BIOLOGY 468/568

---

PRINCIPLES AND APPLICATIONS OF ELECTRON MICROSCOPY

STUDENT NAME:

Mattew Timney

PROJECT TITLE:

The Ultrastructure of the Electric Organ in Torpedo californica .

Species Identification

• Kingdom: Animalia
  
  

  Phylum: Chordata
  
  

  Class: Chondrichthyes
  
  

  Order:
  
  

  Family:
  
  

  Genus: Torpedo
  
  

  Species: Californica

---

ABSTRACT

Torpedo californica, commonly known as the Pacific electric ray, is a relatively abundant fish that exhibits the peculiar ability to produce electricity for feeding and defense. These electric shocks are produced by a specialized organ in the disk region that is made up of many cells called electroplaques. These electroplaques are densely innervated on one side and not innervated on the other side. Because of the large amount of neural activity required to make the electric organ function, the Pacific electric ray has been used extensively as a research animal for neurotransmitter studies. Despite this large amount of research, very little ultrastructural work has been done.

Contained in this paper are electron micrographs which show much of the ultrastructure of the electric organ of T. californica. These micrographs include nuclei, mitochondria, innervated and noninnervated membranes, presynaptic vesicles, and extracellular bundles of collagen.

Key Words:

Torpedo californica, TEM, electric organ.

---

INTRODUCTION

The Pacific Electric Ray, T. californica., is a relatively common ray found on the pacific coast from Baja up to British Columbia (Miller and Lea, 1972). The torpedo ray is usually a charcoal grey color with black spots (see figure1) and can reach lengths up to 1.3 meters and weights of approximately 40 kilograms (Miller and Lea, 1972) The torpedo is primarily a nocturnal animal that is a top level carnivore with no known predators except man (Love, 1991).

T. californica has two kidney shaped electric organs located in the head region just posterior to the brain (see figure 2). These organs can produce up to 240 volts and are used both to stun or kill their prey and for defense (Bray, 1978). The electric organs are made up of dense columns of hexagonal cells, called electroplaques, that are stacked on top of each other with the same orientation (Keynes, 1957). The electroplaques are often three to four millimeters across and only ten microns thick (Mathewson, et.al, 1961). The electric organ is heavily innervated with a nerve making contact with one side of each electroplaque (Keynes, 1957; Mathewson, 1961). In previous studies, Mathewson et.al (1961) have shown that in other members of the same genus, the innervated membrane is highly convoluted while the noninnervated membrane is comparatively smooth. In addition, though each electroplaque is a single cell, they contain multiple nuclei (Keynes, 1957). The electric organs are surrounded by an extracellular gel (presumably for insulation) and are encased in an individual compartment made of connective tissue (Keynes, 1957).

The large number of nerve reactions required to activate the electric organ utilizes a large amount of acetylcholine (McMahon and Nicholls, 1991). It is for this reason that the torpedo ray has been used extensively for research into nerve functions. Acetylcholine, when released, causes the depolarization of, and resulting reversal of, charges in the membrane of each electroplaque (Keynes, 1957). This reversal gives rise to an electric field which is passed through the electroplaques. Since the electroplaques are arranged in series, the results are cumulative, not unlike a series of resistors.

Although the nervous system and the electric organ have been studied extensively from a biochemical standpoint, very little research has been done on the ultrastructure of the electric organ in T. californica.. It is the goal of this study to provide a view of the ultrastructure of the individual electroplaques.

---

MATERIALS AND METHODS

We captured an 16 kg female torpedo ray on 14 November, 1993 at 01:30. The specimen was caught using an otter trawl at a depth of approximately six meters near the east side of Terminal Island Federal Prison, Los Angeles Harbor. We removed the animal from the net and placed her in a holding tank on the boat and transported her back to the lab where we placed her in an aquarium for approximately eight hours. We then removed her from the aquarium and placed her in a dissecting tray. During the removal of the tissue sample, we wore non-conductive rubber gloves and stood on a non-conductive wooden surface. While my assistant held down the ray, I used a surgical scalpel to cut a one cubic centimeter sample from the right electric organ. The animal was returned to the Long Beach harbor.

The tissue sample was immediately placed in 2% gluteraldehyde in a phosphate buffer (pH 7.3) and I attempted to cut it into small sections (1-3mm). The tissue samples were left in gluteraldehyde on a rotary mixer for one hour and then taken through three ten minute rinses of Sorensen's phosphate buffer (pH 7.3), also on the rotary mixer. I placed the sample in 1% osmium tetroxide in Sorensen's buffer (pH 7.2) and refrigerated for one hour. I took the sample through two ten minute rinses in Sorensen's buffer and one two hour rinse in Sorensen's. I then rinsed the sample for ten minutes each in 30%, 50%, and 75% ethanol. The sample was then left in 75% ethanol overnight (approximately 15 hours). I rinsed the sample in 95% ethanol for ten minutes and then in four 15 minute rinses of 100% ethanol. I followed this step with two 15 minute rinses in propylene oxide. I placed the sample in an eight hour rinse of 2:1 propylene oxide:spurr resin and an eight hour rinse of 1:2 propylene oxide:spurr resin. These rinses were followed by two four hour rinses in 100% spurr resin. I placed the specimens in both horizontal and vertical (Beem capsule) embedding molds and baked them in a vacuum oven for 18 hours at 65 c.

The blocks were then trimmed and placed on a ultramicrotome for sectioning. Both semi-thin and ultra-thin sections were cut using glass knives. I observed semi-thin sections under a light microscope after staining with 1% toluidine blue in borax. I placed ultra-thin sections on a copper grid and stained them using a uranyl acetate/lead citrate stain for 10 min/3 min. I viewed the ultra-thin specimens on a JEOL 1200EXII transmission electron microscope. All pictures were taken using the smallest aperture and 40-60 Kv.

---

RESULTS

Figures

Fig 1.



Photograph of T. californica.

Fig 2.



Line drawing showing the location and orientation of electric organs.

Fig 3.



The tissue sample and location.

Fig 4



A section containing a (Nu) nucleus surrounded by (Cy) cytoplasm. Both the (Mi) mitochondria and the (Nm) noninnervated membrane are also visible.

Fig 5.



The (Nu) nucleus with (Cm) cell membrane (probably noninnervated) showing the presence of (Cf) collagen fibers external to the cell

Fig 6.



This micrograph shows the high concentrations of (Cf) collagen fibers near the cells. A (Nu) nucleus is also shown.

Fig 7.



Nucleus (Nu) with visible nuclear pores, (Cy) cytoplasm, and the smooth (Nm) noninnervated membrane. The nucleus is the same nucleus in fig 4.

Fig 8 .



Another portion of the (Nm) noninnervated membrane.

Fig 9.



Both transverse and longitudinal views of collagen fibers shown outside the (Cm) cell membrane. This clearly shows the inconsistent orientation of the collagen fibers.

Fig 10.



Collagen fibers magnified 120,000 times.

Fig 11.



The (Im) innervated membrane showing the convolutions and the (CR) canalicular network.

Fig 12.



Innervated membrane (im) with (star) circular structures - possibly nerve endings.

DISCUSSION

Upon removal, the jelly-like consistency of the tissue sample prevented any fine cutting for fixation. The tissue also did not seem to stain well after sectioning, requiring the use of lower kV for contrast. The tissue sample in the ultra-thin sections was extremely disoriented. These factors, along with the fact that the tissue is also multinucleate and has large cells, did not allow for a complete view of a single cell.




The nuclei of the electroplaques appear to have a standard nuclear ultrastructure but are extremely varied in shape (see figs. 4, 5, and 6). This could be the result of a fixing error or natural conditions. None of the ultra-thin or semi-thin sections were found to have any nucleoli. The mitochondria (fig. 4) have very well defined cristae and are very elongate. According to Mathewson et.al. (1961), this is characteristic of electric cells.




The membranes in the micrographs are broken down into two very distinct categories. The noninnervated membranes are smooth and contain mild convolutions (fig 4, 7, and 8). The innervated membrane, on the other hand, are extremely convoluted (fig. 11 and 12). The innervated membranes appear to have a canalicular network (fig 11) This canalicular network is thought to be -tubes,- made up of cell membrane, which are continuous with the extracellular space. The fact that this extension of extracellular space occurs most prominently on the innervated membrane leads to speculation that its purpose may be to increase the surface area of the membrane for quicker response to stimuli. Although the membrane is continuous, in the micrographs, it appears as many unconnected sack-like projections. This is due to the plane of sectioning. While it is not certain, it is possible that the dark circular structures in figure 14 are nerve endings containing presynaptic vesicles.




A surprising discovery of this study was the presence of collagen in the extracellular space of the electric organ. The collagen fibers are very common and are only found outside of the cell membrane (figs 5, 6, and 9). The collagen fibers have no consistent orientation (fig 9) and have the distinct banding pattern characteristic of collagen




(fig 10). While previous research has found collagen in members of the same family (Mathewson et al., 1961), no research has been done in T. californica. This researcher hypothesizes that the collagen is present as an extracellular matrix to give support to the gelatinous electroplaques.




This idea is supported by research, which shows that the mildly electric fish, Gymnotus, has thick collagen septa dividing the electroplaques into distinct laminae (Couciero, 1961). The lack of consistent orientation is possibly due to fixation techniques which did not prevent structural shifting.




Although the results of this study do provide a limited ultrastructural knowledge of the electric organ of the torpedo ray, there is still much that can be done to improve upon this study and to provide more information. It would be interesting to look at the electric organ in its pre- vs. post-discharge states (not known in this study). In addition, it would provide results more consistent with the true cellular orientation of the organ if the tissue was mildly fixed prior to cutting into cubes. It would also be quite interesting to look at the structure of the nerves, electric organs, and collagen using the SEM on a cryofractured specimen.

ACKNOWLEDGEMENTS

A special thanks to Dr. A.Z. Mason, Dr. R.N. Bray, Tom Douglas, and Adel Rajab for help in the formulation, specimen capture, preparation, viewing, interpretation, and presentation of this study. Without them, this study would not have happened.

CITATIONS

1. Bray, R.N. and M.A. Hixon. (1978). Night shockers: predatory behavior of the Pacific electric ray Torpedo californica. Science 200:333-334.

2.Couciero, A. and De Almeida, D.F. (1961). In: Chagas, C. (Ed.), Bioelectrogenesis, The electrical tissue of some Gymnotidae. pp.3-13, Elsevier publishing, Amsterdam.

3.Keynes, R.D. (1957 ). In: Brown, M.E. (Ed.), The physiology of fishes, vol II, Electric organs. pp. 323-343, Academic Press, New York.

4. Love, R.M. (1991). Possibly more than you wanted to know about the fishes of the Pacific coast. Really big press, Santa Barbara, California.

5.McMahon, H.T. and D.G. Nicholls. (1991). The bioenergetics of neurotransmitter release. Biochimica et Biophysica Acta. 1059:243-264.

6. Mathewson, R., A. Wachtel, and H. Grundfest. (1961). In: Chagas, C. (Ed.) Bioelectrogenesis, Fine structure of electroplaques. pp25-53, Elsevier publishing, Amsterdam.

7. Miller, D.J. and Lea, R.N. (1972). Guide to the coastal marine fishes of California. California dept. of Fish and Game.