Biol. Bull. 212: 1-5. (February 2007)
© 2007 Marine Biological Laboratory
Morphological, Cytochemical, and Cytofluorimetric Features of Supramedullary Neurons of the Fish Solea ocellata
Barbara Cuoghi and
Lucrezia Mola*
Department of Paleobiology Museum and Botanical Garden, Anatomical Museums, University of Modena and Reggio Emilia, 41100 Modena, Italy
* To whom correspondence should be addressed. E-mail: mola.lucrezia{at}unimore.it
 |
Abstract
|
|---|
Various teleost species belonging to different orders possess a particular neuronal system formed by giant supramedullary neurons (SNs). In some species, SNs are scattered along the spinal cord; in others they are organized in a compacted and well-defined cluster located at the boundary between the medulla oblongata and spinal cord. In addition to the many morphological, physiological, and histochemical studies performed both in vivo and in vitro by several authors since the end of the 19th century, quantitative microfluorometric evaluation of the DNA content of SNs has showed that clustered SNs but not aligned SNs have a DNA content much greater than the normal value of 2C. Such a high DNA content is exceptional for vertebrate neurons. In the present study, we extend this analysis of SNs to the fish Solea ocellata. Our results show that the organization of the SNs of S. ocellata is neither strictly aligned nor clustered, but somewhere in between, and that this is also true of both their morphological characteristics and DNA content values. Interspecific differences in the distribution and morphology of SNs may reflect functional differences, possibly related to environmental or behavioral differences among species. In addition, the possible functional significance of endoreplication in SNs is discussed.
The central nervous system of many teleost fish contains large cells, named supramedullary neurons (SNs), located on the dorsal surface of the spinal cord under the external limiting membrane. The SNs of some taxa (Lophiiformes, Tetraodontiformes, Batracoidiformes) are grouped in a cluster on the rostral spinal cord, whereas those in other taxa (Perciformes, Syngnatiformes, Clupeiformes, Scorpaeniformes, Pleuronectiformes) are scattered singly along the spinal cord. The morphology, number, and size of the SNs are characteristic for each species (1), while the ultrastructural features are similar for all species that have been examined and are linked to a high synthetic activity (1). Moreover, electrophysiological studies have shown that the unmyelinated axons of SNs are efferent even though they emerge from the dorsal roots of the spinal cord and are coupled by electrotonic junctions (2,3,4,5,6).
Clustered but not aligned SNs have a differential amplification (endoreplication) of DNA (7,8,9). Endoreplication, in which cells increase their genomic DNA content without dividing, is responsible for the presence of extra copies of genomic DNA and thus for very high C values, a measure of the DNA content as a multiple of the normal haploid genome size (7,8,9). This endoreplication is unusual for vertebrate neurons, which are typically fully differentiated, nondividing cells containing a diploid amount of DNA. Exceptions to this rule include hypothalamic magnocellular neurons in fish (10) and Purkinje cells in cerebellum, especially of mammals (for a review, see ref. 9). In clustered SNs, the amount of DNA in the nucleus is a multiple of the normal diploid level (2C). Moreover, the DNA content of SNs is always correlated with the size of the nucleus, cell, and animal. DNA amount can be more than 500C in Diodon holacanthus (7) or 5000C in Lophius piscatorius (8). For both species, this amplification does not occur for the entire genome but only for specific genes (7,8). Endoreplication of such a magnitude is extremely rare, occurring elsewhere only in giant neurons of molluscs (11,12).
Despite numerous morphological and physiological studies, the role of SNs has been much debated for many years. Recent research has demonstrated that SNs contain signaling molecules such as noradrenaline, a CCK-like peptide, an ACTH-like peptide, and nitric oxide, clearly indicating that SNs are a component of the autonomic nervous system (1). The SNs of the puffer fish Takifugu niphobles have axons that terminate as free nerve endings in the skin near mucous glands (13). Skin mucous cells in puffer fish may play a role in chemical defenses against parasites or predators (14). Therefore the SN system, through mucous secretion, may help protect the animal from infection or predation (15).
The possibility that SNs have a protective function makes the SN system in species of commercial significance of interest; consequently, we examined the SNs of Solea ocellata (Pleuronectiformes, Solenoides). S. ocellata is a commercially valuable species of sole, or flatfish, that is common in the western Mediterranean Sea, where it lives on muddy substrates, normally at depths of 100–300 m, but sometimes in shallower water at 30–40 m. During the day, S. ocellata lies buried in the sand protected by cryptic coloration (Fig. 1); in the evening, it hunts by swimming or gliding over the substrate.
Histological observations of S. ocellata tissues fixed in Bouin’s solution and stained with toluidine blue (0.1% for 3 h at room temperature, after treatment in phosphate-citrate buffer at pH 4.6 for 30 min) showed about 70 SNs in the spinal cord, under the external limiting membrane. The neurons were not regularly aligned, but rather were distributed singly or in small clusters of 2–4 cells in the anterior half of the cord (6–7 cm), with spaces where they were absent. The cells and nuclei had a variety of shapes. Neurons were elliptic, pear-shaped, triangular, and sometimes lobate. Nuclei were round, sub-elliptic, triangular, lobate, or pear-shaped. Nucleoli were intensely basophilic and generally round. The Nissl substance, generally finely granular, was uniformly distributed in the cytoplasm. Vacuoles and endocellular capillaries, sometimes localized near the nucleus or near the axon hillock, were also present in the cytoplasm (Fig. 2A).
View larger version (91K):
[in this window]
[in a new window]
|
Figure 2. Transversal sections of spinal cord in Solea ocellata. (A) Single supramedullary neuron stained with toluidine blue, showing the intensely basophilic nucleolus (arrowhead) and a vacuole (arrow). Bar = 50 µm. (B) Two supramedullary neurons submitted to Feulgen reaction. Nucleoli (arrowheads) are easily recognizable in the stained nuclei.
|
|
Cytometric data from 400 SNs showed a wide variation in the dimensions of cells and nuclei. The SNs could be roughly divided into three groups: 5% of the cells were large, with mean diameters of 175 µm (major diameter) x 117 µm (minor diameter) and mean nuclear diameters of 93 x 58 µm; 65% of the cells were medium sized, with cell diameters of 150 x 86 µm; the remaining 30% of the cells were smaller, with mean diameters of 70 x 47 µm and mean nuclear diameters of 28 x 23 µm. Due to the extreme variability in dimensions, it is not possible to define a statistsically valid mean size for S. ocellata SNs.
Unlike the cell and nuclei, which varied markedly in size, the nucleoli were more uniform: from 9 x 7 µm to 21 x 16 µm. Because the range of nucleoli sizes was not large, it is not possible to divide the data into three groups (Table 1).
Vacuoles and endocellular capillaries are normally absent in aligned SNs, which are smaller (1) and thus able to carry out cellular functions without internal capillaries for metabolic exchanges. Because the SNs of S. ocellata were large and had both vacuoles and endocellular capillaries, they were more similar to clustered SNs (1,16) than to aligned SNs (1).
Sections of spinal cords fixed in 10% neutral formalin and submitted to the Feulgen reaction (according to ref. 17) showed an intense positive staining of SN nuclei (Fig. 2B) and thus a high amount of Feulgen-DNA.
To extend the description of the SNs, cytological imprints were made, by gently touching the surface of slides with a small fragment of spinal cord. These slides were fixed in methanol/acetic acid (3:1, v:v) for 5 min at 4 °C; rinsed with 0.1 mol 1–1 phosphate buffer solution (PBS) at pH 7.4 for 2 min at room temperature; stained with ethidium bromide 0.04% in PBS, pH 7.4, for 30 min in the dark; and finally rinsed and mounted in PBS. Fluorescence emission was evaluated with a Zeiss Photomicroskop III equipped with a Photometer 03 microfluorimeter, HBO 100-W mercury vapor light source, dichroic filter (FT 580), exciter filter (PB 546/12), and barrier filter (LP 590). Emission of DNA fluorescence was evaluated for 300 SN nuclei (easily recognized due to their remarkable size) and for diploid small glial cells surrounding the SNs.
This cytofluorimetric evaluation revealed a DNA content ranging from 6C in the smaller SNs to 100C in the larger ones (Table 1). Thus, the DNA content varies widely and is correlated with cellular and nuclear size. These results demonstrate that DNA endoreplication occurs in S. ocellata SNs, as it does in the clustered SNs of Lophius piscatorius (8) and Diodon holacanthus (7). These giant neural cells, which are larger than the aligned SNs, might develop a high DNA content relative to the clustered SNs of other species as a means to compensate for a high metabolic rate. DNA amplification occurs in L. piscatorius and D. holacanthus in different ways: SNs amplification involves GC-rich sequences in L. piscatorius, but not in D. holacanthus SNs (7,8,9). This does not reflect different functions of SNs but only a difference in the type of genes that are amplified passively by virtue of their proximity to the functionally important ones. This result could simply be due to differences in the genomic organization of these two fish species, which are phylogenetically distant. The same hypothesis may hold for S. ocellata SNs: whatever type the amplification may be, it is related to the genome organization of the species and not to alternative SN functions.
We found the SNs of S. ocellata to be immunopositive (Fig. 3) when tested with CCK-39 antibodies (for methods, see ref. 18). In this respect, they are similar to both aligned and clustered SNs of all species previously examined (1). Thus, all SNs contain CCK-like molecules.
View larger version (159K):
[in this window]
[in a new window]
|
Figure 3. Transversal section of Solea ocellata spinal cord showing three supramedullary neurons immunopositive to anti-CCK-39. Note also peripheral vacuoles and capillaries (arrows).
|
|
Previous morphological studies on SNs from Pleuronectiformes described these neurons as aligned along the spinal cord (19,20). In particular, in Solea impar, Tagliani (20) reported about 80 SNs with a mean diameter of 200 µm. In contrast, our results indicated that the SNs of S. ocellata show characteristics intermediate between the clustered and the aligned, in that they extend for about half the length of the spinal cord scattered in small groups, not forming an authentic cluster (Fig. 4). Moreover, like clustered SNs, the SNs of S. ocellata have vacuoles, endocellular capillaries, and DNA amplification.
View larger version (24K):
[in this window]
[in a new window]
|
Figure 4. Different distribution of supramedullary neurons (SNs) in various orders of teleosts. (A, B) Typical distribution of SNs in Perciformes, Syngnatiformes, Clupeiformes, and Scorpaeniformes: (A) dorsal view of a spinal cord segment with many aligned SNs (arrow); (B) transversal section of the same spinal cord segment with a supramedullary neuron (arrow). (C, D) Distribution of SNs in Solea ocellata: (C) dorsal view of a spinal cord segment with several small groups of supramedullary neurons (arrow); (D) transversal section of the same spinal cord segment with three supramedullary neurons (arrow). (E, F) Typical distribution of SNs in taxa like Lophiiformes, Batracoidiformes, and Tetraodontiformes: (E) dorsal view of a rostral spinal cord segment with many supramedullary neurons grouped in a cluster (arrow); (F) transversal section of the same spinal cord segment with the clustered supramedullary neurons (arrow).
|
|
On the whole, our results on S. ocellata suggest that the partitioning of SNs into "aligned" and "clustered" categories is artificial, because this neuronal system shows a wide morphological variability in different species, and ranges in a graded fashion from clustered to scattered. This variability may reflect functional differences related to the environment and behavior of each species. For example, S. ocellata uses camouflage that enables it to live cryptically in sand and mud, where it is exposed to parasites and microrganisms; therefore it may be that the mucous cutaneous glands innervated by SNs function to prevent infections from these sources.
|
Footnotes
|
|---|
Received 29 June 2006; accepted 14 October 2006.
|
Literature Cited
|
|---|
- Mola, L., and B. Cuoghi. 2004. The supramedullary neurons of fish: present status and goals for the future. Brain Res. Bull. 64:195–204.[ISI][Medline]
- Bennett, M. L. V., S. M. Crain, and H. Grundfest. 1959. Electrophysiology of supramedullary neurons in Sphaeroides maculatus. I. Orthodromic and antidromic responses. J. Gen. Physiol. 43:159–188.[Abstract/Free Full Text]
- Bennett, M. L. V., S. M. Crain, and H. Grundfest. 1959. Electrophysiology of supramedullary neurons in Sphaeroides maculatus. II. Properties of the electrically excitable membrane. J. Gen. Physiol. 43:189–219.[Free Full Text]
- Bennett, M. L. V., S. M. Crain, and H. Grundfest. 1959. Electrophysiology of supramedullary neurons in Sphaeroides maculatus. III. Organization of the supramedullary neurons. J. Gen. Physiol. 43:221–250.[Medline]
- Bennett, M. V. L., Y. Nakajima, and G. D. Pappas. 1967. Physiology and ultrastructure of electrotonic junctions. I. Supramedullary neurons. J. Neurophysiol. 30:161–179.[Free Full Text]
- Zottoli, S. J., D. E. W. Arnolds, N. O. Asamoah, C. Chevez, S. N. Fuller, N. A. Hiza, J. E. Nierman, and L. A. Taboada. 2001. Dye coupling evidence for gap junctions between supramedullary/dorsal neurons of the cunner, Tautogolabrus adspersus. Biol. Bull. 201:277–278.
- Mola, L., B. Cuoghi, M. Mandrioli, and M. Marini. 2001. DNA endoreplication in the cluster supramedullary neurons of the pufferfish Diodon holacanthus L. (Osteichthyes). Histochem. J. 33:59–63.[ISI][Medline]
- Sassi, D., G. C. Manicardi, L. Mola, and I. Benedetti. 1995. Cytofluorimetric evidence for differential genome endoreplication in the cluster of Lophius piscatorius L. (Osteichthyes, Lophiiformes). Eur. J. Histochem. 39:117–126.[Medline]
- Mola, L., M. Mandrioli, B. Cuoghi, and D. Sonetti. 2006. DNA endoreplication: what did you not expect from neurons. Pp. 39–60 in New Trends in Brain Research, F. J. Chen, ed. Nova Science Publishers, New York.
- Benedetti, I., D. Sassi, G. Mescoli, and G. C. Manicardi. 1999. High values of DNA content in the hypotalamic neurons of Lophius piscatorius and Diodon holacanthus (Osteichthyes). Caryologia 52:141–146.
- Gillette, R. 1991. On the significance of neuronal giantism in gastropods. Biol. Bull. 180:234–240.[Abstract]
- Manicardi, G. C., D. Bizzaro, D. Sonetti, F. Lombardo, and U. Bianchi. 1992. Amplification of GC-rich DNAs in neuronal nuclei of Planorbarius corneus (L.) (Mollusca, Pulmonata), Eur. J. Histochem. 36:303–309.[ISI][Medline]
- Funakoshi, K., T. Kadota, Y. Atobe, M. Nakano, R. C. Goris, and R. Kishida. 1998. Gastrin/CCK-ergic innervation of cutaneous mucous gland by the supramedullary cells of the puffer fish Takifugu niphobles. Neurosci. Lett. 258:171–174.[ISI][Medline]
- Hirazawa, N., S. Oshima, T. Mitsuboshi, and S. Yamashita. 2003. Mucus pH of the tiger puffer Takifugu rubripes is an important factor for host identification by the monogenean Heterobothrium okamokoi. Parasitology 127:225–230.[Medline]
- Zottoli, S. J., F. R. Akanki, N. A. Hiza, D. A. Ho-Sang, Jr., M. Motta, X. Tan, and K. M. Watts. 1999. Physiological characterization of supramedullary/dorsal neurons of the cunner, Tautogolabrus adspersus. Biol. Bull. 197:239–240.[ISI][Medline]
- Nakajima, Y., G. D. Pappas, and M. V. L. Bennett. 1965. The fine structure of the supramedullary neurons of the puffer with special reference to endocellular and pericellular capillaries. Am. J. Anat. 116:471–492.[ISI][Medline]
- Bernocchi, G., G. F. De Stefano, F. Porcelli, C. A. Redi, and M. G. Manfredi Romanini. 1976. Feulgen reaction hydrolysis kinetics in the interphase and metaphase. Nucleus (Calcutta) 19:141–149.
- Benedetti, I., and L. Mola. 1988. Survey of neuropeptide-like immunoreactivity in supramedullary neurons of Coris julis (L.). Brain Res. 449:373–376.[ISI][Medline]
- Dahlgren, U. 1897. The giant ganglion cells in the spinal cord of the order Heterosomata COPE (Anacanthini Pleuronectoidei Guenther). Anat. Anz. 13:281–293.
- Tagliani, G. 1899. Ueber die Riesennervenzellen im Rückenmarke von Solea impar. Anat. Anz. 15:234–237.
TOP
Abstract
Literature Cited
