ganglion into sets of identified neurons. Electrophysiological criteria included whether the command element invoked flexion, extension, or inhibition and whether the motor roots were excited, inhibited, or suppressed. Output was measured at two ganglia, and for those command elements that evoked excitation we noted whether the strength of output was biased and, if so, whether the bias was rostral or caudal. Morphological criteria included whether an axon was present and, if so, whether it was bipolar, unipolar rostral, or unipolar caudal, whether the dendrites were noticeably thickened, whether they crossed the midline, and whether the cell body was ipsilateral or contralateral to the axon. For bipolar axons, we noted whether the rostral and caudal axons were the same diameter. In addition to these criteria, we inspected both the tracings and records of the motor output of the interneurons and qualitatively judged the extent of similarity. Using the above criteria, we recognized 14 unique, that is, identified, neurons in abdominal ganglion three (A3), of which 9 were present once in our catalogue, 1was present twice, 3 were present four times each, and 1was present six times, for a total of 29 neurons (Larimer and Pease '88). Similarly, there are 19 identified neurons in our catalogue of A4 and 16 in our catalogue of A5. In the present study we compared each of the 14 identified command elements in our catalogue of A3 t o the 19 identified command elements in our catalogue of A4 and to the 16 identified command elements from A5, and, additionally, we compared the identified command elements of A4 t o those of A5. In making these comparisons, we used the same criteria as were used by Larimer and Pease ('88) to recognize identified command elements within each ganglion. We therefore scored as the same those identified command elements from different ganglia that would have been regarded as the same identified command element had they occurred in a single ganglion. For each pair of ganglia, we recorded the number of identified cells that were the same. It should be recognized, however, that cells that we refer to as
UNEXPECTED DIVERGENCE OF IDENTIFIED NEURONS
23
c
Fig. 1. Examples of intersegmental identities of abdominal positioning interneurons in crayfish. A,B. Tracings of flexion-producing interneurons (FPIs) located in abdominal ganglia five (A5) and four (A4), respectively. C , D Profiles of
flexion-producing cells (FPIs) from A4 and A3, respectively. E,F: Two additional examples of FPIs from A5 and A4, respectively. The first and second roots are labeled (1and 2) in A. Root three is not visible. Bar = 200 pm.
the same or identical show small morphological and electrophysiological differences. Because the catalogue is incomplete, the observed percent identified interneurons that were the same between two ganglia underestimates the true amount of similarity between the abdominal ganglia. Thus all of the command elements in a given ganglion that are identical to those in other ganglia will generally not be present in our catalogue. We used the mathematical procedure given in Appendix A to estimate the true percent of cells that are the same. Briefly, the results of Larimer and Pease ('88) tell us that our catalogue is incomplete since it contains 14 of the estimated 17 identified command elements in A3, 19 of 30 in
A4, and 16 of 43 in A5. The more complete the catalogue is, the less the computed identities underestimate the true identities. The major assumption of the methods developed in Appendix A is that sampling was random. We discuss elsewhere (Larimer and Pease, '88) why some interneurons might be more easily impaled than others and thus why some samples may be nonrandom. In the previous study, we concluded that the nonrandom sampling did not substantially alter our conclusions.
RESULTS Using the above criteria, our catalogue was found t o contain four identified interneurons that are the same between ganglia A3 and A4, two
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J.L. LARIMER AND C.M. PEASE
Fig. 2. Additional examples of intersegmental identities of abdominal positioning interneurons in crayfish. A,B: Profiles of extension-producing interneurons (EPIs) from A4 and A3, respectively. C,D. FPIs from A3 and A4, respectively.
E-G Profiles of intersegmental identities with mixed motor
that are the same between A4 and A5, and one that is the same between A3 and A5. These command elements are shown in Figures 1 and 2. Deciding which command elements are similar enough morphologically t o be scored as the same is to a certain extent arbitrary, since, as noted above, no two cells in our catalogue are exactly the same. Figure 3 shows two pairs of interneurons that are somewhat similar but have sufficient differences so that we do not regard them as the same. In the first pair the neurite and cell body project caudally in one cell and rostrally in the other. In the second pair there is an unfused loop in the axon of one cell, while the axon of the other cell is straight. We found several cases in which a pair of command elements from different ganglia was remarkably similar morphologically but differed electrophysiologically. Three pairs of such cells are shown in Figure 4. We did not consider the cells in these pairs t o be the same. In the upper-
most pair the cell on the left excited the flexion roots and evoked reciprocity, while the cell on the right suppressed both flexion and extension. In the middle pair the left cell excited flexion, while the cell on the right suppressed extension. Finally, the left cell of the bottom pair excited flexion, while the cell on the right excited extension. Using the statistical procedure outlined in Appendix A, we calculated the percent of identity in abdominal positioning interneurons occurring between pairs of ganglia. There are six percentages, including the percent of command elements in A3 identical t o an interneuron in A4, the percent of elements in A4 identical t o one in A3, and similar percentages for the comparisons of A3 with A5 and of A4 with A5. If we let hij be the fraction of command elements in ganglion i that are identical t o a cell in ganglion j, then the calculations outlined in Appendix A show that hS4 = 0.45, h43 = 0.26, h35 = 0.19, h53 = 0.08, h45 = 0.28, and hs4 = 0.20. The average of these six values is 0.24,
output FPI-EPI from A3, A4, and A5, respectively. The first and second roots are labeled in A; however, root three is not visible. Bars = 200 &m.
UNEXPECTED DIVERGENCE OF IDENTIFIED NEURONS
-
\r
25
Fig. 3. Examples of abdominal positioning interneurons that were considered t o be almost intersegmental identities, but were not scored as such. The outputs of each pair were nearly identical but differed in morphology to a greater extent than one might expect from the same cells from two
individuals. A,B: profiles of EPIs from A5 and A4, respectively. Note the positions of the somata. C , D FPIs drawn from A5 and A3, respectively. Note the unfused loop in the axon of the cell shown in C and the general locations of the somata. Bars = 200 km.
meaning that when a pair of ganglia is considered, only about one-fourth of the abdominal positioning interneurons are the same (Table 2). Because of sampling errors in the estimates of the various percent identities, no biological significance should be attached to the differences between these numbers.
Table 1 summarizes some of the evidence published by others for serially similar abdominal structures other than the controlling interneurons. Although some of the studies summarized in Table 1employed as many and as broad a range of criteria as did ours (e.g., Paul and Mulloney, '85; Bastiani and Mulloney, ' S S ) , others did not. One concern is that studies that used few criteria might overestimate the true extent of identity. In many cases the studies we quote in Table 1 were conducted for purposes other than examining intersegmental similarity. We found that there is more similarity between the interneurons of adjacent ganglia than between interneurons of more distant ganglia. This result is consistent with what previous workers have found for the abdominal structures controlled by these interneurons. For example, the relative lengths and positions of the two receptor organ muscles differ slightly from one segment t o the next, but the differences between adjacent ganglia are not as great as those between more dis-
DISCUSSION In the present study we used a large number of morphological and electrophysical criteria t o determine whether abdominal position interneurons from different ganglia are the same. The basic observation of the present paper is that different ganglia contain morphologically and physiologically different abdominal position interneurons. This is in contrast t o the longobserved fact that the skeleton, the tonic muscles, and many of the sensory and motor neurons involved in abdominal positioning behaviors are quite similar from one ganglion t o the next. We will discuss aspects of the genetics and development of serial homology, as well as its evolution.
J.L. LARIMER AND C.M. PEASE
26
/IA3
. Fig. 4. Examples of abdominal positioning interneurons that meet the morphological criteria of intersegmental identities but differ in function. A Profile of an FPI. B: A similar looking cell that suppresses both flexion and extension. The
FPI in C is similar in morphology to the suppressor shown in D. The FPI shown in E is also similar in morphology to the EPI in F. Bars = 200 pm.
TABLE 2. Data used to compute intersegmental identities 1
j
Si
sj
Bij
Ti
Tj
hij
3 4 3
4 3 5 3 5 4
14 19 14 16 19 16
19 14 16 14 16 19
4 4 1 1 2 2
17 30 17 43 30 43
30 17 43 17 43 30
0.45 0.26 0.19 0.08 0.28 0.20 0.24
5 4 5 Mean
UNEXPECTED DIVERGENCE OF IDENTIFIED NEURONS
tant ones (Pilgrim and Wiersma, '63). Additionally, the first and last ganglia are frequently modified. For example, the terminal ganglion A6 results from fusion of two neuromeres and controls the uropods and telson, while A1 (in males) controls the copulatory appendages. Similarly, ganglia A2, A3, and A4 have the same number of serotonin-producing cells, but this number differs from those present in both A1 and A5 (Beltz and Kravitz, '83). The functional and structural similarity between adjacent ganglia may reflect the simple fact that they tend t o control related movements. Ideally, we would like t o know the developmental origin of each abdominal positioning interneuron. For example, evidence that several segmentally repeated neurons have a common developmental origin would support the hypothesis that they were serially homologous even if they did not retain the same form or function as adult neurons. In this regard, some information is available on the early development of the nervous system of crayfish and related arthropods. The overall pattern of early neurogenesis in the crayfish Procambarus is very similar to that of several insects (Thomas et al., '84). For example, crayfish embryos have neuroblasts (NBs) and midline precursors (MPs) that are homologous t o those of grasshopper, Drosophila, and cockroach embryos. Although the number of neuronal precursor cells differ somewhat among arthropod species, they are all organized in a similar pattern. In addition, as development proceeds, the identified embryonic neurons extend their growth cones in specific and like pathways in various arthropod embryos, including crayfish, to form connectives and commisures (Thomas et al., '84). This however, constitutes only a general embryonic description and does not allow us to trace identified adult neurons in crayfish back to their embryonic progenitors. There is evidence that approximately equal numbers of command elements for abdominal positioning are present in ganglia A3, A4, and A5 (Larimer and Pease, '88).Thus the ancestral similarity was probably lost through morphological and functional divergence rather than through a variable number of cell additions occurring in the various ganglia or from differential cell death. It is instructive t o consider the genetic and developmental mechanisms whereby originally identical neurons in two ganglia could become different. The studies of Pearson et al. ('85) show how serially homologous cells, although of com-
27
mon developmental origin, may come to look and function differently in adults. They investigated three identified auditory neurons in locusts that are present in three different segments. These identified auditory neurons arise from the same segmentally repeated neuroblasts or at the same division. In adults, however, these three neurons develop distinctly different morphologies and synaptic connections. Evidence from Drosophila suggests that segmentation and homeotic genes may be involved in altering form and function of certain identified neurons. There are three subclasses of segmentation genes, gap (e.g., hunchback and Kriippel), pair-rule (e.g., fushi tarazu and even-skipped), and segment polarity (e.g., engrailed and gooseberry). For example, in Drosophila carrying a mutant of the pair-rule gene fushi tarazu (ftz), the projecting axon of one of the identified embryonic precursor neurons, RP2, courses posteriorly and contralaterally rather than anteriorly and ipsilaterally as in the wild type (Doe et al., '88). However, mutant fiz individuals have an abnormal RP2 neuron in all their segments, whereas to explain a loss of serial homology, a mutant would have to be expressed in only a subset of segments. In this regard, the previous example notwithstanding, most pair-rule, gap, and homeotic genes are not expressed equally in all segments. In some identified cells that express these genes, the extent of expression varies depending on which segment the cell is in (Doe et al., '88). Segmentation and homeotic genes act by binding to DNA and evidently by regulating gene expression (Doe and Scott, '88). We suggest that in crayfish similar sets of genes may be responsible for altering the form or function of originally identical command elements in different ganglia. A consideration of the biochemistry of transmitters helps one understand how functional differences between neurons could easily evolve. Significantly, two cells can secrete different transmitters if they differ biochemically by only one enzyme. For example, the generally excitatory transmitter glutamate is the substrate for the enzyme glutamic acid decarboxylase, which catalyzes its conversion t o the inhibitory transmitter y-aminobutyric acid. Similarly, the transmitter/modulator octopamine is converted to the transmitter norepinephrine by a single catecholforming enzyme, and norepinephrine can in turn be converted t o the transmitter/hormone epinephrine by the single enzyme phenylethanolamine-N-methyl transferase (Cooper et al., '86).
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J.L. LARIMER AND C.M. PEASE
Thus it is entirely possible that two neurons could become functionally unique by secreting different transmitters even if they are otherwise very similar in morphology, soma size and position, axonal projection, or other criteria. The set of neurons shown in Figure 4 is of interest in this context. They are from different ganglia and are quite similar in morphology but are totally different in function. They may represent originally identical cells in which some functional differences could have arisen that are as minor as a single enzyme or as major as its synaptic connections with other cells. Assuming that in the primitive state the command elements were identical across ganglia, why have so many become different? We offer three hypotheses. First, although the interneurons of the various ganglia appear to perform superficially similar functions, there may be subtle differences between the functions performed by the various ganglia that require corresponding differences in the morphology and physiology of the command elements. Such differences might arise, for example, from the fact that the more rostra1 cells in A3 interact with different sets of neurons than the more caudal command elements. Second, it is known that many of the abdominal positioning command elements are involved in other behaviors, for example, swimmeret beating (Murchison and Larimer, '86). Because of this pleiotropy, any selective pressure acting on swimmerets may also act on abdominal positioning behavior. Similarly, many of the abdominal positioning interneurons send their axons forward into thoracic and cerebral ganglia (Larimer and Moore, '84). Thus their evolution could be determined in part by the interactions at these distant sites as well as those occurring in the abdominal ganglia. Third, it may be that the differences that have evolved between originally identical command elements have relatively little functional significance. The command elements are organized into functional groups, and it is known that deleting a single neuron from such a group produces only a minor deficit in the motor output (Larimer, '88). Under such a scenario the morphological differentiation of originally identical neurons would be analogous to genetic drift that occurs at the molecular level.
ACKNOWLEDGMENTS We thank K. Kalthoff and B. Kruszewska for reading an early version of the manuscript. We thank J. Jellies and D. Moore for gathering the
original data. This work was supported by NIH grant NS 05423 (J.L.L.). and NSF grant BSR8706729 (C.M.P.)
LITERATURE CITED Alexandrowicz, J.S. (1951) Muscle receptor organs in the abdomen of Homarus vulgaris and Palinurus vulgaris. Q.J. Microsc. Sci., 92:163-199. Bastiani, M.J., and B. Mulloney (1988) The central projections of the stretch receptor neurons of crayfish: Structure, variation and postembryonic growth. J. Neurosci., 8: 1254- 1263. Beltz, B.S., and E.A. Kravitz (1983) Mapping of serotoninlike immunoreactivity in the lobster nervous system. J. Neurosci., 3.585-602. Cooper, J.R., F.E. Bloom, and R.H. Roth (1986) The Biochemical Basis of Neuropharmacology, 5th ed. Oxford University Press, New York. Doe, C.Q., Y. Hiromi, W.J. Gehring, and C.S. Goodman (1988) Expression and function of the segmentation gene fushi tarazu during Drosophila neurogenesis. Science, 239:170-175. Doe, C.Q., and M.P. Scott (1988) Segmentation and homeotic gene function in the developing nervous system of Drosophila. Trends Neurosci., 11: 101-106. Evoy, W.H. (1976) Modulation of proprioceptive information in crustacea. In: Neural Control of Locomotion. R.M. Herman, s.Grillner, P. Stein, and D. Stuart, eds. Plenum Press, New York, pp. 617-645. Heitler, W.J., and S. Darrig (1986) The segmental giant neurone of the signal crayfish Pacifastacus leniusculus and its interactions with abdominal fast flexor and swimmeret motor neurones. J. Exp. Biol., 121~55-75. Jellies, J., and J.L. Larimer (1985) Synaptic interactions between neurons involved in the production of abdominal posture in the crayfish. J . Comp. Physiol. A., 256:861-873. Jellies, J., and J.L. Larimer (1986) Activity of crayfish abdominal-positioning interneurons during spontaneous and sensory-evoked movements. J . Exp. Biol., 120:173-188. Johnson, G.E. (1924) Giant nerve fibers in crustaceans with special reference t o Cambarus and Palaemonetes. J . Comp. Neurol., 36:323-373. Kennedy, D., and K. Takeda (1965) Reflex control of abdominal flexor muscles in the crayfish. 11. The tonic system. J. Exp. Biol., 43:229-246. Kondoh, Y., and M. Hisada (1986) Neuroanatomy of the terminal (sixth abdominal) ganglion of the crayfish Procambarus clarkii (Girard). Cell Tissue Res., 243:273-288. Larimer, J.L. (1988) The command hypothesis: A new look using a n old example. Trends Neurosci., 11:506-510. Larimer, J.L., and J . Jellies (1983) The organization of flexion-evoking interneurons in the abdominal nerve cord of the crayfish, Procambarus clarkii. J. Exp. Zool., 226:341-351. Larimer, J.L., and D. Moore (1984) Abdominal positioning interneurons in crayfish: Projections to and synaptic activation by higher CNS centers. J . Exp. Zool., 23O:l-10. Larimer, J.L., and C.M. Pease (1988) A quantitative study of command elements for abdominal positioning behavior in the crayfish Procambarus clarkii. J. Exp. Zool., 247:45-55. Mittenthal, J.E., and J.J. Wine (1978) Segmental homology and variation in flexor motoneurons of the crayfish abdomen. J. Comp. Neurol., 177:311-334. Moore, D., and J.L. Larimer (1987) Neural control of a cyclic
UNEXPECTED DIVERGENCE OF IDENTIFIED NEURONS postural behavior in the crayfish Procambarus clarkii: The pattern-initiating interneurons. J. Comp. Physiol., 149: 145- 162. Murchison, D., and J.L. Larimer (1986) Dual motor output interneurons. SOC.Neurosci. Abstr., 12:1299. Otsuka, M., E.A. Kravitz, and D.D. Potter (1967) Physiological and chemical architecture of a lobster ganglion with particular reference to gamma-aminobutyrate and glutamate. J . Neurophysiol., 30:725-752. Paul, D.H., and B. Mulloney (1985) Nonspiking local interneuron in the motor pattern generator for the crayfish swimmeret. J . Neurophysiol., 54.28-39. Pearson, K.G., G.S. Boyan, M. Bastiani, and C.S. Goodman (1985) Heterogeneous properties of segmentally homologous interneurons in the ventral nerve cord of locusts. J. Comp. Neurol., 233:133-145. Pilgrim, R.L.C., and C.A.G. Wiersma (1963) Observations on the skeleton and somatic musculature of the abdomen and thorax of Procambarus clarkii (Girard), with notes on the thorax of Panulirus interruptus (Randall) and Astacus. J. Morphol., 113:453-487. Reichert, H., M.R. Plummer, G. Hagiwara, R.L. Roth, and J.J. Wine (1982) Local interneurons in the terminal abdominal ganglion of the crayfish. J . Comp. Physiol., 149:145-162. Siwicki, K.K., and C.A. Bishop (1986) Mapping of proctolinlike immunoreactivity in the nervous systems of lobster and crayfish. J. Comp. Neurol., 243:435-453. Stewart, W.W. (1978) Functional connections between cells as revealed by dye-coupling with a highly fluorescent naphthalimide tracer. Cell, 14:741-759. Thomas, J.B., M.J. Bastiani, M. Bate, and C.S. Goodman (1984) From grasshopper to Drosophila: A common plan for neuronal development. Nature, 310:203-207. Velez, S.J., and R.J. Wyman (1978) Synaptic connectivity in a crayfish neuromuscular system. I. Gradient of innervation and synaptic strength. J . Neurophysiol., 41 :75-84. Wilson, J.A. (1979a) The structure and function of serially homologous leg motor neurons in the locust. I. Anatomy. J. Neurobiol., 10:41-65. Wilson, J.A. (197913) The structure and function of serially homologous leg motor neurons in the locust. 11. Physiology. J. Neurobiol., 10:153-167. Wine, J.J., and G. Hagiwara (1977) Crayfish escape behavior. I. The structure of efferent and afferent neurons involved in abdominal extension. J. Comp. Physiol., 121:145-172. Wine, J.J., J.E. Mittenthal, and D. Kennedy (1974) The structure of tonic flexor motorneurons in crayfish abdominal ganglia. J . Comp. Physiol., 93:315-335.
APPENDIX A: ESTIMATING THE PERCENT IDENTITY OF COMMAND ELEMENTS BETWEEN TWO GANGLIA-CORRECTING FOR INCOMPLETE SAMPLES Because our catalog is incomplete, the percent identity between command elements in our catalog underestimates the true percentage. For example, there may be some neurons in our catalog of one ganglion that are the same as uncatalogued cells in a second ganglion. The procedure given below corrects for this bias.
29
The following quantities can be measured directly: Si,number of identified cells in the catalog of ganglion i; Sj, number of identified cells in the catalog of ganglion j; and B,, number of identified cells present both in the catalog of ganglion i and in the catalog of ganglion j. We can estimate the following quantities from our data set using the maximum likelihood and Lincoln Index methods discussed by Larimer and Pease ('88): Ti, total number of identified cells in ganglion i (both catalogued and uncatalogued); and Tj, total number of identified cells in ganglion j (both catalogued and uncatalogued). We want to estimate: hij, proportion of all the cells in ganglion i that are the same as a cell in ganglion j; and hji,proportion of all the cells in ganglion j that are the same as a cell in ganglion i. The quantity hijTiis the total number of identified cells in ganglion i that are the same as an identified cell in ganglion j. The fraction Si/Ti is the probability that a particular one of these cells is present in our sample of ganglion i, and Sj/Tj is the probability that this same cell is present in our sample of ganglion j. Assuming random sampling, note that in order t o contribute to Bij, an identified cell must be present both in our sample of ganglion i and in our sample of ganglion j: Bij
=
hijTi(Si/Ti)(Sj/Tj).
We can solve this equation for hij in terms of quantities that can be measured from our data: hij
=
BijTj/(SiSj).
Because Bij = Bji, it is also true that hji = BijTi/ (SiSj). As a specific example of how the percent identities were computed from the data, consider the calculation of h34, which is the percent of neurons in A3 that are identical to a neuron in A4 (See Table 2). Our sample contains 14 identified neurons in A3, so S3 = 14, and since there are 19 identified neurons in our sample of A4, S4 = 19. Four of the known identified neurons in A3 are identical t o a cell in A4; thus, B34 = 4. From Larimer and Pease ('88) we estimate that there are also three uncatalogued cells in A3, giving a total of 17 identified command elements in A3 and a total of 30 command elements in A4 (19 catalogued plus 11 uncatalogued); thus, T3 = 17 and T4 = 30. Substituting these values of S3,S4, B34,T3, and T4 into equation 1 shows that h34 = 0.45.
Unexpected Divergence Among Identified Interneurons in Different Abdominal Segments of the Crayfish Procambarus clarkii JAMES L. LARIMER AND CRAIG M.PEASE Department of Zoology, University of Texas, Austin, Texas 78712 ABSTRACT The command elements that initiate and coordinate the abdominal movements in crayfish show little similarity between the various abdominal segments. Our criteria for similarity among interneurons were based on both cell morphology and electrophysiology. By contrast, previously published evidence shows much greater intersegmental similarity in the skeletal, muscular, motoneuronal, and sensory components of the abdominal system in crayfish, structures that are controlled by or send information to the command elements. Therefore, unlike the command elements, these structures have retained nearly identical form and function in the various segments. We also found in different ganglia examples of interneurons involved with abdominal positioning behavior that have similar morphology but different function and vice versa. Such interneurons could represent divergent pairs of serial homologues. It is unknown why so many of the abdominal positioning interneurons have become different. The various ganglia may perform subtly different functions, requiring differences in the positioning interneurons but not in the motor neurons or muscles. Alternatively, some of the abdominal positioning interneurons underlie more than one behavior; consequently, selection acting on these multiple functions may have changed these interneurons through evolution.
Serial homology is a fundamental consequence It is known that in crayfish the skeletal and of the metameric body plan. In the primitive con- muscular structures as well as many motor and dition, for example, in the annelids, serial homol- sensory neurons involved in abdominal positionogy is apparent since the segments resemble each ing behaviors are quite similar among different other in detail. Evolutionary modification and segments. We show, however, that the command specialization of serially homologous structures elements that coordinate abdominal positioning has created much of the morphological diversity behaviors are not very similar among different found in the annelids, arthropods, and verte- ganglia. This result is of interest, because the command elements are situated between the senbrates. Before presenting the data, it is instructive to sory and motor neurons, which are similar from consider the concept of serial homology itself. Spe- segment t o segment. In the discussion, we suggest cial problems arise in defining serial homology, as how this divergence among the command eleopposed to homology of structures in different ments could have evolved. We compiled a large catalogue of identified species. Two structures can be serially homologous either because they are the products of a neurons that are involved in crayfish abdominal determinant cell lineage present more than once positioning behavior (Larimer, '88; Larimer and in the developing animal or because they were Pease, '88). Detailed cellular morphology and subjected to a developmental cue present in two physiology were gathered for each of the 79 intersegments. Unfortunately, in crayfish individual neurons in this study. The data in this catalogue adult neurons have not yet been traced back were gathered over a period of more than 5 years, through development to their origins, and we do and the catalogue was used.in a previous study not know the critical developmental cues; hence (Larimer and Pease, '881, which, like the present serial homologs cannot be recognized with either one, quantified aspects of the organization of the of these criteria. Because of this difficulty we in- crayfish abdominal positioning system. We will vestigated the percentage of cells that retain similar form and function. Received January 20, 1989; revision accepted June 9, 1989. 0 1990 WILEY-LISS, INC.
UNEXPECTED DIVERGENCE OF IDENTIFIED NEURONS
21
TABLE 1 . Intersegmental similarities of abdominal structures in crayfish and related crustaceans 1.Immunohistochemical maps show that ganglia A2 to A4 have the same number of serotonergic neurons and that these neurons have similar soma and tract locations. However, the ganglia at the extremes of the series, i.e., A1 and A5, do exhibit different numbers of serotonergic neurons. Lobster Homarus americanus. (Beltz and Kravitz, '83) 2. Immunohistochemical maps of proctolin cells show that approximately the same number of such neurons are present in A2 to A5 and that these neurons have similar soma and tract locations. As indicated above, ganglia A1 and A6 are different, however. Lobster H . americanus and crayfish Procambarus clarkii. (Siwicki and Bishop, '86) 3. A segmentally repeated giant neuron is situated between the phasic flexor motor neurons and the lateral and medial giant interneurons. Crayfish Pacifastacus leniusculus and other crustaceans. (Heitler and Darrig, '86) 4.A local, nonspiking interneuron involved in swimmeret control is present in A3, A4, and A5 and shows very similar morphology and function in these ganglia. Crayfish Pa. leniusculus. (Paul and Mulloney, '85) 5 . Lateral giant fibers are formed by end to end gap junctions of segmentally repeated neurons that are similar in morphology and position. Crayfish Cambarus limosus and other crustaceans. (Johnson, '24) 6. Soma maps of the motor neurons controlling abdominal flexor and extensor muscles are similar in ganglia A2 and A3, and the GABA maps of these motoneurons are the same. Lobster H . americanus. (Otsuka et al., '67) 7. Ganglia A1 to A4 have the same number of tonic and phasic extensor motor neurons, and soma maps obtained by backfilling these motoneurons are very similar for these ganglia. However, A5 has a different number of extensor motoneurons. Four efferent neurons to the muscle receptor organs have similar structure in ganglia A1 t o A5. Crayfish Pr. clarkii. (Wine and Hagiara, '77) 8. The superficial flexor muscles of each hemisegment form a sheet of about 40 fibers. They are innervated by five excitor and one inhibitor neurons on each side in each segment. These motor neurons are readily identifiable in any segment from their impulse amplitudes, and each one innervates the sheet in a unique, gradient fashion. Crayfish Pr. clarkii (Kennedy and Takeda, '65; Velez and Wyman, '78) 9. The same number of tonic flexor motor neurons innervate the superficial flexor muscles of each segment and have similar structures in the various ganglia. Crayfish Pr. clarkii. (Wine et al. '74) 10. The soma maps, axon courses, and branching patterns of the tonic flexor motor neurons are similar in each abdominal ganglion. The phasic flexors are also somewhat similar, but vary in number from ganglion to ganglion. Ganglia A2 to A4 are more alike than A1 and A5. Crayfish Pr. clarkii. (Mittenthal and Wine, '78) 11. In spite of the fact that the terminal ganglion, A6, is derived from the fusion of two neuromeres, each abdominal ganglion, i.e., A1 through A6, contains a nearly constant number of neurons. Several counts estimate there are approximately 650 neurons per ganglion. Crayfish Pr. clarkii. (Reichert et al., '82; Kondoh and Hisada, '86) 12. Detailed anatomy of the superficial extensor muscles and the receptor muscles of the muscle receptor organs of the two most anterior abdominal segments are very similar. Crayfish Pr. clarkii (Pilgrim and Wiersma, '63) 13. Two pairs of muscle receptor organs (MRO) are present in each abdominal segment of crayfish and lobsters. The MROs in each segment consist of similar receptor muscles, receptor neurons, motor neurons, and accessory control neurons, and they have similar innervation patterns. Lobster Homarus vulgaris and Palinurus vulgaris, also found in cravfish and other crustaceans. (Alexandrowicz. '51: Evov. '76: Bastiani and Mullonev. '88)
compare the intersegmental similarity between these interneurons with data already published on segmentally repeated sensory and motor neurons, muscles, limbs, and skeletal structures in the abdomen of crayfish and their close relatives. An extensive literature shows that crayfish abdominal segments 2 through 5 have retained a great deal of their ancestral similarity (see Table 1).
At the gross anatomy level the segments of the carapace look alike, and each has a pair of swimmerets. Additionally, at the internal anatomical level, the muscles controlling the swimmerets, and particularly those that are used in tonically positioning the abdominal segments, are clearly similar. At the cellular level each ganglion contains almost exactly the same number of neurons, and the roots emerging from the surface of each
22
J.L. LARIMER AND C.M. PEASE
ganglion contain the same classes, if not the exact number, of axonal processes. The motor neurons in each ganglion innervating the fast and slow flexor muscles, as well as the fast and slow extensor muscles, are quite similar in number and function among ganglia. For comparison, remarkable serial similarity also occurs among the leg motor neurons in locusts (Wilson, 1979a,b). The two pairs of large stretch receptor neurons of each segment look remarkably the same, and each is associated with very similar receptor muscles and motor and inhibitory neurons. The well-known lateral giant fibers in crayfish and lobsters are clearly segmental. Finally, at the molecular level, the immunocytochemical maps for serotonin (in the lobster CNS) and for proctolin (in the crayfish CNS) are alike. Prior t o this study, the interneurons controlling abdominal positioning behavior had not been investigated for evidence of similarity. One might expect that the interneurons occurring in different ganglia that are involved in these behaviors would be as similar as those cells and structures described above. We will show, however, that only about 25% of these command elements are similar in form and function to a cell in another ganglion and will suggest how this divergence might have come about.
MATERIALS AND METHODS The data utilized in the present study were originally gathered to survey the command elements involved in abdominal positioning behavior in the crayfish Procambarus clarkii (Larimer and Jellies, '83; Larimer and Moore, '84; Jellies and Larimer, '85, '86; Moore and Larimer, '87). A complete description of the data base is given by Larimer and Pease ('88). Briefly, an abdominal ganglion was probed with a dye-filled microelectrode, impaling a single neuron. This neuron was depolarized, and the output of tonic abdominal flexion and extension motor roots of two ganglia were recorded. Neurons that produced motor output in response to depolarization were considered command elements, and only these neurons were included in the data base. Such neurons were filled with Lucifer dye (Stewart, '78), and their morphology was traced using a Zeiss drawing tube. Each crayfish contributed only one, or at most two, neurons t o the data base. The catalogue used for the present study consists of 79 neurons, including 29 from the third abdominal ganglion (A3), 30 from the fourth (A4), and 20 from the fifth (A5).
This catalogue is known t o be incomplete, since new command elements are still encountered occasionally in all the ganglia. We presented in a previous paper a statistical procedure t o estimate the total number of command elements, both seen and unseen, in these ganglia (Larimer and Pease, '88). To accomplish this, we used both morphological and electrophysiological criteria to recognize identified neurons. We then grouped the cells from each ganglion into sets of identified neurons. Electrophysiological criteria included whether the command element invoked flexion, extension, or inhibition and whether the motor roots were excited, inhibited, or suppressed. Output was measured at two ganglia, and for those command elements that evoked excitation we noted whether the strength of output was biased and, if so, whether the bias was rostral or caudal. Morphological criteria included whether an axon was present and, if so, whether it was bipolar, unipolar rostral, or unipolar caudal, whether the dendrites were noticeably thickened, whether they crossed the midline, and whether the cell body was ipsilateral or contralateral to the axon. For bipolar axons, we noted whether the rostral and caudal axons were the same diameter. In addition to these criteria, we inspected both the tracings and records of the motor output of the interneurons and qualitatively judged the extent of similarity. Using the above criteria, we recognized 14 unique, that is, identified, neurons in abdominal ganglion three (A3), of which 9 were present once in our catalogue, 1was present twice, 3 were present four times each, and 1was present six times, for a total of 29 neurons (Larimer and Pease '88). Similarly, there are 19 identified neurons in our catalogue of A4 and 16 in our catalogue of A5. In the present study we compared each of the 14 identified command elements in our catalogue of A3 t o the 19 identified command elements in our catalogue of A4 and to the 16 identified command elements from A5, and, additionally, we compared the identified command elements of A4 t o those of A5. In making these comparisons, we used the same criteria as were used by Larimer and Pease ('88) to recognize identified command elements within each ganglion. We therefore scored as the same those identified command elements from different ganglia that would have been regarded as the same identified command element had they occurred in a single ganglion. For each pair of ganglia, we recorded the number of identified cells that were the same. It should be recognized, however, that cells that we refer to as
UNEXPECTED DIVERGENCE OF IDENTIFIED NEURONS
23
c
Fig. 1. Examples of intersegmental identities of abdominal positioning interneurons in crayfish. A,B. Tracings of flexion-producing interneurons (FPIs) located in abdominal ganglia five (A5) and four (A4), respectively. C , D Profiles of
flexion-producing cells (FPIs) from A4 and A3, respectively. E,F: Two additional examples of FPIs from A5 and A4, respectively. The first and second roots are labeled (1and 2) in A. Root three is not visible. Bar = 200 pm.
the same or identical show small morphological and electrophysiological differences. Because the catalogue is incomplete, the observed percent identified interneurons that were the same between two ganglia underestimates the true amount of similarity between the abdominal ganglia. Thus all of the command elements in a given ganglion that are identical to those in other ganglia will generally not be present in our catalogue. We used the mathematical procedure given in Appendix A to estimate the true percent of cells that are the same. Briefly, the results of Larimer and Pease ('88) tell us that our catalogue is incomplete since it contains 14 of the estimated 17 identified command elements in A3, 19 of 30 in
A4, and 16 of 43 in A5. The more complete the catalogue is, the less the computed identities underestimate the true identities. The major assumption of the methods developed in Appendix A is that sampling was random. We discuss elsewhere (Larimer and Pease, '88) why some interneurons might be more easily impaled than others and thus why some samples may be nonrandom. In the previous study, we concluded that the nonrandom sampling did not substantially alter our conclusions.
RESULTS Using the above criteria, our catalogue was found t o contain four identified interneurons that are the same between ganglia A3 and A4, two
24
J.L. LARIMER AND C.M. PEASE
Fig. 2. Additional examples of intersegmental identities of abdominal positioning interneurons in crayfish. A,B: Profiles of extension-producing interneurons (EPIs) from A4 and A3, respectively. C,D. FPIs from A3 and A4, respectively.
E-G Profiles of intersegmental identities with mixed motor
that are the same between A4 and A5, and one that is the same between A3 and A5. These command elements are shown in Figures 1 and 2. Deciding which command elements are similar enough morphologically t o be scored as the same is to a certain extent arbitrary, since, as noted above, no two cells in our catalogue are exactly the same. Figure 3 shows two pairs of interneurons that are somewhat similar but have sufficient differences so that we do not regard them as the same. In the first pair the neurite and cell body project caudally in one cell and rostrally in the other. In the second pair there is an unfused loop in the axon of one cell, while the axon of the other cell is straight. We found several cases in which a pair of command elements from different ganglia was remarkably similar morphologically but differed electrophysiologically. Three pairs of such cells are shown in Figure 4. We did not consider the cells in these pairs t o be the same. In the upper-
most pair the cell on the left excited the flexion roots and evoked reciprocity, while the cell on the right suppressed both flexion and extension. In the middle pair the left cell excited flexion, while the cell on the right suppressed extension. Finally, the left cell of the bottom pair excited flexion, while the cell on the right excited extension. Using the statistical procedure outlined in Appendix A, we calculated the percent of identity in abdominal positioning interneurons occurring between pairs of ganglia. There are six percentages, including the percent of command elements in A3 identical t o an interneuron in A4, the percent of elements in A4 identical t o one in A3, and similar percentages for the comparisons of A3 with A5 and of A4 with A5. If we let hij be the fraction of command elements in ganglion i that are identical t o a cell in ganglion j, then the calculations outlined in Appendix A show that hS4 = 0.45, h43 = 0.26, h35 = 0.19, h53 = 0.08, h45 = 0.28, and hs4 = 0.20. The average of these six values is 0.24,
output FPI-EPI from A3, A4, and A5, respectively. The first and second roots are labeled in A; however, root three is not visible. Bars = 200 &m.
UNEXPECTED DIVERGENCE OF IDENTIFIED NEURONS
-
\r
25
Fig. 3. Examples of abdominal positioning interneurons that were considered t o be almost intersegmental identities, but were not scored as such. The outputs of each pair were nearly identical but differed in morphology to a greater extent than one might expect from the same cells from two
individuals. A,B: profiles of EPIs from A5 and A4, respectively. Note the positions of the somata. C , D FPIs drawn from A5 and A3, respectively. Note the unfused loop in the axon of the cell shown in C and the general locations of the somata. Bars = 200 km.
meaning that when a pair of ganglia is considered, only about one-fourth of the abdominal positioning interneurons are the same (Table 2). Because of sampling errors in the estimates of the various percent identities, no biological significance should be attached to the differences between these numbers.
Table 1 summarizes some of the evidence published by others for serially similar abdominal structures other than the controlling interneurons. Although some of the studies summarized in Table 1employed as many and as broad a range of criteria as did ours (e.g., Paul and Mulloney, '85; Bastiani and Mulloney, ' S S ) , others did not. One concern is that studies that used few criteria might overestimate the true extent of identity. In many cases the studies we quote in Table 1 were conducted for purposes other than examining intersegmental similarity. We found that there is more similarity between the interneurons of adjacent ganglia than between interneurons of more distant ganglia. This result is consistent with what previous workers have found for the abdominal structures controlled by these interneurons. For example, the relative lengths and positions of the two receptor organ muscles differ slightly from one segment t o the next, but the differences between adjacent ganglia are not as great as those between more dis-
DISCUSSION In the present study we used a large number of morphological and electrophysical criteria t o determine whether abdominal position interneurons from different ganglia are the same. The basic observation of the present paper is that different ganglia contain morphologically and physiologically different abdominal position interneurons. This is in contrast t o the longobserved fact that the skeleton, the tonic muscles, and many of the sensory and motor neurons involved in abdominal positioning behaviors are quite similar from one ganglion t o the next. We will discuss aspects of the genetics and development of serial homology, as well as its evolution.
J.L. LARIMER AND C.M. PEASE
26
/IA3
. Fig. 4. Examples of abdominal positioning interneurons that meet the morphological criteria of intersegmental identities but differ in function. A Profile of an FPI. B: A similar looking cell that suppresses both flexion and extension. The
FPI in C is similar in morphology to the suppressor shown in D. The FPI shown in E is also similar in morphology to the EPI in F. Bars = 200 pm.
TABLE 2. Data used to compute intersegmental identities 1
j
Si
sj
Bij
Ti
Tj
hij
3 4 3
4 3 5 3 5 4
14 19 14 16 19 16
19 14 16 14 16 19
4 4 1 1 2 2
17 30 17 43 30 43
30 17 43 17 43 30
0.45 0.26 0.19 0.08 0.28 0.20 0.24
5 4 5 Mean
UNEXPECTED DIVERGENCE OF IDENTIFIED NEURONS
tant ones (Pilgrim and Wiersma, '63). Additionally, the first and last ganglia are frequently modified. For example, the terminal ganglion A6 results from fusion of two neuromeres and controls the uropods and telson, while A1 (in males) controls the copulatory appendages. Similarly, ganglia A2, A3, and A4 have the same number of serotonin-producing cells, but this number differs from those present in both A1 and A5 (Beltz and Kravitz, '83). The functional and structural similarity between adjacent ganglia may reflect the simple fact that they tend t o control related movements. Ideally, we would like t o know the developmental origin of each abdominal positioning interneuron. For example, evidence that several segmentally repeated neurons have a common developmental origin would support the hypothesis that they were serially homologous even if they did not retain the same form or function as adult neurons. In this regard, some information is available on the early development of the nervous system of crayfish and related arthropods. The overall pattern of early neurogenesis in the crayfish Procambarus is very similar to that of several insects (Thomas et al., '84). For example, crayfish embryos have neuroblasts (NBs) and midline precursors (MPs) that are homologous t o those of grasshopper, Drosophila, and cockroach embryos. Although the number of neuronal precursor cells differ somewhat among arthropod species, they are all organized in a similar pattern. In addition, as development proceeds, the identified embryonic neurons extend their growth cones in specific and like pathways in various arthropod embryos, including crayfish, to form connectives and commisures (Thomas et al., '84). This however, constitutes only a general embryonic description and does not allow us to trace identified adult neurons in crayfish back to their embryonic progenitors. There is evidence that approximately equal numbers of command elements for abdominal positioning are present in ganglia A3, A4, and A5 (Larimer and Pease, '88).Thus the ancestral similarity was probably lost through morphological and functional divergence rather than through a variable number of cell additions occurring in the various ganglia or from differential cell death. It is instructive t o consider the genetic and developmental mechanisms whereby originally identical neurons in two ganglia could become different. The studies of Pearson et al. ('85) show how serially homologous cells, although of com-
27
mon developmental origin, may come to look and function differently in adults. They investigated three identified auditory neurons in locusts that are present in three different segments. These identified auditory neurons arise from the same segmentally repeated neuroblasts or at the same division. In adults, however, these three neurons develop distinctly different morphologies and synaptic connections. Evidence from Drosophila suggests that segmentation and homeotic genes may be involved in altering form and function of certain identified neurons. There are three subclasses of segmentation genes, gap (e.g., hunchback and Kriippel), pair-rule (e.g., fushi tarazu and even-skipped), and segment polarity (e.g., engrailed and gooseberry). For example, in Drosophila carrying a mutant of the pair-rule gene fushi tarazu (ftz), the projecting axon of one of the identified embryonic precursor neurons, RP2, courses posteriorly and contralaterally rather than anteriorly and ipsilaterally as in the wild type (Doe et al., '88). However, mutant fiz individuals have an abnormal RP2 neuron in all their segments, whereas to explain a loss of serial homology, a mutant would have to be expressed in only a subset of segments. In this regard, the previous example notwithstanding, most pair-rule, gap, and homeotic genes are not expressed equally in all segments. In some identified cells that express these genes, the extent of expression varies depending on which segment the cell is in (Doe et al., '88). Segmentation and homeotic genes act by binding to DNA and evidently by regulating gene expression (Doe and Scott, '88). We suggest that in crayfish similar sets of genes may be responsible for altering the form or function of originally identical command elements in different ganglia. A consideration of the biochemistry of transmitters helps one understand how functional differences between neurons could easily evolve. Significantly, two cells can secrete different transmitters if they differ biochemically by only one enzyme. For example, the generally excitatory transmitter glutamate is the substrate for the enzyme glutamic acid decarboxylase, which catalyzes its conversion t o the inhibitory transmitter y-aminobutyric acid. Similarly, the transmitter/modulator octopamine is converted to the transmitter norepinephrine by a single catecholforming enzyme, and norepinephrine can in turn be converted t o the transmitter/hormone epinephrine by the single enzyme phenylethanolamine-N-methyl transferase (Cooper et al., '86).
28
J.L. LARIMER AND C.M. PEASE
Thus it is entirely possible that two neurons could become functionally unique by secreting different transmitters even if they are otherwise very similar in morphology, soma size and position, axonal projection, or other criteria. The set of neurons shown in Figure 4 is of interest in this context. They are from different ganglia and are quite similar in morphology but are totally different in function. They may represent originally identical cells in which some functional differences could have arisen that are as minor as a single enzyme or as major as its synaptic connections with other cells. Assuming that in the primitive state the command elements were identical across ganglia, why have so many become different? We offer three hypotheses. First, although the interneurons of the various ganglia appear to perform superficially similar functions, there may be subtle differences between the functions performed by the various ganglia that require corresponding differences in the morphology and physiology of the command elements. Such differences might arise, for example, from the fact that the more rostra1 cells in A3 interact with different sets of neurons than the more caudal command elements. Second, it is known that many of the abdominal positioning command elements are involved in other behaviors, for example, swimmeret beating (Murchison and Larimer, '86). Because of this pleiotropy, any selective pressure acting on swimmerets may also act on abdominal positioning behavior. Similarly, many of the abdominal positioning interneurons send their axons forward into thoracic and cerebral ganglia (Larimer and Moore, '84). Thus their evolution could be determined in part by the interactions at these distant sites as well as those occurring in the abdominal ganglia. Third, it may be that the differences that have evolved between originally identical command elements have relatively little functional significance. The command elements are organized into functional groups, and it is known that deleting a single neuron from such a group produces only a minor deficit in the motor output (Larimer, '88). Under such a scenario the morphological differentiation of originally identical neurons would be analogous to genetic drift that occurs at the molecular level.
ACKNOWLEDGMENTS We thank K. Kalthoff and B. Kruszewska for reading an early version of the manuscript. We thank J. Jellies and D. Moore for gathering the
original data. This work was supported by NIH grant NS 05423 (J.L.L.). and NSF grant BSR8706729 (C.M.P.)
LITERATURE CITED Alexandrowicz, J.S. (1951) Muscle receptor organs in the abdomen of Homarus vulgaris and Palinurus vulgaris. Q.J. Microsc. Sci., 92:163-199. Bastiani, M.J., and B. Mulloney (1988) The central projections of the stretch receptor neurons of crayfish: Structure, variation and postembryonic growth. J. Neurosci., 8: 1254- 1263. Beltz, B.S., and E.A. Kravitz (1983) Mapping of serotoninlike immunoreactivity in the lobster nervous system. J. Neurosci., 3.585-602. Cooper, J.R., F.E. Bloom, and R.H. Roth (1986) The Biochemical Basis of Neuropharmacology, 5th ed. Oxford University Press, New York. Doe, C.Q., Y. Hiromi, W.J. Gehring, and C.S. Goodman (1988) Expression and function of the segmentation gene fushi tarazu during Drosophila neurogenesis. Science, 239:170-175. Doe, C.Q., and M.P. Scott (1988) Segmentation and homeotic gene function in the developing nervous system of Drosophila. Trends Neurosci., 11: 101-106. Evoy, W.H. (1976) Modulation of proprioceptive information in crustacea. In: Neural Control of Locomotion. R.M. Herman, s.Grillner, P. Stein, and D. Stuart, eds. Plenum Press, New York, pp. 617-645. Heitler, W.J., and S. Darrig (1986) The segmental giant neurone of the signal crayfish Pacifastacus leniusculus and its interactions with abdominal fast flexor and swimmeret motor neurones. J. Exp. Biol., 121~55-75. Jellies, J., and J.L. Larimer (1985) Synaptic interactions between neurons involved in the production of abdominal posture in the crayfish. J . Comp. Physiol. A., 256:861-873. Jellies, J., and J.L. Larimer (1986) Activity of crayfish abdominal-positioning interneurons during spontaneous and sensory-evoked movements. J . Exp. Biol., 120:173-188. Johnson, G.E. (1924) Giant nerve fibers in crustaceans with special reference t o Cambarus and Palaemonetes. J . Comp. Neurol., 36:323-373. Kennedy, D., and K. Takeda (1965) Reflex control of abdominal flexor muscles in the crayfish. 11. The tonic system. J. Exp. Biol., 43:229-246. Kondoh, Y., and M. Hisada (1986) Neuroanatomy of the terminal (sixth abdominal) ganglion of the crayfish Procambarus clarkii (Girard). Cell Tissue Res., 243:273-288. Larimer, J.L. (1988) The command hypothesis: A new look using a n old example. Trends Neurosci., 11:506-510. Larimer, J.L., and J . Jellies (1983) The organization of flexion-evoking interneurons in the abdominal nerve cord of the crayfish, Procambarus clarkii. J. Exp. Zool., 226:341-351. Larimer, J.L., and D. Moore (1984) Abdominal positioning interneurons in crayfish: Projections to and synaptic activation by higher CNS centers. J . Exp. Zool., 23O:l-10. Larimer, J.L., and C.M. Pease (1988) A quantitative study of command elements for abdominal positioning behavior in the crayfish Procambarus clarkii. J. Exp. Zool., 247:45-55. Mittenthal, J.E., and J.J. Wine (1978) Segmental homology and variation in flexor motoneurons of the crayfish abdomen. J. Comp. Neurol., 177:311-334. Moore, D., and J.L. Larimer (1987) Neural control of a cyclic
UNEXPECTED DIVERGENCE OF IDENTIFIED NEURONS postural behavior in the crayfish Procambarus clarkii: The pattern-initiating interneurons. J. Comp. Physiol., 149: 145- 162. Murchison, D., and J.L. Larimer (1986) Dual motor output interneurons. SOC.Neurosci. Abstr., 12:1299. Otsuka, M., E.A. Kravitz, and D.D. Potter (1967) Physiological and chemical architecture of a lobster ganglion with particular reference to gamma-aminobutyrate and glutamate. J . Neurophysiol., 30:725-752. Paul, D.H., and B. Mulloney (1985) Nonspiking local interneuron in the motor pattern generator for the crayfish swimmeret. J . Neurophysiol., 54.28-39. Pearson, K.G., G.S. Boyan, M. Bastiani, and C.S. Goodman (1985) Heterogeneous properties of segmentally homologous interneurons in the ventral nerve cord of locusts. J. Comp. Neurol., 233:133-145. Pilgrim, R.L.C., and C.A.G. Wiersma (1963) Observations on the skeleton and somatic musculature of the abdomen and thorax of Procambarus clarkii (Girard), with notes on the thorax of Panulirus interruptus (Randall) and Astacus. J. Morphol., 113:453-487. Reichert, H., M.R. Plummer, G. Hagiwara, R.L. Roth, and J.J. Wine (1982) Local interneurons in the terminal abdominal ganglion of the crayfish. J . Comp. Physiol., 149:145-162. Siwicki, K.K., and C.A. Bishop (1986) Mapping of proctolinlike immunoreactivity in the nervous systems of lobster and crayfish. J. Comp. Neurol., 243:435-453. Stewart, W.W. (1978) Functional connections between cells as revealed by dye-coupling with a highly fluorescent naphthalimide tracer. Cell, 14:741-759. Thomas, J.B., M.J. Bastiani, M. Bate, and C.S. Goodman (1984) From grasshopper to Drosophila: A common plan for neuronal development. Nature, 310:203-207. Velez, S.J., and R.J. Wyman (1978) Synaptic connectivity in a crayfish neuromuscular system. I. Gradient of innervation and synaptic strength. J . Neurophysiol., 41 :75-84. Wilson, J.A. (1979a) The structure and function of serially homologous leg motor neurons in the locust. I. Anatomy. J. Neurobiol., 10:41-65. Wilson, J.A. (197913) The structure and function of serially homologous leg motor neurons in the locust. 11. Physiology. J. Neurobiol., 10:153-167. Wine, J.J., and G. Hagiwara (1977) Crayfish escape behavior. I. The structure of efferent and afferent neurons involved in abdominal extension. J. Comp. Physiol., 121:145-172. Wine, J.J., J.E. Mittenthal, and D. Kennedy (1974) The structure of tonic flexor motorneurons in crayfish abdominal ganglia. J . Comp. Physiol., 93:315-335.
APPENDIX A: ESTIMATING THE PERCENT IDENTITY OF COMMAND ELEMENTS BETWEEN TWO GANGLIA-CORRECTING FOR INCOMPLETE SAMPLES Because our catalog is incomplete, the percent identity between command elements in our catalog underestimates the true percentage. For example, there may be some neurons in our catalog of one ganglion that are the same as uncatalogued cells in a second ganglion. The procedure given below corrects for this bias.
29
The following quantities can be measured directly: Si,number of identified cells in the catalog of ganglion i; Sj, number of identified cells in the catalog of ganglion j; and B,, number of identified cells present both in the catalog of ganglion i and in the catalog of ganglion j. We can estimate the following quantities from our data set using the maximum likelihood and Lincoln Index methods discussed by Larimer and Pease ('88): Ti, total number of identified cells in ganglion i (both catalogued and uncatalogued); and Tj, total number of identified cells in ganglion j (both catalogued and uncatalogued). We want to estimate: hij, proportion of all the cells in ganglion i that are the same as a cell in ganglion j; and hji,proportion of all the cells in ganglion j that are the same as a cell in ganglion i. The quantity hijTiis the total number of identified cells in ganglion i that are the same as an identified cell in ganglion j. The fraction Si/Ti is the probability that a particular one of these cells is present in our sample of ganglion i, and Sj/Tj is the probability that this same cell is present in our sample of ganglion j. Assuming random sampling, note that in order t o contribute to Bij, an identified cell must be present both in our sample of ganglion i and in our sample of ganglion j: Bij
=
hijTi(Si/Ti)(Sj/Tj).
We can solve this equation for hij in terms of quantities that can be measured from our data: hij
=
BijTj/(SiSj).
Because Bij = Bji, it is also true that hji = BijTi/ (SiSj). As a specific example of how the percent identities were computed from the data, consider the calculation of h34, which is the percent of neurons in A3 that are identical to a neuron in A4 (See Table 2). Our sample contains 14 identified neurons in A3, so S3 = 14, and since there are 19 identified neurons in our sample of A4, S4 = 19. Four of the known identified neurons in A3 are identical t o a cell in A4; thus, B34 = 4. From Larimer and Pease ('88) we estimate that there are also three uncatalogued cells in A3, giving a total of 17 identified command elements in A3 and a total of 30 command elements in A4 (19 catalogued plus 11 uncatalogued); thus, T3 = 17 and T4 = 30. Substituting these values of S3,S4, B34,T3, and T4 into equation 1 shows that h34 = 0.45.
