Brain and Sense Organ Anatomy and Histology of the ... - Lannoo Lab

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BRAIN AND SENSE ORGANS OF ELEGINOPS MACLOVINUS 87 ended in Cape Town, South Africa on July 17, 2004. During a stop at Stanley (51842 0 S; 57851 0 W) in the Falkland Islands, we captured Eleginops maclovinus (Cuvier and Valenciennes, 1830) with a 30-m long beach seine in Fish Creek (water temperature 58C), Port Louis, East Falkland Island on May 29. These specimens were 29–49 cm TL, 24–43 cm standard length (SL) and most were males since they had not yet reached the length at which transformation to females typically occurs. In describing brain nuclei, we follow the nomenclature of authors included in Northcutt and Davis (1983) and Davis and Northcutt (1983) with the exception of the lateral line nerves. What have been traditionally termed the anterior and posterior lateral line nerves of fishes are actually ‘‘complexes’’ consisting of several distinct cranial nerves (Northcutt, 1989; Northcutt and Bemis, 1993). We follow Northcutt (1989) in distinguishing the anterodorsal and anteroventral lateral line nerves as innervating both canal and superficial neuromasts on the head. When describing ocular structures and vasculature, we use the nomenclature of Nicol (1989). Histology We fixed specimens onboard ship by transcardial perfusion of Bouin’s fixative. After anesthetization in a solution of 3-aminobenzoic acid ethyl ester (MS-222, Sigma), the heart and bulbus arteriosus were exposed. Notothenioid saline solution (O’Grady et al., 1982) was prepared, adjusted with NaCl to a concentration of 330 mOsm/L, maintained at ambient seawater temperature, and perfused through the ventral aorta. Saline was followed by Bouin’s fixative. During this perfusion, the gills were periodically irrigated with seawater of ambient temperature (58C). Three hours after perfusion, we removed brains and other tissues of interest and postfixed them in Bouin’s. After several days of fixation, we transferred tissues to 70% ethanol for storage and transport. We subjected brains to the following protocol: dehydration in alcohol, clearing in butanol, and embedding in paraffin according to standard procedures (Kiernan, 1990). Embedded brains and spinal cords were cut in a transverse plane on a rotary microtome to produce sections 10–12-lm thick. Sections were mounted on slides, dried, deparaffinized, stained with hematoxylin and eosin, dehydrated, and coverslipped using Cytoseal 60 as the mounting medium. We tried the traditional neurological stain for Nissl substance, 0.1% aqueous cresyl violet acetate, on these brains but slides would not adequately destain, probably because of the effects of fixation in Bouin’s solution. We cut histological sections of the eyes of five specimens. In examining the eyes, we took dorsoventral strips from the central retina immediately temporal (lateral) to the optic disk. We also took transverse sections of the ventral retina in the area of the choroid fissure. We employed the histological protocol outlined earlier except that CitriSolv (Fisher Scientific) was substituted for butanol and sections were cut at 7 lm. In measuring retinal thickness, we excluded the optic nerve fiber layer. Sections were stained with hematoxylin and eosin, phloxine B, and methylene blue, Gomori’s one step trichrome, or Bodian’s Protargol for 24 h at 508C. We counted olfactory lamellae in six specimens of Eleginops maclovinus. We also examined transverse and longitudinal serial histological sections of the olfactory apparatus and of the head, with an emphasis on the membranous canals of the cephalic lateral line system. We used the same histological techniques outlined earlier. Other Morphological Procedures We examined the cephalic lateral line canal system to determine whether or not Eleginops maclovinus has membranous canals branching off of the bony canals. For this, we used specimens that had been cleared and stained with alizarin red S (Taylor, 1967) dissolved in 75% ethyl alcohol (Springer and Johnson, 2000). The head skin was left intact to preserve the membranous canals. We used yellow Microfil 1 (Flow Tech, Carver, MA), a liquid silicon rubber injection compound, to demonstrate ocular blood vessels. We injected fish in the caudal vein with 0.3 ml of heparinized (20 mg/ml) notothenioid saline and then returned them to the holding tank for 15 min. After anaesthetization, we placed the specimens ventral side-up on an iced surgical platform. We cut the bulbus-ventricle junction and cannulated the ventral aorta with a 34-cm length of either PE-50 tubing (0.96 mm OD) or PE-160 tubing (1.57 mm OD). We blunted the free end on a hot plate and secured the cannula with two sutures. The cannula was in turn connected to a 23-G or 18-G needle, an 84-cm extension tube and a 20-ml syringe. The entire apparatus was surrounded by ice to maintain a body temperature of 58C. We placed the syringe in either a Sage model 341B or a KD Scientific model 100 syringe pump and began the perfusion with notothenioid saline followed by Microfil. Flow rate was about 0.5–1.0 ml/min. We allowed the Microfil to polymerize while maintaining the specimen on ice for about 1 h, and then preserved the specimen in 10% formalin with subsequent storage in 70% ethanol. Since Microfil did not fill hyaloid arteries at the vitreoretinal interface in Eleginops maclovinus, another means of assessing the presence or absence of these vessels was necessary. We removed a block of skull containing the eyes from four Microfil specimens and dissected away the cornea, lens, vitreous body, and some of the vitreous membrane. We introduced a reversible stain (a mixture of 1 part of 1% aqueous aniline blue and 10 parts of saturated aqueous picric acid) into the vitreous chamber for 12 min. We then rinsed with water and returned the specimen to 70% ethanol for additional dissection, examination, and storage. If present, the hyaloid arteries stain more darkly than the background and can be seen radiating from the falciform artery at the optic disk. RESULTS Brain and Cranial Nerve Anatomy In gross view, the brain of Eleginops maclovinus exhibits a mixture of reduced and hypertrophied structures (see Fig. 3). The olfactory bulbs are large and positioned rostral to the telencephalon. The telencephalic lobes are about twice the volume of the olfactory bulbs and roughly half the volume of the tectal lobes. Lobules of the telencephalon are pronounced, with dorsodorsal, dorsomedial, and dorsolateral nuclei prominent. The tectal lobes (vision) are small- to medium-sized, as are the inferior lobes (Fig. 3A). The corpus cerebellum (motor skill) is large, dorsally expanded, and caudally directed. The eminentia granularis and crista cerebellares, two structures associated with mechanoreceptive lateral line inputs, are well developed. A decussation of the crista cerebellares is present, bridging the fourth ventricle (Fig. 3B). Cranial nerves exhibit proportions atypical for perciforms but in accordance with Eleginops brain regions. The optic nerve is the largest-diameter cranial nerve and is pleated, but is relatively small for a perciform brain, only slightly larger than the olfactory nerve. The olfactory nerve is relatively large and exhibits a prominent bulge immediately rostral to the olfactory bulb (Fig. 3A). The anterior dorsal and anterior ventral lateral line nerve complexes are more than twice the diameter of the Journal of Morphology DOI 10.1002/jmor
88 J.T. EASTMAN AND M.J. LANNOO Fig. 3. Brain and cranial nerves of Eleginops maclovinus (SL 5 34.5 cm) in left lateral (top) and dorsal (bottom) views 34.3. To illustrate features of the rhombencephalon in dorsal view, the drawing was made from a slightly oblique dorsocaudal angle. Therefore structures such as the corpus of the cerebellum are not perfectly aligned in the two views of the brain. ADLL, anterodorsal lateral line nerve complex; AVLL, anteroventral lateral line nerve complex; CC, crista cerebellaris of the rhombencephalon; CCb, corpus division of the cerebellum; Dl, dorsolateral subdivision of the telencephalon; Dd, dorsodorsal subdivision of the telencephalon; Dm, dorsomedial subdivision of the telencephalon; EG, eminentia granularis division of the cerebellum; IL, inferior lobe of the diencephalon; OB, olfactory bulb; Pit, pituitary gland; PLL, posterior lateral line nerve complex; SN1, first spinal nerve; SN2, second spinal nerve; SV, saccus vasculosus; Tec, tectum of the mesencephalon; Tel, telencephalon; I, olfactory nerve; II, optic nerve; III, oculomotor nerve; IV, trochlear nerve; V, trigeminal nerve; VII, facial nerve; VIII, auditory/vestibular nerve; IX, glossopharyngeal nerve; X, vagus nerve. posterior lateral line nerve complex. The vagus nerve is large. The octaval nerve (hearing and balance), oculomotor, trochlear, and abducens nerves (eye movement), as well as the spinal nerves are proportional. Brain and Spinal Cord Histology Olfactory nerve and telencephalon. In gross view, the olfactory bulbs are sessile, positioned at the rostral base of the telencephalon. Before entering the olfactory bulbs, the olfactory nerves exhibit expansions. Histologically, these expansions consist of fiber bundles interspersed centrally with sparse groups of small cells (Fig. 4A). Cell types characteristic of the olfactory bulbs, including glomerular cells and the medial smaller cell layers (not labeled), appear caudal to the olfactory nerve expansions and rostral to the leading edge of the telencephalon (Fig. 4B). The rostral portion of the telencephalon is positioned over the caudal portion of the olfactory bulbs and consists primarily of the dorsal division, characterized by uniform, small cells. Cell groups present but not particularly prominent include the dorsodorsal, dorsomedial, and dorsolateral nuclei (Fig. 4C). At the level of the anterior commissure, the dorsodorsal, dorsomedial, dorsolateral, and dorsocentral nuclei are prominent, as are nuclei in the ventral division, including the ventrodorsal, Journal of Morphology DOI 10.1002/jmor
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