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JEZ 842 THE JOURNAL OF EXPERIMENTAL ZOOLOGY 279:243–253 (1997) Muscle Receptor Organs of the Crayfish, Cherax destructor: Organisation of Central Projections of Stretch Receptor Neurons DAVID L. MACMILLAN* AND PAUL J. VESCOVI Department of Zoology, University of Melbourne, Parkville Vic. 3052, Australia ABSTRACT The stretch receptor neurons of the abdominal muscle receptor organs of crayfish enter the ventral nerve cord and branch to send axons to the brain and to the last abdominal ganglion. They mediate local reflexes in the ganglion of entry and adjacent ganglia but evidence from previous cobalt filling and histology suggested that local branching is limited and variable and does not accord closely with physiological evidence. We examined the fine structure of the branching in a large number of preparations from large animals of the crayfish Cherax destructor, which is also a large species, to determine whether the increased resolution afford by size would permit us to detect previously undetected patterns. We found some predictable features in the pattern of fine branching in the ganglion of entry and adjacent ganglia that could explain some of the apparent anomalies. We also examined the relative positions of the hook-shaped projections from different segments where they terminate in the last abdominal ganglion by differentially staining the stretch receptors with Co++ and Ni++. We found evidence for somatotopic organisation in the longitudinal position of the endings. J. Exp. Zool. 279:243–253, 1997. © 1997 Wiley-Liss, Inc. Allen (1864) first described some simple sensory cells which were later named muscle receptor organs (MROs) by Alexandrowicz (’51) in his studies on the abdomen of the lobsters Homarus vulgaris and Palinurus vulgaris. Since Alexandrowicz’s description there have been numerous studies carried out on the microanatomy, neurophysiology, and neuropharmacology of the MRO. MROs have since been identified in more than ten species of decapod crustacean (Pilgrim, ’60; Fields, ’76). Each MRO consists of a stretch receptor neuron (SR) with its dendrites embedded in a thin muscle bundle in parallel with the dorsal extensor musculature. They are found in pairs on either side of the midline in each abdominal segment. The MROs that make up a pair have distinct anatomical and physiological characteristics and respond to different features of abdominal movement. The medial organ has a SR with a broad dendritic area and responds tonically to maintained stretch (MRO1 and SR1). The lateral receptor organ has a shorter, thinner muscle and its conically-shaped SR responds phasically to maintained stretch (MRO2 and SR2). There is a considerable amount of evidence from extracellular studies that the SRs are involved in a number of local reflexes (Eckert, ’61; Fields and Kennedy, ’65; Fields, ’66; Fields et al., ’67; © 1997 WILEY-LISS, INC. Sokolove, ’73; Nja and Walloe, ’75) involving the ganglion of entry and adjacent ganglia. These appear to be present in the range of crayfish species studied (for review see Fields, ’76) and some of them have been confirmed and further investigated with intracellular techniques (Hausknecht, ’96, and in preparation). One might have predicted, therefore, that there would be clear and consistent patterns in the branching and projections of the SRs in these ganglia. Histological studies of the abdominal ganglia of Procambarus clarkii failed to find substantial branches (Liese et al., ’87; Skinner, ’85a,b) or stereotyped projection patterns. Bastiani and Mulloney (’88a) also examined this aspect of the SRs in their study of the morphology of the projections anteriorly and posteriorly in the cord. They stained the SRs by cobalt infusion into the cut peripheral ends of the axons and noted a number of small branches in the ganglion of entry and in other ganglia through which the axons passed but could find no consistency in the position or shape of those branches. While this result was suggestive, it was not con- *Correspondence to: Dr. D.L. Macmillan, Department of Zoology, University of Melbourne, Parkville, Vic. 3052, Australia. E-mail: email@example.com Received 15 November 1996; revision accepted 10 June 1997. 244 D.L. MACMILLAN AND P.J. VESCOVI clusive because this aspect of the work was subordinate to the main aim of the project which was to visualise the terminations. To achieve this they used small animals (2–3 cm) which meant that details of the fine branching in the ganglia in between were difficult to determine and may not represent the mature condition. One of the objectives of the experiments described here was to use large animals and target the local ganglia specifically so that all details of even the finest branches would be apparent following intensification of the cobalt filled preparations. The SRs from each MRO pair enter the ventral nerve cord in the second root of the next anterior segmental ganglion. Bastiani and Mulloney (’88a) demonstrated that Alexandrowicz’s (’51) conjecture about the possible fate of the SR axons was correct. Upon entering the ganglion, the sensory axons bifurcate and run in opposite directions. They demonstrated further that one branch travels all the way to the brain and the other to the last abdominal ganglion, A6. A6 is the major integrating centre for the motor system controlling the various appendages that comprise the tailfan (Dumont and Wine, ’87a,b). The sensory axons of the SRs travel the entire length of the body, passing through every ganglion and running together in a single bundle in the dorsal medial tract (DMT) (Wiersma and Hughes, ’61). The sensory axons from all the abdominal muscle receptors terminate in the same dorsal area of A6 with a characteristic, hooked structure that is symmetrically disposed across the midline. The hook is formed near the posterior end of the interconnective fissure where the axons turn medially across the midline in commissures A6 DCII or A7 DCII (terminology from Kondoh and Hisada, ’86) and, after crossing the midline, run anteriorly to the end of the core of A6. This labelled-line input and extensive branching in A6 suggests that MRO input plays some important role in the tailfan control. The tailfan is a highly specialised terminal structure in the crayfish, formed by a fusion of the telson and the paired appendages of the sixth abdominal segment, the uropods. Many of the neurons in the terminal ganglion and muscles of the tailfan have been characterised morphologically and physiologically and some of their functional relationships have been determined (Larimer and Kennedy, ’69; Wine and Krasne, ’82; Hisada et al., ’84a,b; Wine, ’84; Dumont and Wine, ’87a,b; Kondoh and Hisada, ’87; Takahata and Hisada, ’85; Vescovi et al., ’97). Bastiani and Mulloney (’88a) recorded intracellularly from a number of neurons of different classes in A6 that were postsynaptic to SR axons in P. clarkii. They found local interneurons, plurisegmental interneurons and motor neurons that responded to SR activation. Vescovi et al. (’97) identified some of those neurons in Cherax destructor. Bastiani and Mulloney (’88b) also found that SR input from posterior abdominal segments was more likely to cause larger EPSPs in unidentified terminal ganglion neurons than input from anterior segments of P. clarkii and demonstrated evidence of an anterior-posterior gradient in the strength of the response. Vescovi et al. (’97) found that similar gradients occur in C. destructor, but not in any of the neurons they identified. Studies in insects have demonstrated some highly ordered projections from mechanosensory afferents, with somatotopic relationships between the location of the sensory structure and the projection in the central nervous system (Teugels and Ghysen, ’83; Johnson and Murphey, ’85; Kent and Levine, ’88; Murphey et al., ’89). We hypothesized that the physiological gradients in some postsynaptic cells in both P. clarkii and C. destructor could be related to some kind of somatotopic order of the SR endings in A6, perhaps echoing the topological organisation of hair projections from different tailfan nerves into the last abdominal ganglion described by Kondoh and Hisada (’87). The search for evidence for such organisation was a second objective of this study. MATERIALS AND METHODS Australian freshwater crayfish, Cherax destructor, from the Murray River system were obtained from a commercial supplier and kept indoors at 22°C in polystyrene aquaria. They remained active and in good condition for long periods when provided with shelters, normal light-dark cycles and a change of water following a weekly feeding with dry-pellet cat food. Seventy five inter-moult specimens of both sexes were used in the experiments. Specimens with a carapace length of 6–10 cm were used. Animals were anaesthetised by immersion in ice for ten minutes and the brain destroyed. Specimens were immobilised and dissected to expose the nerves to be stained. Details of SR morphology were visualised by infusing CoCl2 intracellularly through the cut ends of the appropriate axons for periods of up to 24 hours. Some of the infusions were done in whole preparations, others were done in vivo by dissecting nerves and ganglia free and pinning the preparations in a Sylgard-lined (Dow-Corning, Midland, MI) dish flooded with physiological saline (modi- CRAYFISH ABDOMINAL STRETCH RECEPTOR PROJECTIONS fied van Harreveld’s (’36) solution: 11.68 g/l NaCl; 0.40 g/l KCl; 0.55 g/l MgCl2.6H2O; 1.51 g/l Trisma; made up in distilled H2O, pH = 7.4; developed by Pasztor and Macmillan, ’90). In most cases, 1.5% CoCl2 was used but a range of concentrations up to 5% CoCl2 solution was used to reduce the probability of missing details of fine axonal and dendritic branches because of factors associated with ionic concentration and penetration. The time and temperature were varied to achieve a balance that best suited the part of the neuronal morphology under examination. Once determined, a particular regime was used over and over to examine any area in detail because typically a regime designed for one purpose was unsuitable for others e.g., a regime to show the detail of the endings in A6 produces an overstained preparation in the ganglion of entry. A typical filling regime would be 10–12 hours at 4°C. The cobalt ions were precipitated with 0.1% ammonium sulphide (four drops per 10 ml) and left for 10 minutes. The tissue was then washed in several changes of saline over a period of 30 minutes and fixed in Bouins for 2 hours. The preparation was dehydrated and cleared and mounted in methyl salicylate. Some preparations were also silver intensified using the Timm’s method (Bacon and Altman, ’77; Altman and Tyrer, ’80; Altman, ’81) before being cleared and mounted. The mounted preparations were drawn using a drawing tube attached to a Zeiss compound microscope (Thornwood, NY). To improve our ability to differentiate between SRs from different segments, in some experiments we used NiCl2 to fill the SR from one segment and CoCl2 to fill from the other. The infused ions were then precipitated with Rubeanic Acid to obtain fills with colour differentiation (Altman, ’81; Jones and Page, ’83). These preparations were always drawn before and after intensification because the colour difference disappears following the silver intensification process. RESULTS The general morphology of the abdominal MROs in C. destructor is essentially the same as that described in other species (Alexandrowicz, ’51; Macmillan and Field, ’94; Pilgrim, ’60, ’64). Upon entering the abdominal ganglion the SRs bifurcate and send a branch to the head and a branch to A6 as in P. clarkii (Bastiani and Mulloney, ’88a). In all cases (n = 72), the tonic and phasic SR filled simultaneously and there were no obvious differences in the point of bifurcation, although the phasic SR usually has a slightly larger diameter than 245 the tonic SR. To determine whether there was an as yet undisclosed pattern to the fine branching in the ganglion of entry we conducted a number of fills (n = 24) of the second nerve of the third abdominal ganglion (A3) in large animals. As in P. clarkii (Bastiani and Mulloney, ’88a), no branches are found in consistent positions in C. destructor and all branches are short and fine. Because of the larger size of our preparations, we are able to provide further details of the branching. In C. destructor, the point of bifurcation of the two axons is invariably very close and there are numerous fine branches from both axons (Fig. 1). The branches are all short and run perpendicular to the main axon with very little branching and are found along the axons throughout the whole central region of the ganglion from the point of entry of the first nerve to slightly posterior to the entry of the second nerve. We also used large animals (n = 24) to examine the branching patterns of the SRs in the adjacent anterior (A2) and posterior (A4) ganglia. There are always some branches in both the anterior and posterior ganglion but they occur over a shorter length of the axons in the central region of the ganglia. It should be noted in respect of this last observation, however, that in C. destructor the distance between exit points of the first and second nerves is commonly greater in A3 than in A2 or A4. In the anterior ganglion the branches are similar to those in the ganglion of entry but there are far fewer of them. In the posterior ganglion there are also fewer branches but they commonly give rise to long transverse secondary branches running parallel and in close lateral proximity to the main axon (Fig. 2B). In many of the experiments designed to reveal SR branching in the posterior ganglion we also filled an accessory neuron (Fig. 2B) (Alexandrowicz, ’51, ’67; Wine and Hagiwara, ’77). The projections of the SRs into the last abdominal segment (A6) in C. destructor (Macmillan and Field, ’94) are closely similar in position and form to those described in P. clarkii (Bastiani and Mulloney, ’88a). The hook-shaped endings of the phasic and tonic units from the same segment are almost identical and in some preparations are intertwined (Fig. 3A). We filled pairs of SRs entering in different nerves of segments A3, A4, and A5 simultaneously to study the spatial relationship between them in A6 (n = 48). Simultaneous SR fills from both sides of segments A3, A4, and A5 showed that the longitudinal position of the hooked part of the pro- 246 D.L. MACMILLAN AND P.J. VESCOVI Fig. 1. Two examples of branching pattern of the SR axons in the ganglion of entry. Cobalt backfill of SRs into abdominal ganglion A3. The axons pass medially into the ganglion and then bifurcate to send a branch anteriorly to the brain and also posteriorly to the last abdominal ganglion, A6. The phasic and tonic axons branch at the same point. Both axons give rise to many short fine branches in the region between the first and second nerves but no single branch is consistent in position or shape. jection in A6 is always the same so that the pairs from the same segments are bilaterally symmetrical in this regard (Fig. 3B). Most of the secondary branching also matches closely although the finest branches show differences. In some preparations in which we filled the second nerves of A5, we stained a neuron with a contralateral cell body in A6 (Fig. 3C). To investigate the relationship between the A6 projections of the SRs from different segments we filled SRs from different segments simultaneously with Co++ and Ni++. Although we were technically able to fill more than two segments simultaneously, we found that we could not interpret the branching patterns in A6 unambiguously because of the tangle of projections. We also concentrated CRAYFISH ABDOMINAL STRETCH RECEPTOR PROJECTIONS 247 Fig. 2. Examples of branching patterns of the SR axons in the adjacent ganglia. A: Branching pattern in A4 of cobalt filled SRs entering second nerve of A3. There are typically fewer branches but some of these give rise to long transverse secondary branches that lie parallel and close to the main axons. B: In some preparations we filled accessory neurons as well as SRs. Branching pattern and accessory neuron in A4 stained in a fill of the ipsilateral second nerve of A3. on contralateral pairs because we found the preparations were easier to interpret than ipsilateral ones and on segments A3, A4, and A5 because to fill from A1 or A2 it was necessary to use such small animals that the relationship in A6 could not be determined with any reliability. The results reported here are based on 23 successful fills of combinations of SRs from segments 3 and 4, 4 and 5, and 3 and 5. In all cases examined the longitudinal position of the transverse part of the SR hook in A6 reflected the antero-posterior order of the segments of SR origin. The transverse branches from adjacent segments are 5–10 µ apart longitudinally (Figs. 4, 5A) and simultaneous fills of A3 and A5 express a multiple of this spatial order (Fig. 5B). 248 D.L. MACMILLAN AND P.J. VESCOVI Fig. 3. Projections of filled SR axons in the last abdominal ganglion (A6). A: Example of the hook-shaped endings of the phasic and tonic units that enter the ipsilateral second nerve of A5. Note the closely similar branching pattern in the phasic and tonic SRs. B: Example of a simultaneous fill of both pairs of SRs from A5. The transverse branch that forms the hook is always at the same longitudinal position in contralateral pairs. In some preparations we stained a neuron with a cell body in A6 when we filled the second nerves of A5. CRAYFISH ABDOMINAL STRETCH RECEPTOR PROJECTIONS 249 Fig. 4. Two examples of pairs of filled SR axons from contralateral sides of the adjacent segments A4 and A5. DISCUSSION Early extracellular recordings from abdominal extensor motor neurons indicated, and later intracellular studies confirmed, that the SRs excite the motor neurons in the ganglion of entry more strongly than they excite those in other ganglia (Eckert, ’61; Wine. ’77; Wine and Hagiwara, ’77; Hausknecht, ’96 and in preparation). The detailed 250 D.L. MACMILLAN AND P.J. VESCOVI Fig. 5. Examples of pairs of filled SR axons from contralateral sides of the (A) the adjacent A3 and A4 segments and (B) the A3 and A5 segments which are two segments apart. CRAYFISH ABDOMINAL STRETCH RECEPTOR PROJECTIONS information provided to us by Hausknecht (’96) from her thesis about the morphology of the extensor motor neurons in C. destructor, shows that most of them, including number 2 (for numbering details see Kennedy and Takeda, ’65; Fields, ’66; Sokolove and Tatton, ’75), which responds reflexly, have long side branches running parallel to the SR axon through the ganglionic regions close to the SR axons where the SR short branches lie (Liese et al., ’87). These branches are thus essentially perpendicular to the short SR branches and there is a considerable longitudinal overlap where connections between them or their associated neurons could occur. It may be this aspect of the relationship that explains why neurons that have been shown to have a close functional relationship (some are probably monosynaptic) do not have a stereotyped morphological one. It is possible that the precise contact points of the SRs or their associated neurons on the dendritic tree of the motor neurons are not important so long as there are sufficient at the right distance from the integrating sites. The difference in the number of fine branches found in this study of C. destructor in comparison with the study on P. clarkii (Bastiani and Mulloney, ’88a) could reflect species difference or perhaps a difference in the maturity of the animals used. Given the physiological background to the SR motor neuron relationship, our finding that there are always more fine SR branches in the ganglion of entry would not be surprising except that a detailed histological study by Liese et al. (’87) found that the total length of axonal branches from SR axons was always greatest in the adjacent posterior ganglion. While this could be a species difference between Pacifastacus leniusculus, which they used, and C. destructor, which we used, some of the other possible explanations could prove more interesting. There are typically long secondary parallel branches in the posterior ganglion of C. destructor and in any measurement of the total length of SR branches these might well compensate for any effect that a greater number of short branches in the ganglion of entry might have. From morphological analyses in other species (Liese et al., ’87) and intracellular dye fills of single neurons in C. destructor (Hausknecht, ’96 and in preparation) we conclude that the accessory neuron that we filled was Accessory 1 (Acc1). We found, like Liese et al. (’87) in their study of the Acc-1 projections, that the dendritic tree in the ganglion with the soma is not extensive. In C. destructor, however, as in the case of the ex- 251 tensor motor neurons, there are again long secondary branches from the main neurite that run parallel to the long axis of the cord in regions where the short and long parallel SR branches are found. In C. destructor the dendritic tree is more extensive in the ganglion of entry and there are even more parallel branches (Hausknecht, ’96 and in preparation) which might be expected given the strong intra and interganglionic reflex connections between the SRs and the accessory neurons. In a number of preparations in which we filled the second nerve of A5 we stained a neuron with a soma in A6 (n = 16). The morphology of the neuron is consistent with its being an accessory and, based on Hausknecht’s (’96 and in preparation) individual staining of all four accessory neurons in more anterior ganglia of C. destructor, it resembles Acc-2 most closely. The result was completely unexpected because no accessory neurons have been found associated with the A5 nerves in other species in spite of specific searching. Larimer and Kennedy (’69b) could not detect physiological evidence for their presence when they looked for an MRO-accessory reflex associated with the SR entering A5 in P. clarkii. Wine and Hagiwara (’77) confirmed that conclusion when they filled the second nerve of A5 stating that “Larimer and Kennedy concluded that the accessory efferent neuron is missing in the terminal segment; our anatomical evidence is consistent with that conclusion.” It is possible that we have described a species difference, it is also possible that it is a very difficult neuron to fill from A5 in P. clarkii. It did not fill in some C. destructor preparations, even ones in which the SRs projections filled well in the same time over the same distance. The assumption is that if this is indeed an accessory neuron, it is an accessory from the ganglion posterior to A5. But because A6 is a fused ganglion (Dumont and Wine, ’87a), the simplest supposition would be that it was originally associated with the anterior primordial ganglion. If this is so, it is surprising that the soma of the neuron lies so posteriorly in A6, almost certainly in the domain of the second primordial ganglion. Dumont and Wine (’87a,b; P. clarkii) and Vescovi and Macmillan (this volume; C. destructor) both found that the relative position of the motor neurons associated with the two primordial ganglia are generally maintained. This anomaly warrants further investigation for what it may reveal about the evolution of both A6 and the MRO-accessory complex. Somatotopic organisation of mechanosensory projections has been described in a number of situ- 252 D.L. MACMILLAN AND P.J. VESCOVI ations in insects (e.g., Murphey et al., ’80, ’89; Johnson and Murphey, ’85). Kondoh and Hisada (’87) also showed that the projections of the different nerves associated with the last abdominal ganglion in P. clarkii are topologically organised but such organisation remains to be documented at the single cell level in crustaceans. Liese et al. (’87) looked for evidence for its presence in their detailed study of sensory projections in crayfish abdominal ganglia but found none. Our data suggest that the segment of origin of each SR may determine where its terminal ending is positioned in A6. The strength of that case is dependent on a number of considerations. First, we were limited by technical considerations to pairs of neurons, so the argument is based on the finding that in no case was the projection of an SR from the more anterior segment ever found posterior to its filled pair. Second, our assumption of longitudinal position is based on the position of the transverse part of the hook and there is no a priori reason for arguing that this is representative of the functional position of the neuron. We analysed other structural features such as prominent branches, the length between branches, and the number of secondary branches to see if we could detect any other markers for longitudinal position but the variation in the level of secondary branching (also remarked by Bastiani and Mulloney, ’88a) obscured any order that might be present. Third, we found analysing bilateral pairs improved our ability to compare the neurons but this means that any interpretation of the relative longitudinal position of two filled neurons from different segments will depend to an extent on the relationship between bilateral homologues as well. We are reasonably confident that this issue does not put the conclusion about the order at risk because all bilateral homologues that we filled crossed the midline at the same point and where we did fill ipsilateral pairs of cells from different segments, the outcome was the same as that predicted by the rest of our results. Fourth, to see the relationship of the neurons in A6 clearly we were limited to large animals and because of this were only able to fill reliably from A3 back so we cannot be sure that the apparent order we found applies to the rest of the abdominal segments. Even with these caveats, the evidence appears to point to somatotopic organisation, but even if only three of the ganglia are involved, the result remains interesting, particularly in light of the physiological evidence of gradients in the strength of SR synapses in A6 (Bastiani and Mulloney, ’88b; Vescovi et al., ’97). ACKNOWLEDGMENTS Supported by a grant from the Australian Research Council to D.L.M. 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