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Whitney Laboratory for Marine Bioscience, University of Florida
1 Department of Physiology and Functional Genomics, University of Florida
2 Department of Neuroscience, University of Florida
* To whom correspondence should be addressed at Whitney Laboratory for Marine Bioscience, 9505 Ocean Shore Blvd., St. Augustine, Fl 32080. E-mail: paa{at}whitney.ufl.edu
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Abstract |
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 TOP Abstract IntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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Introduction |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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A variety of ultrastructural (for review, see Westfall, 2004) and histological (Anderson et al., 1992; Golz, 1994) studies have reported on the association between nerves and cnidocytes, and it is now clear that cnidocytes are innervated by both sensory neurons and interneurons (Westfall, 2004). Ultrastructural studies of the innervation of cnidocytes in anemones (Anthozoa) have revealed the presence of a variety of types of synaptic vesicles, including both dense- and light-cored vesicles, suggesting the presence of a variety of transmitters, including Antho-RFamide peptides (Westfall, 2004). Here we expand on this understanding of the role of the peptidergic innervation of cnidocytes by demonstrating, in representatives of the three other cnidarian classes (Hydrozoa, Scyphozoa, and Cubozoa), a common pattern of peptidergic innervation of cnidocytes. This consists of a network or basket of peptidergic neurons that surrounds the base of clustered cnidocytes, together with a finite number of peptidergic sensory neurons that send processes to the surface of the tentacle.
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Materials and Methods |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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Excised tissue was then prepared for immunocytochemistry as described by Grimmelikhuijzen (1985). Briefly, tissues were fixed in 4% phosphate-buffered paraformaldehyde (pH 7.0) overnight at 4 °C, and rinsed (6 x 1 h) in phosphate-buffered saline containing 0.25% Triton X-100 (PBS-T). Samples were then incubated overnight at 4 °C in the presence of either an anti-FMRFamide antibody (Diasorin, Inc.) or an anti-RFamide antibody (146III provided by C. J. P. Grimmelikhuijzen, Univ. of Copenhagen) diluted 1:250 in PBS-T supplemented with 0.25% goat serum (PBS-T-G). After rinsing (4 x 1 h) with PBS-T, the samples were incubated overnight at 4 °C in secondary goat-anti-rabbit antibody conjugated to either FITC (Boehringer Mannheim) or Cy3 (Jackson Immunoresearch), diluted 1:150 with PBS-T-G. After a final round of rinsing, the samples were transferred to a drop of 90% glycerol in PBS containing 1 mg/ml O-phenylenediamine HCl on a microscope slide. The edges of the coverslip were then sealed with clear nail polish. Control samples were prepared in exactly the same manner, except that the secondary antibody was omitted. Samples were examined with either a Leica DM1RBE fluorescence microscope or a Leica laser scanning confocal microscope.
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Results |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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Equally common in all but Aiptasia were assemblies of immunoreactive neurons associated with cnidocytes. This organization was best illustrated in tentacles of Chrysaora (Fig. 1A), where cnidocytes clusters are more widely scattered than in the other species. In Chrysaora, immunoreactive nerve nets were associated with clusters of as few as two or three cnidocytes as well as with the larger assemblies of cnidocytes. These assemblages of immunoreactive neurons had the appearance of baskets composed of immunoreactive neurons 1–2 µm in diameter. At higher power (Fig. 1B), the immunoreactive neurons in Chrysaora could be seen to be located primarily at the base of the cnidocytes. However, individual processes emerged from this dense plexus and partially enveloped individual cnidocytes (Fig. 1B).
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In the two siphonophore species examined, Porpita and Physalia, the tentacles bear large assemblies of cnidocytes, called cnidosacs, that appear as a line of hemispheres or spheres arranged along one edge of each tentacle. In a well-relaxed tentacle, this organization gives the appearance of a string of small blue beads. In Porpita, the cnidosacs almost form self-contained spheres that are attached to the tentacle through a small stalk (Fig. 1D). The central core of each cnidosac was inevitably filled with large numbers of immunoreactive processes that were connected to the shaft of the tentacle by additional immunoreactive neurons. Cnidocytes from Porpita are remarkably long, and much of their length is taken up by a long (80 µm), narrow, basally directed cytoplasmic projection (Fig. 1D, inset). Thus, although the immunoreactivity in Porpita was located in the central core of the cnidosac, its location was still ectodermal, around the base of the long cytoplasmic projections.
Several sensory neurons were observed to extend to the surface of each cnidosac (Fig. 1D, E). These cells had large ovoid somata (Fig. 1E), which were typically located near the surface of the tentacle; many had a single fine process that extended into the extracellular medium (Fig. 1E). Occasionally, cells that projected to the surface of the cnidosac had more centrally located somata (Fig. 1D). The cnidosacs in Porpita were small enough to be fully reconstructed from confocal optical slices and provided a measure of the overall density of sensory neurons. A typical cnidosac contained up to 6–7 sensory neurons.
In Physalia, the cnidosacs are more hemispherical and the ectoderm thinner than Porpita. When cnidosacs were viewed from the surface, a dense plexus of immunoreactivity surrounding the dark cores of the cnidocytes was apparent (Fig. 1F). When viewed tangentially, it could be seen that the immunoreactivity was largely restricted to the base of the ectoderm of the cnidosac, around the base of the cnidocytes (Fig. 1F), but that occasional neurons emerged from the plexus and extended to the surface of the tentacle (Fig. 1F).
In the case of the hydroid Cladonema, the FMRFa-like innervation of the tentacle was particularly evident. Immunoreactive processes located in what appeared to be the base of the ectoderm covered the entire tentacle (Fig. 1G). This nerve net was particularly dense, however, at the base of the capitate portion of the tentacle, immediately under the cluster of cnidocytes (Fig. 1G, H). This organization was very reminiscent of that in the two siphonophores and of that reported for another hydroid, Coryne (Golz, 1994). Once again, single neurons occasionally emerged from this plexus and extended through the cnidocytes to the surface of the tentacle (Fig. 1H).
In the cubomedusa Chiropsalmus, the bulk of tentacular cnidocytes exist in regular bands that encircle the entire tentacle (Fig. 1I). Once again, these cnidocyte bands were associated with a dense plexus of immunoreactive neurons that was located in the ectoderm of the tentacle, at the base of the cnidocytes (Fig. 1I). Single immunoreactive cells could be seen to emerge from this plexus to the surface (Fig. 1J).
The pattern of immunoreactivity in the anthozoan Bunodosoma differed markedly from that of the other species examined. As noted above, while the anti-FMRFamide antibody was only partially effective, the anti-RFamide antibody revealed a dense network of multipolar neurons located at the base of the ectoderm. When the edges of tentacles were viewed tangentially, single ciliated sensory cells were evident (Fig. 1K) and processes from these merged with the immunoreactive nerve net. The cilia of these sensory cells emerged from the surface of the tentacle, but seemed to extend no further than the mucus layer on the surface.
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Discussion |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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The results presented here indicate that cnidocytes in both these species, together with representatives from at least two other cnidarian classes (Scyphozoa, Cubozoa) are innervated by neurons that are immunoreactive to antibodies against the peptide FMRFamide. Irrespective of the organization of the cnidocytes, be they in small planar clumps (Chrysaora), broad circumtentacular bands (Chiropsalmus), single capitate bulbs (Cladonema), or the linear arrangement of nearly cnidosacs found in Physalia and Porpita, a dense plexus of FMRFamide IR neurons is present around the base of the cnidocytes. The basal location of this plexus is best exemplified by Porpita, where the long cytoplasmic extension at the basal end of each cnidocyte creates a considerable separation between the cyst and the FMRFamide IR neurons. The basal location of this plexus is consistent with the site of neuro-cnidocyte synapses reported by other investigators (Holtmann and Thurm, 2001; Westfall, 2004), although there is evidence—in Chrysaora at least—that FMRFamide IR processes extend partway up the length of some cnidocytes, presumably forming synapses around the midpoint of the cell.
The sea anemone Bunodosoma may appear to be the exception to this pattern of peptidergic innervation. While the tentacles did contain a dense plexus of FMRFamide and RFamide immunoreactive neurons, it was not possible to discern any obvious association between these neurons and cnidocytes in the tentacles. EM studies of cnidocytes in the anemone Aiptasia (Westfall, 2004), on the other hand, have revealed the presence of RFamide immunoreactive dense-cored vesicles at neuro-cnidocyte synapses, suggesting that cnidocytes in anemones are indeed innervated by peptidergic neurons. The lack of a clear association between the immunoreactive nerve nets and cnidocytes may, therefore, simply be a consequence of the anemone’s anatomy. In Bunodosoma and other anemone species, the ectoderm of the tentacles is filled, almost uniformly, with cnidocytes and, unlike the representatives of the other classes examined, there are no discontinuities in cnidocyte coverage that could be mapped to interruptions or breaks in the underlying peptidergic nerve net. Thus, the presence of a peptidergic nerve net over the entire tentacle could merely be a reflection of the uniform distribution of cnidocytes in the tentacles.
A consistent feature of the FMRFamide IR plexus in all species examined was the presence of what appear to be peptidergic sensory neurons that emerge from the plexus. Their classification as sensory neurons is based on the peripheral location of the cell body and the presence, in many of them, of a very fine process that extends into the external medium, criteria that are consistent with those used by other investigators (Saripalli and Westfall, 1996). Cells in the capitate tentacles of the hydroid Coryne and classified as sensory cells (Holtmann and Thurm, 2001) contain dense cored vesicles that are consistent with their being peptidergic. Although the sensory cells described here are relatively rare (a single 200-µm-diameter cnidosac in Porpita contains only 6–7), the average density is on the order of one sensory process per 8,000–10,000 µm2 of cnidocyte-rich tentacle surface. However, because those sensory cells are afferent to a dense nerve net, or plexus, the result is that this organization has the potential to directly or indirectly activate a great many cnidocytes. It is not clear whether all cnidocytes in a cluster receive synaptic input directly. It is possible that only a finite number of cnidocytes are innervated but that the afferent input is conveyed to other cnidocytes through signaling systems such as nitric oxide (Salleo et al., 1996; Colasanti et al., 1997; Moroz et al., 2004) or by way of gap junctions, which are very abundant in hydrozoans (Josephson and Schwab, 1979; Spencer, 1981) and may also be present in anthozoans (Germain and Anctil, 1996; Mire et al., 2000). However, while agents that uncouple gap junctions affect the responses of hydrozoan (Price and Anderson, unpubl. data) and anthozoan (Mire et al., 2000) cnidocytes, cnidocytes in the hydroid Stauridiosarsia (Brinkmann et al., 1996) are not dye-coupled to one another or to adjacent cells, making the possible role of gap junctions equivocal.
It is not altogether surprising that these cnidocyte-specific plexuses were revealed using antibodies to FMRFamide. RFamide peptides are exceedingly common in the Cnidaria (for review, see Grimmelikhuijzen et al., 2002). They have been isolated from representatives of all classes, with the exception of the Cubozoa where peptide studies have not yet been conducted; and they have been shown to be functionally important for development (Katsukura et al., 2003) and physiological activity (McFarlane et al., 1987, 1991). However, cnidarians do not possess FMRFamide per se, but rather a variety of other peptides that terminate in the sequence RFamide peptides. The fact that FMRFamide antibodies were only minimally effective in Bunodosoma while RFamide antibodies were effective may reflect this point, and suggests that the use of the antiRFamide antibody with the other species might reveal more details of the peptidergic innervation of the cnidocytes.
It must be stressed, however, that the presence of an RFamide-like innervation of cnidocytes does not preclude the involvement of other neurotransmitter pathways. While the evidence for other types of neurotransmitter in the phylum Cnidaria is not as conclusive as for peptides, evidence for other transmitters is growing (for review, see Anderson, 2004). In addition, some evidence (Kass-Simon and Scappaticci, 2004) suggests that glutamatergic pathways may also be involved in the regulation of Hydra cnidocytes, and that dopamine is involved in modulating the discharge of cnidocytes from Corynidae (Thurm et al., 1998). Furthermore, the finding that the threshold for cnidocyte discharge is raised in satiated animals (Sandberg et al., 1971; Smith et al., 1974) suggests that regulatory pathways must be present—ones that somehow inhibit or down-regulate cnidocyte discharge.
As noted earlier, application of an extract of fish mucus to Physalia tentacles triggers bursts of electrical activity that can be recorded as synaptic events in single cnidocytes (Purcell and Anderson, 1995). That synaptic input does not, however, evoke discharge of the impaled cnidocytes, or any adjacent ones. This suggests, therefore, that the synaptic activity may serve to somehow prime the cnidocyte in preparation for the subsequent mechanical stimulus that would indicate physical contact has been made with the source of the odorant. Given the organization of the cnidocyte-associated peptidergic nerve nets presented here and the presence of what appear to be peptidergic afferent sensory neurons, it is tempting to speculate that the synaptic activity that can be recorded from the cnidocytes of Physalia (Purcell and Anderson, 1995) and Cladonema (Price and Anderson, unpubl. data) arises, directly or indirectly, from these peptidergic nerve nets. While there is no direct physiological or pharmacological evidence that peptides are involved in the cnidocyte response, there is no evidence that peptides are not involved. Furthermore, electron microscopic immunocytochemistry has revealed the presence of RFamide peptides in the nerve terminals of neurocnidocyte synapses in anemones (Westfall, 2004). Thus, given their prevalent role in many aspects of cnidarian biology and their clear association with cnidocytes in all cnidarian classes, it would be surprising if neuropeptides were not actively involved in some aspect of the regulation of cnidocyte discharge.
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Acknowledgments |
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Footnotes |
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Literature Cited |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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