Prepared by Judith Handlinger

1. Systematic Fish PathologyPart 12. Pathology and diseases of the musculoskeletal and nervous systemsSection B: Nervous systemPart I: Anatomy and microanatomy of the nervous system and sensory organs Prepared by Judith Handlinger With the support of Animal Health Laboratory, Department Of Primary Industries, Parks, Water and Environment, Tasmania, for TheAustralian Animal Pathology Standards (AAPSP) program

2. Course Outline A. Systematic Fish Pathology 1.Consider the Fish: An evolutionary perspective on comparative anatomy and physiology 2. Pathology of the kidney I – interstitial tissue Part A 3. Pathology of the kidney II – interstitial tissue Part B 4. Pathology of the kidney III – the nephron 5. Pathophysiology of the spleen 6. Fish haematology 7. Fish immunology – evolutionary & practical aspects 8. Pathology of the digestive system I – the oesophagus, stomach, & intestines. 9. Pathology of digestive system II – the liver and pancreas, swim bladder, peritoneum. Section A (this presentation) – general & non-infectious pathology Section B (following presentation): Pathology of infectious causes. 10. Pathology of fish skin 11. Pathology and diseases of circulatory / respiratory system – heart, gills and vessels 12. Pathology of the musculoskeletal system and nervous systems A: Musculoskeletal system B: Nervous system (this presentation) 13. Pathology of gonads and fry

3. Anatomy & microanatomy • Fish brains show less complex grey matter than higher mammals, reflected as less surface folding, a smaller telencephalon, usually well developed olfactory lobes (anterior to the dissected area in this photo), & large optic lobes that usually form the largest part of the brain . Telencephalon Optic lobes Cerebellum Lower jaw

4. Sampling - brain access : There are two approaches to the brain, choice dependent on size / practicality & the samples required. Trans-section of the head as shown exposes the brain for collection of non-site critical samples such as bacteriology, virology, PCR, but is usually less suitable for broad scope histological examination. Note the small brain size (lower photo) in 3 fish transsected at different levels. [The periorbital tissues are congested in all 3 fish (lower photo). Marked unilateral exopthalmia (‘pop-eye’) due to disease is present in one fish (above & right).] (Gloves should be worn!) Brain Peri-orbital tissues (congested)

5. The other approach is progressive removal of overlying bone. (These brains are also congested).

6. Exposed brain of striped trumpeter (fixed specimen) Relative lobe size does vary between fish species. Predators and fish with more complex social interactions tend to have larger brains. Brain size also tends to decrease with depth (perhaps because these factors also decline with depth). Olfactory lobes Telencephalon Optic lobes Cerebellum Striped trumpeter brain dissected to show cerebellum & spinal cord Photos, G Grossel, used with permission

7. Striped trumpeter brain – ventral aspect: Upper photo shows the ventral aspect of the above brain, including the pituitary (arrowed) and anterior to that the severed optic chiasma. In the lower photo, the pituitary and optic nerves have been removed, better exposing the highly vascular saccusvasculosusposterior to the pituitary. In some fish this is hidden between the two inferior lobes of the brain (on the ventral side of the diencephalon). Pituitary Saccusvasculosus Photos, G Grossel, used with permission

8. Histological appearance of the brain of Rainbow trout fry. (Triploid, normal except for electrocution trauma to some fish, including this one - note haemorrhage in the third ventricle.) Fragment of saccusdorsalis Cerebellum 3rd ventricle Saccusvasculosus Pituitary

9. The histological appearance of the fish brain is similar to that of mammals, but the functional aspects are much less studied (and seldom of concern to pathologists, except perhaps more recently as models for human disease). The reduced cortical folding compared to higher vertebrates, and the results of most problem solving experiments, has lead to the assumption that the fish brain is more “primitive” and less complex. While this is no doubt true with regard to problem solving “intelligence”, the extent of complexity is only now being explored (e.g. Mueller, 2012) Cartoon source unknown, widely circulated on Facebook.

10. Histological appearance of the spinal cord is similar to that of other vertebrates (3 g Atlantic salmon).

11. Spinal cord in cross-section (10 g Atlantic salmon).

12. Spinal cord continued: histology. In fish with abundant and obvious eosinophilic granular cells [ECGs], such as this Angel Fish, these may be seen in association with peri-spinal tissue, and in this case within the cord – significance unknown. (No signs of infectious disease in these fish, some losses following mixing of groups with some ocular haemhorrhages suggesting social stress & possibly other water quality factors.)

13. Cohort of Rainbow trout triploid above. The saccusvasculosus, posterior to the pituitary, has a single layer of columnar to cuboidal cells (called coronet cells) lining blood vessel rich villus-like projections into a spacious lumen. Note also the bony depression housing the pituitary. 3rd ventricle Saccusvasculosus Pituitary

14. Brain appendages continued: Atlantic salmon, 15 g triploid fry, killed with a blow to the head (otherwise normal). The above section shows the pituitary and saccusvasculosus in close proximity. This rare plane of section shows these to be joined. Detail of the saccusvasculosusof a similar fish, showing the simple folded & vascular structure. The complexity of folding (or not) does vary with species. Association of this organ with the pituitary is consistent with the recent finding that the saccusvasculosus (whose function has long been a mystery despite much speculation), has seasonal sensor functions similar to the pars tuberalis of mammalian and bird pituitary glands (Nakane et al, 2013). (The fish pituitary as shown does not contain an anatomically distinct pars tuberalis.) They concluded this organ acts as a sensor of seasonal changes in day length based on expression in the coronet cells of key genes regulating seasonal reproduction in higher vertebrates (that is, the rhodopsin gene family, rhodopsin being the “visual purple” of mammalian rods, responsible for monochromatic vision in the dark).

15. Same slide as saccusvasculosus above. Detail of the pituitary, showing the different secretory zones. Inset: pituitary overview. Neurohypophysis Detail of secretory zones (adenohypophysis). The rostral pars distalis is composed mainly of prolactin secreting cells, though ACTH secreting cells (corticotrophs) are also present. Growth hormone, gonadotrophin are produced in the proximal pars distalis. Thyroid stimulating hormone is reported in either rostral or proximal pars distalis, in different species Melanophorestimulatin hormone is produced in the pars intermedia. Pars intermedia Rostral pars distalis

16. Brain appendages continued: The extent of pituitary protection varies between species (and probably age), that of this mature Blackmore goldfish being much deeper.

17. Brain appendages continued: Another cohort of the two previous fish (15 g Atlantic salmon), this time showing the dorsal surface of the brain and the saccusdorsalis, a similar sac-like organ with highly vascular internal folds, covered by simple epithelium. Anatomically this is a rostro-dorsal evagination of the diencephalic roof, communicating with the cerebro-spinal fluid, and often folded round the small pineal gland. No examples of pineal gland in section in our collection. Photos of pineal gland by US & Wildlife service available on line @ http://histology-world.com/photoalbum/displayimage.php?album=73&pid=1933#top_display_media These show a typical acinar structure. Like other vertebrates, the pineal gland pinealocytes, which have similarities with and are believed to have evolved from photoreceptor cells such as retinal cells, produces melatonin, which functions primarily to maintain circadian rhythms, but also has significant interactions with the immune system. Pinealocytes retain photoreceptor activity in fish (and in some other vertebrates including amphibia, reptiles and birds). References: Klein, 2006; Falcon et al, 2010; Esteban et al, 2013).

18. Brain appendages continued: The saccusdorsalis tissue (usually smaller and less obvious) is visible in the exposed striped trumpeter brain, though it appears pale rather than highly vascular due to fixation. Exposed brain of striped trumpeter (fixed specimen) Saccusdorsalis tissue Photos, G Grossel, used with permission

19. Anatomy of the sensory organs • Atlantic salmon head showing the relationship of the eye to the brain, the spherical optic lens, retina, optic nerve; and the nares (arrowed). Nares

20. The spherical fish lens has a refractive index gradient that compensates for spherical aberration, and fish focus by changing the position of the lens, rather than its shape, moving the lens closer or further from the retina. (This one is cloudy, due to cataract).

21. Choroid plexus Orbital cartilages Sensory organs (eye) continued: Eye of a larger rainbow trout eye, lens not in section (often removed at cut-in when examination not critical, as can be difficult to cut). The highly vascular choroid plexus is present only in those fish with pseudobranchs, and covers only part of the orbit. The known function of the pseudobranch / choroid plexus complex is to bring oxygenated blood from the gills to the pseudobranch, where further oxygen uptake occurs, then to the choroid plexus to supply hyper-oxygenated blood to the eye. Loss of pseudobranch function (in those species that have them), leads to gradual loss of eye function. Iris Cornea

22. The “eye spot” is not present in the upper margins of the eye. The cornea is similar to other vertebrates, with highly aligned collagen fibres forming much of the corneal stroma, covered by a thin uncornifed epithelium (often cuboidal, 1 to a few cells thick), allowing light transmission. Detail of the iris at the lower margin of the eye. Note the blood filled sinus (no specific name) in the base of the iris, lower margin of the eye. This is visible grossly as a red spot (inset). It is often much enlarged with infections and is often mistaken for haemorrhage, but at least the initial reaction is congestion of a normal blood sinus.

23. Detail of a salmonid eye (rainbow trout, marine environment). Teleost fish generally have no eyelids (sharks do, though they don’t need to blink). Teleosts cannot contract or dilate the pupil (sharks can). Note the very faint “red spot” on the lower margin.

24. Detail of the above rainbow trout eye section showing the relationship of orbital cartilage, choroid plexus and retina. The several layers of the retina are generally similar to those of mammals, with both rods and cones. Basement membrane is adjacent to the choroid. They are (top to bottom of photo): pigment epithelium; layer of rods & cones – separated by an external limiting membrane (arrow) from the outer nuclear layer of the rods & cones; outer plexiform layer; inner nuclear layer; ganglion cell layer; nerve fibre layer. Diurnal reef fish can often see a wider spectrum of light than can humans. Some fish can discern polarized light. Note the thick layer of melanin that prevents light from reflecting within the eye & disrupting vision. Nocturnal fish (which are thought to have evolved from deep water low-light fish), often have very large eyes but see only in black & white.

25. Detail of the choroid plexus, same section, showing this to be a typical plexus of closely applied vessels. While this facilitates oxygenation of the eye, it forms a common site for bacterial entrapment during septicaemias. Detail of the margin of the choroid plexus, where vessels coalesce.

26. Detail of another salmonid eye, showing the optic nerve and margin of the chorid plexus.

27. Choroid plexus of a normal aquarium Platy fish (Xiphophorusmaculatusor X. variatusor hybrids), cut in transverse section.

28. N Sensory organs (nares) Two examples of simple nares (N) or nostrils (i.e. olfactory organs) inAtlantic salmon fry. These are open to the water, though this is only obvious in low magnification view (left). Note too the tooth shown below (T) N N T

29. Sensory organs (nares) continued: Two examples of more nares with more complex turbinate-like folding in a Danio species fish. Detail of nares showing ciliated surface sensory cells. Most fish have a very well developed sense of smell.

30. Detail of auditory canals within head cartilage, showing the tufts of sensory hairs, similar to the lateral line. The otolith, a small bone that transmits the sound to these sensory cells, is not in section. Sensory organs (auditory canals): Head of young Atlantic salmon, showing auditory canals within the head cartilage (arrow). These are in many ways the most anterior extensions of the lateral line system. Although fish do not have vocal chords they do make noises, and need to be aware of other noises, which travel faster in water than in air. Sharks retain a small opening from the water to the inner ear, but in bony fish the initial transmission of sound is through bone.

31. Another head of Atlantic salmon fry, showing auditory canals. The system consists of 3 canals and one larger sac, all with foci of sensory epithelium (photographed with x 4 objective). Detail (x 20 objective).

32. Otolithgrowth varies with seasonal food supply, thus forming the equivalent of tree growth rings. They are often, therefore, used to age fish, but their accuracy is dependent on a seasonal pattern of growth. The otolith is a small bone (not fully decalcified in this slide), that lies over one of the sensory tufts, making contact with this when impacted by sound waves. Composite of another head of a 2g triploid Brook trout, showing the interconnecting auditory chambers, including the otolith (arrow), and their relationship to head bones.

33. Auditory canal of a cohort of the previous fish, showing the close relationship of these canals with the lateral line system (arrow). Some fish also have connections between ears and swim bladder, and use the latter to amplify sound, or to produce sounds by swim bladder vibration, to call other fish.

34. Sensory organs (lateral line): A reminder that the lateral line canals (this example, Atlantic salmon) are also sensory receptors, detecting water current as they impact on the “hair” cells (ciliated sensory cells) of the sensory tufts. See also Skin, Part 10.

35. References re nervous system of fish: • References nervous system anatomy & physiology: • Mueller, T. 2012. What is the thalamus in zebrafish? Review Article. Frontiers in Neuroscience. 6:64. doi:10.3389/fnins.2012.00064 • http://www.frontiersin.org/Neurogenesis/10.3389/fnins.2012.00064/full • Nakane, Y. et al. The saccusvasculosus of fish is a sensor of seasonal changes in day length. Nat. Commun. 4:2108 doi: 10.1038/ncomms3108 (2013). [21 authors] • Klein, D. 2006; Evolution of The Vertebrate Pineal Gland: The Aanat Hypothesis. Chronobiology International, 2006, Vol. 23, No. 1-2 , Pages 5-20 (doi:10.1080/07420520500545839). • Falcón J, Migaud H, Muñoz-Cueto JA, Carrillo M. 2010. Current knowledge on the melatonin system in teleost fish. Gen Comp Endocrinol. 2010 Feb 1;165(3):469-82. doi: 10.1016/j.ygcen.2009.04.026. Epub 2009 May 4. • Esteban, M A, Cuesta, A, Chaves-Pozo, E and Meseguer, J. 2013. Influence of Melatonin on the Immune System of Fish: A Review. Int. J. Mol. Sci. 2013, 14, 7979-7999; doi:10.3390/ijms14047979 [OPEN ACCESS] • Re infectious diseases of the nervous system of fish: • Munday B.L., Kwang J. & Moody N. (2002). Betanodavirus infections of teleost fish: a review. J. Fish Dis., 25, 127–142. • Anon, 2013. VIRAL ENCEPHALOPATHY AND RETINOPATHY. CHAPTER 2.3.11. of the Aquatic Animal Health Code. OIE. http://www.oie.int/fileadmin/Home/eng/Health_standards/aahm/current/2.3.11_VER.pdf

36. References nervous system – infectious diseases, continued: • Munday B.L., O’Donoghue P.J., Watts M., Rough K. & Hawkesford T. (1997) Fatal encephalitis due to the scuticociliateUronemanigricans(Mueller) in sea-caged, southern bluefin tuna, Thunnusmaccoyii(Castelnau). Diseases of Aquatic Organisms 30, 17–25. • Munday B.L., Sawada, Y., Cribb, T., & Hayward, C.J. 2003. Review. Diseases of tunas, Thunnus spp. Journal of Fish Diseases 26, 187–206 • Nowak, B., Johnston, C., Hayward, C., Aiken, H., Adams, M., Evans, D., Deveney, M., Carson, C., Jones, B., Evans, R., Dyková, I., Porter, M., Naeem, S., Kruesmann, M., Bayly, T. & Pitney, C. 2006. Southern Bluefin Tuna Health. (AquafinCRC, on disc) B Nowak (Ed). University of Tasmania. ISBN 978-1-86295-374-1 • Miller, TL and Adlard, RD. 2012. Brain infecting kudoids of Australia's coral reefs, including a description of Kudoalemniscati n. sp. (Myxosporea: Kudoidae) from Lutjanuslemniscatus (Perciformes: Lutjanidae) off Ningaloo Reef, Western Australia. Parasitology International, Volume 61, Issue 2, June 2012, Pages 333–342 • Langdon, J.S. (1990) Observations on new Myxobolus species and Kudoa species infecting the nervous system of Australian fishes. Journal of Applied Ichtyology 6: 107-116. • G.W. Grossel, I. Dyková, J. Handlinger and B.L. Munday. 2003 Pentacapsulaneurophilasp.n. (Multivalvulida) from the central nervous system of striped trumpeter, Latrislineata (Forster). Journal of Fish Diseases, 26, 315-320. • Whipps CM, Grossel G, Adlard RD, Yokoyama H, Bryant MS, Munday BL, Kent ML. 2004. Phylogeny of the multivalvulidae (Myxozoa: Myxosporea) based on comparative ribosomal DNA sequence analysis. J Parasitol. 90(3):618-22.

37. References nervous system continued: • G.W. Grossel , J. Handlinger, S. Battaglene and B.L. Munday. 2005. Diagnostic polymerase chain reaction assay to detect Kudoaneurophila(Myxozoa: Multivalvulida) in a marine finfish hatchery. Diseases in Aquatic Organisms 64, 141-149. • Grossel, G. 2005. Kudoaneurophila in striped trumpeter: identification, diagnostic development and histopathology. PhD Thesis. University of Tasmania April. 2005. Re general pathology, eyes: • Stephens F.J., Cleary J.J., Jenkins G., Jones B., Raidal S.R. & Thomas J.B. 2001. Pathogenesis and epidemiology of spontaneous exophthalmos in the West Australian dhufish, Glaucosomahebraicum Richardson. Journal of Fish Diseases 24, 515–522. • Stephens F.J., Cleary J.J., Jenkins G., Jones B., Raidal S.R. & Thomas J.B. 2002. Haemoglobin and oxygen transport of the West Australian dhufish, GlaucosomahebraicumRichardson, and other species. Journal of Fish Diseases 25, 409–414. • Verde, C., Vergara, A., Giordano, D., Mazzarella, L., and diPrisco. G. 2007. The Root effect - a structural and evolutionary perspective. Antarctic Science 19 (2), 271–278. DOI: 10.1017/S095410200700034X