International Journal of Pharma and Bio Sciences ULTRASTRUCTURE OF THE OLFACTORY ORGAN IN THE STRIPED SNAKEHEAD OPHIOCEPHALUS STRIATUS (BLOCH).

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1 Research Article Neurobiology International Journal of Pharma and Bio Sciences ISSN ULTRASTRUCTURE OF THE OLFACTORY ORGAN IN THE STRIPED SNAKEHEAD OPHIOCEPHALUS STRIATUS (BLOCH). S.C. MASRAM AND V.V. BAILE* PG Department of Zoology, Division of Fish and Fisheries, RTM Nagpur University, Nagpur , India. Professor PG department of Zoology, Division of Fish and Fisheries, Mahatma Phule Educational Campus, RTM Nagpur University, Nagpur , India ABSTRACT Olfactory organ of Ophiocephalus striatus Bloch consists of quadrangular shaped olfactory rosette, long olfactory nerve and sessile olfactory bulb. Olfactory rosette comprises of elongated olfactory lamellae arranged in rostro-caudal direction. In olfactory lamellae, sensory epithelium consists of olfactory receptor cells, supporting cells, basal cells and goblet cells. Olfactory receptor cells are of two types microvillous and ciliated. Cells of olfactory bulb are organized in four concentric layers. Outer layer is olfactory nerve layer formed byaxons of olfactory receptor cells. Inner to it is glomerular layer where axons of olfactory receptor cells synapse with dendrites of mitral cells. Next towards deeper part is mitral cell layer which comprises larger multipolar mitral cells. Central core is formed by granular cell layer with small granular cells. KEY WORDS: Olfactory organ, olfactory receptor neurons, Ophiocephalus striatus, granular cells, mitral cells. V.V. BAILE Professor PG department of Zoology, Division of Fish and Fisheries, Mahatma Phule Educational Campus, RTM Nagpur University, Nagpur , India *Corresponding author B - 955

2 INTRODUCTION The olfactory organs of teleosts are of great biological importance as they are the major sites of receiving external cues and play an important role in various behaviours including food searching, migration, predator avoidance and reproduction 1. Each olfactory organ comprises of olfactory rosette and olfactory bulb. Olfactory rosettes are placed in the snout region behind mouth and consist of numerous lamellae. The nature of olfactory epithelial cells of olfactory lamellae in a number of fish representatives of diverse groups have widely been described, viz., in rainbow trout Salmo gairdneri (Richardson) 2, Schizothorerichthys progastus (McClelland) and Schizothorax richardsonii (Gray) 3, stinging catfish H. fossilis (Bloch) 4, spotted snakehead Ophiocephalus punctatus (Bloch) 5, Indian major carp Labeo rohita (Hamilton) 6 and in bronze featherback Notopterus notopterus (Palas) 7. Though ultrastructural details of olfactory cells are studied extensively in different species but the studies on fine structure of olfactory bulb cells are available only on a few species. Present study explores the details of cells of olfactory rosette and olfactory bulb in a striped snakehead, O. striatus at light and ultrastructral levels. MATERIALS AND METHODS In this study, fishes for experiments were used in accordance with the Institutional Animal Ethics Committee, P G T D of Zoology, RTM Nagpur University, Nagpur (Registration no. 478/01/a/CPCSEA). For the present work adults of either sex of O. striatus (n=6) were collected in and around the water bodies of Nagpur City for a period of 2 years ( ). They were brought to the laboratory and acclimatized in aquarium. After acclimatization, fishes were anesthetized and olfactory organs were dissected out and immediately fixed in Bouin s fixative for 18 to 24 hours cut in transverse and saggital planes at 8 to 10 µm, stained by Kluver Barrera technique and observed under compound microscope. Scanning electron microscopy The fishes were anesthetized with an overdose of 2-phenoxy ethanol and perfused transcardially with phosphate buffer (ph 7.4) followed by 2.5% glutaraldehyde containing 2% paraformaldehyde in 0.1M phosphate buffer (ph 7.4, primary fixative). The olfactory organs were dissected out and kept in the same fixative overnight. After fixation, tissues were washed with phosphate buffer for 2-3 hours. Then tissues were dehydrated in the ascending series of acetone (from the 30% to the absolute acetone), dried in CO 2 at critical point, and shadowed with gold. The samples were examined at 15 kv in Leo 435 VP low vaccum scanning electron microscope. Transmission electron microscopy Adults of O. striatus were perfused and the olfactory organs were dissected out and fixed in ice cold 2.5% gluteraldehyde containing 2 % paraformaldehyde in 0.1 M phosphate buffer at ph 7.4. The tissues were washed in phosphate buffer and postfixed in 1 % osmium tetraoxide (secondary fixative) for 2 hours at 4⁰C. After post fixation, the tissue pieces were washed in phosphate buffer, dehydrated through ascending grades of alcohol, cleared in toluene and embedded in epoxy resin. Semithin sections of 1µm were stained with toluidine blue and examined by light microscopy. Ultrathin sections (silver to gold) were stained with uranyl acetate followed by lead citrate and examined under a Philips-10 transmission electron microscope. The electron microscopy work was carried out at the National Electron Microscope Laboratory, All India Institute of Medical Sciences, New Delhi, India. RESULTS Snake head O. striatus possesses a pair of olfactory chambers situated dorso laterally in the snout, anterior to the eyes. Olfactory chambers are connected to the external environment through separate external nasal apertures. Anterior incurrent apertures open B - 956

3 through a short muscular tube just above the upper jaw while the posterior inner one opens directly through a slit. Olfactory system of this fish is composed of olfactory rosette (OR), olfactory nerve (ON) and olfactory bulb (OB) (Fig. 1a). Long olfactory nerve connects the OR with the olfactory bulb. Olfactory bulbs terminate on area ventralis telencephali of the forebrain and are sessile. Light microscopic structure of olfactory lamellae and olfactory bulb Olfactory lamellae bear non sensory epithelium and sensory epithelium. Non-sensory epithelium occupies distal area while sensory epithelium covers proximal and distal portions (Fig. 1b). Sensory epithelium consists of supporting cells, olfactory receptor cells and basal cells (Fig. 1c) In O. striatus, olfactory bulb comprises four layers from periphery towards the inner side as observed under light microscope: olfactory nerve layer (ONL), glomerular layer (GL), mitral cell layer (MCL) and granular cell layer (GCL) (Fig.1d). Figure 1 a Dissected head of O. straitus showing olfactory rosette (OR), olfactory nerve (ON) and olfactory bulb(ob), scalebar 1cm; b transverse section of olfactory rosette showing nonsensory (NSE) and sensory epithelium. At the base of olfactory lamellae, axons coming from ORCs seen to form fascicles (arrow), scalebar 400 µm; c t. s. of sensory epithelium showing olfactory receptor neuron (ORN), supporting cell (SC), basal cell (BC) and central core (arrow), scalebar 40 µm; d t. s. of olfactory bulb exhibiting olfactory nerve layer (ONL), glomerular layer (GL), mitral cell layer (MCL) and granular cell layer (GCL), scalebar 1mm. B - 957

4 SEM structure of olfactory epithelium Olfactory rosettes (OR) are situated on the floor of nasal chambers and are quadrangular in shape (Fig. 2a) and possess crescent-shaped olfactory lamellae (OL) which are arranged parallel to each other in rostro-caudal direction(fig. 2b). Sensory epithelium is composed of olfactory receptor cells (ORC) and supporting cells (SC) arranged in alternate rows. The apical surface of the supporting cells is broad, flat and provided with fingerlike microridges (Fig. 2c). The ORCs are of two types: ciliated olfactory receptor cells (corc) and microvillous olfactory receptor cells (morc) which are intermingled and distributed throughout the olfactory epithelium. The apical surface of morc is broad and consists of numerous microvilli (Fig. 2d) while the apical surface of corc is raised in small round olfactory knob and is provided with cilia (Fig. 2e). Non-sensory epithelium shows a dense mat of cilia of ciliated non sensory cells (Fig. 2f). Apical surface of supporting cells in nonsensory epithelium are provided with zigzag pattern microridges (Fig. 2g). Many mucus or goblet cell s openings with secreted mucus balls have been observed throughout the epithelium (Fig. 2h). Figure 2 a SEM microphotograph a quadrangular olfactory rosette scalebar 10 mm; b olfactory lamellae (curved tailed arrow) separated from each other by trough (arrow), scalebar 2mm; c supporting cells of sensory epithelium, scalebar 10 µm; d microvilli of microvillous receptor neurons (arrow), scalebar 1 µm; e olfactory knob (arrow) of ciliated olfactory receptor neurons, scalebar 10 µm; f dense mat of cilia over nonsensory epithelium, scalebar 10 µm; g pits (arrow) and microridges (curved tailed arrow) observed over nonsensory epithelium, scalebar 5 µm; h mucus ball (arrow) over nonsensory epithelium, scalebar 2 µm. B - 958

5 TEM structure of olfactory epithelium Under transmission electron microscope, corc appear columnar and bipolar cells bearing a cell body and posterior axon. It bears elongated nucleus with denser nucleoplasm and nucleoli. Long axonal process arises from cyton and runs towards the basal lamina while dendrite extended towards outer side with 7-8 long cilica. morc are also columnar but without cilia (Fig. 3a). Goblet cells are large, oval, glandular cells found alongwith sensory and non-sensory epithelium. Apical part of the goblet cells is filled with secretory vesicles. Nucleus occupies basal part of the goblet cell (Fig. 3b). Basal cells are small, oval in shape with a prominent nucleus lying in deeper part of the epithelium just above the basal lamina. In basal cell, nucleus is quite large and occupies maximum area leaving very little space for the cytoplasm (Fig. 3b). White cell present in the epithelium towards apical part. Large nucleus with distinct nuclear membrane and nucleolus is present in central part. Vesicles are less as secretory organelles are scanty but it comprises several mitochondria (Fig. 3d). Figure 3 TEM microphotograph of olfactory lamellae a ciliated olfactory neurons (arrowhead) and microvillous olfactory neurons (arrow), scalebar 2 µm; b goblet cell (arrow) and white cell (arrowhead), scalebar 6 µm; c basal cell (arrowhead), scalebar 5 µm; d white cell exhibiting nucleus (arrow) and mitochondria (arrowhead), scalebar 1 µm. Supporting cells are elliptical to columnar in shape without dendrite and axonal process.these cells show rounded nucleus and dense nuceloplasm (Fig. 4a). The nucleus of supporting cell shows numerous pores ( Fig. 4b). Away from nucleus, smooth endoplasmic reticulum too is well discernible. In close proximity of the smooth endoplasmic reticulum, lipid filled vesicle is noticed (Fig. 4c). Besides ER, Golgi complex with its parallel arranged cisternae are observed (Fig. 4d) together with large number of vesicles. Mitochondria could not be observed in supporting cells. B - 959

6 Figure 4 TEM microphotograph a supporting cell (arrow) with spherical nucleus (arrowhead), scalebar 2 µm; b round nucleus of supporting cell showing double layered nuclear membrane (arrow) and nuclear pore (arrowhead), scalebar 1 µm; c smooth endoplasmic reticulum of supporting cell (arrow), dark lipid vesicle (arrowhead) are also seen, scalebar 1 µm; d golgi apparatus of supporting cell with prominent cisternae (arrow) and newly formed vesicles (arrowhead), scalebar 1 µm. TEM structure of olfactory bulb Four concentric layers as noted in light microscopy are evident in ultrastructural details in the olfactory bulb. Outermost olfactory nerve layer (ONL) is formed of thin, unmyelinated axons extending from olfactory rosette and entering the bulb from the anterior side. Axons of olfactory nerves synapse with the dendrites of mitral cells (Fig. 5a). Mitral cells of MCL are multipolar and clearly show numerous dendrites arising from its cyton extending towards the GL. Nucleus of bigger mitral cell is triangular, darkly stained with dense nucleoplasm and nucleoli (Fig. 5b). Synaptic bulb of mitral cell axon is clearly seen to have vesicles filled with neurotransmitter (Fig. 5c). Mitral cell axons are surrounded by protecting and supporting neuroglial cells (Fig. 5c). GCL shows number of small bipolar granular cells. Nucleus of granular cell also occupies maximum part of cell body. Along with the granular cell, glial cells are seen throughout the GCL (Fig. 5d). B - 960

7 Figure 5 TEM microphotograph of olfactory bulb a glomerular layer where telodendrite of olfactory receptor neuron (arrowhead) synapses with dendrite (arrow) of mitral cell, scalebar 4 µm; b large mitral cell (arrow) with dendrites (arrowhead), scalebar 2 µm; c axon of mitral cell (arrow) bearing synaptic bulb (curved tailed arrow) with neurotransmitter filled synaptic vesicles (arrowhead), note axon of mitral cell seen surrounded by neuroglial cells (triangle), scalebar 1 µm; d granular cell (arrow) with round nucleus (arrowhead), scalebar 1 µm. DISCUSSION The olfactory organ, almost in all the teleosts occupies a constant and fixed position lodged in a shallow depression in the ethmoid bone of the skull. Depending upon the families or groups, characteristic minor variations occur in details of the nostrils, the rosette and olfactory cavity. In common carp, Cyprinus carpio (Linnaeus) 8, L. rohita 9 (Bhute et al., 2007), in N. notopterus 7, olfactory rosette bears olfactory lamellae radiating outward from a long median central raphe. In O. striatus on the contrary, long olfactory lamellae are arranged in parallel fashion. Sensory region is at the proximal end and basal regions of lamellae in N. notopterus 7 B - 961

8 while in L. rohita, sensory region occupies middle of the lamellae and nonsensory region is at the proximal and distal regions on either side of sensory region of lamellae 9. Similar to N. notopterus, in O. striatus sensory region is at the proximal and basal part of the olfactory lamellae. SEM and TEM studies of the olfactory epithelium in H. fossilis 10,4 and Japanese catfish Parasilurus asotus (Linnaeus) 11 show the lamellar surface of olfactory with three clearly distinct areas; sensory epithelium, ciliated non-sensory epithelium and non-ciliated non-sensory epithelium. In O. striatus, nonsensory epithelium does not show such separation in ciliated and non-ciliated areas. Sensory epithelium in O. striatus is formed of olfactory receptor cells (ORCs), supporting cells, goblet cells and basal cells. Two major types of ORCs, ciliated olfactory receptor cells (corcs) and microvillous olfactory receptor cells (morcs) are found this fish. These ORCs are also reported in Salmo gairdneri 2. Another type of ORCs called as rod cells are reported in Salmoniformes 12 and Siluriformes 13. Crypt ORCs are reported in olfactory sensory epithelium of fishes 14. Both these cells however, could not be observed in the sensory epithelia of O. striatus suggesting that they are not the regular type of cells. corcs and morcs are distributed in the sensory area of O. striatus. A ciliated cell ontogenetically precedes the microvillous receptor cells in S. gairdneri 2. Dense gathering of golgi complex in the receptor cells in O. striatus indicates its secretory nature. Presence of stimulatory neuropeptide GnRH in the ORCs and their projections to the olfactory bulb are revealed in 14 major carp Cirrhinus mrigala (Hamilton) suggesting the role of ORCs in transduction of environmental cues and further transmitting it through brain-pituitary-gonadal axis. ORCs are also characterized in O. striatus, whose neuroanatomy will help to understand the role of olfactory system in the signal transduction in further areas of brain and their subsequent role in reproduction in this fish. Supporting cells with rounded nuclei are seen placed on the side of ORCs. These cells are reported to perform several functions; secretory, absorbing and glial 15,16. Ciliated supporting cells can be distinguished from the ciliated receptor cells because of the round shape of nucleus and absence of olfactory knob in ciliated supporting cells. Presence of large number of vesicles and golgi complex suggests the secretory nature of supporting cells and absence of mitochondria indicates slow metabolic activities in these cells in this fish. Basal cells in the fish under study are present towards the basal region just above the basal lamina. These cells work as stem cells for regeneration of lost or damaged non-sensory and goblet cells 18,19. The basal cells can also be the progenitors of receptor cells 19,20. Elongated goblet cells are distributed in the distal and surface layer of the lamellae. These cells are filled with secretory vesicles. Secretion of goblet cells helps in facilitating the odorant removal 20. Presence of large number of mucous cells is a noteworthy feature of the olfactory epithelium of freshwater teleostean fishes. Such cells make the epithelium highly secretory. Mucus is distributed uniformly over the olfactory epithelium. Acid mucopolysaccharides in the epithelium may have some antitoxin effects and may protect the epithelium from various types of infection. The mucus secreted by these gland cells in the olfactory epithelium of Ophiocephalus species may help to keep their olfactory lamellae clear of mud and other fine sediments which would otherwise clog them, because these fishes usually inhabit muddy waters of ponds and shallow pools 21. Occurrence of white cell is reported in the neuropeithlium of O. mykiss 22. Similar kind of cell is described in olfactry epithelium of H. fossilis (Datta & Bandopadhyay, 1997). Olfactory bulb is either pedunculated or sessile. Pedunculated olfactory bulb is rostrally attaches with olfactory rosette. Pedunculated olfactory bulb is found in C. auratus, elephantnose fish Gnathonemusi petersi (Gunther) 23, L. rohita 9 and N. notopterus 7. Sessile olfactory bulb is attached directly to the telencephalon. Sessile olfactory bulb is reported in O.mykiss, A.angulla, three spined stickleback Gasterosteus aculeatus (Linnaeus) 23 and O. punctata 5. TEM studies elaborate the pattern of internal structure of olfactory bulb in O. striatus which was observed B - 962

9 under the light microscope. Olfactory nerve fibers entered the olfactory bulb and peripherally form the olfactory nerve layer in N. notopterus 7. These axons march inward and synapse with the dendrite of mitral cell in glomerular layer (GL) 24. In mykiss, the glomerular layer appears to contain nine discrete terminal fields, each of which receives convergent input from all rosettes in the olfactory epithelium 25. In O. striatus, axons of olfactory nerves synapse with dendrites of multipolar mitral cells forming GL. Distal dendrites of mitral cells synapse with fibers of olfactory nerve layer while proximal dendritic shaft makes contact with granule cell dendrites 24. Axons of mitral cells originate in the basal part of the soma, become myelinated after some distance and projected in the medial and lateral olfactory tract 26. In pedunculated olfactory bulb of C. carpio, some slight but significant morphological differences between medially and laterally located mitral cells are reported 27. These morphological differences might be correlated with different physiological properties 28. CONCLUSION Sensory epithelium comprising supporting cells, olfactory nerve cells and basal cells occupies proximal and basal part of olfactory lamellae in O. striatus. Two types of olfactory nerve cells viz., cryptic and microvillous are reported in present study. Besides olfactory nerve cells other cells like goblet cells and basal cells are observed. White cells are prominently seen but significance of white cells in the physiology of olfaction is still remaining unexplored. In olfactory bulb, mitral cells, granular cells along with synpases in glomerular layer are studied by electron microscope. By studying the anatomical details of olfactory organs present study will open new avenues for further investigation of chemical neuroanatomy, sensory signal processing. REFERENCES 1. T.J. Hara. Mechanism of olfaction. In: T J Hara (ed.), Fish chemoreception, Chapman and Hall, London, 1992, pp Zielinski BS and Hara TJ, Morphological and physiological development of olfactory receptor cell in rainbow trout (Salmo gairdneri) embryo. J Com Neurol, 271: , (1988). 3. Singh N, Bhatt KC, Bahuguna MK and Kumar D, Fine structure of olfactory epithelium in Schizothoraicthys progastus McClelland and Schizothorax richardsonii Gray (Cyprinidae: Teleostei) from Garhwall Himalaya(India). J Biosci, 20: , (1995). 4. Datta NC and Bandopadhyay SK, Ultrastructure of cell types of the olfactory epithelium in a catfish, Heteropneustes fossilis (Bloch). J Biosci, 22: , (1997). 5. Mandal DK, Roy D and Ghosh L, Structural organization of the olfactory epithelium of a spotted snakehead fish, Channa punctatus. Acta Ichthyol Piscatoria, 35: 45-50, (2005). 6. Bhute YV and Baile VV, Organization of the olfactory System of the Indian Major Carp Labeo rohita (Ham.) : A Scanning and Transmission Electron Microscopy study, J Evol Biochem Physiol, 43: , (2007). 7. Baile VV, Raut IN and Bhute YV, Organization of olfactory system, forebrain and pituitary gland of a teleost, Notopterus notopterus, Annals of Neuroscience, 15: 43-50, (2008). 8. Chakrabarti P and Choudhary SH, The fine structural organization of the olfactory epithelium of Cyprinus carpio (Lin.): a scanning electron microscopic study, Folia Morphol, 66: 10-14, (2007). 9. Bhute YV, Masram SC, Raut IN and Baile VV, Cytoarchitectonic pattern of the olfactory system, forebrain and pituitary gland of the Indian Major Carp, Labeo B - 963

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