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Animal Physiology lab 4B Date Performed: Date Submitted: Activity 3 Animal Nervous System I. Earthworm Dissection Materials and Methods Materials: Earthworm Dissection tray Dissection pins Forceps Scalpel Scissors Probe Water bath Water Ice

Group 2 Benson, Jonah (leader) Catayas, Keth Laurel Legarda,Criscire

Reagents: Ringers Solution - 6g NaCl - 0.12g KCl - 0.2g CaCl2 - 0.1g NaHCO3 - bring to 1 liter distilled water

After the preparation of materials and reagents, the earthworm was euthanized in the water bath with ice for 15 minutes. After 15 minutes, the body length and weight of the worm was obtained using a ruler and an analytical balance, respectively. Photos were taken for documentation. Dorsal blood vessel

Figure 1. Dorsal side of the earthworm The euthanized earthworm was placed on the dissecting pan, dorsal side up. The dorsal side was determined using two ways: first, by locating the dorsal side of the clitellum (without gonopores) and second, by finding the dorsal blood vessel which was a very prominent dark line that ran throughout the worms body (Figure 1).

Then, the worm was pinned first through the prostomium (first segment). This step was done carefully since the cerebral ganglion is located in the third segment which was very near to the prostomium. To completely put the worm in place, the anal (last segment) was pinned (Figure 1).

Figure 2. Incision to the worms skin

Figure 3. Incision towards the anus

With forceps, the dorsal skin was lifted. The scissors was inserted at the base of the forceps to make a small slit on the skin (Figure 2). Beginning on the slit, the skin was cut towards the anus (Figure 3). The separated body wall was pinned through the dissecting pan to hold the worm firmly while continuously cutting the skin of the worm towards the prostomium. After cutting through the entire worm, the septa was severed using the scalpel, and the separated body wall was again pinned to the tray (Figure 4). Once the internal organs were fully exposed, Ringers solution was poured to keep the organism from dehydrating and free from dirt and blood (Figure 5).

Figure 4. Cutting of septa

Figure 5. Application of Ringers solution

After cleaning the worm, the cerebral ganglion (brain) was found near the anterior and dorsal part of the pharynx. This structure was not that evident since it encircled the pharynx. The brain was isolated by carefully cutting the pharynx and removing the excess tissue trapped in the middle of it.

Figure 6. Removal of internal organs

Figure 7. Removal of the thin film

Next, the pharynx, esophagus, and other internal organs were pushed aside to expose the ventral nerve cord and segmental ganglia which looked like white bulges (Figure 6). Ringers solution was again poured to the worm. Using a scalpel, a thin layer of film above the nerve cord was scraped to make the cord more visible, thus making the isolation easier (Figure 7). The nerve cord and segmental ganglia were simultaneously isolated by separating the nerve cord from the underlying ventral body wall using a probe (Figure 8). This step was done until the last segmental ganglion was separated. The isolated nervous system was placed in a petri dish with Ringers solution (Figure 9).

Figure 8. Isolation of ventral nerve cord

Figure 9. Nerve cord in Ringers solution

After the dissection, the materials and the laboratory area were cleaned. The remains of the earthworm were buried on the area assigned previously by the teacher. B. Results and Discussion It was known that the body size of any organism is relatively 30-fold of that the size of the brain. Due to this information, the length and weight of the earthworm were

obtained. The model organism measured 11.5 cm long (Figure 10a) and weighed 1.23 g (Figure10b).

Figure 10. Measurements obtained from the earthworm (a) length (b) weight After dissection, the nervous system of the earthworm was easily identified due to its prominent white color. The major organs isolated were the cerebral ganglion and the segmental ganglia along the ventral nerve cord. These segmental ganglia were characterized due to their bulged appearance along the entire length of the nerve cord. Peripheral nerves also arose on the sides of the nerve cord which looked like thin hairlike projections (Figure 11). The central nervous system is primarily composed of a bilobed cephalic ganglion and a ventral nerve cord, with one ganglion per segment, extending through the whole length of the body. The segmental ganglionic enlargements vary in size, shape, and approximation at different parts (Clarke, 1856). The two lobes of the cerebral ganglion are connected by a pair of circumpharyngeal connectives to the most anterior of the ventral ganglia, the subpharyngeal ganglion (Mill, 1982). The cephalic or cerebral ganglion rests on the beginning of the pharynx, beneath the dorsal part of the third ring. Each lobe is a pyriform sac, which is very thick and convex posteriorly. The convex portion is opaque-white in color and filled with oval, round and pyriform cells, of various sizes. The anterior half, where the lobes are joined are composed with a line of lamina cells. The interior of this portion is entirely fibrous. The cephalic nerves are attached to the upper part of the ganglion. Roots of the nerves immediately separate into two trunks; a lower and upper. The former runs above the mouth, to the underside of the first segment, or upper lip (Clarke,1856)

. Subpharyngeal ganglion

Cerebral ganglion

Circumpharyngeal connective

Peripheral nerves Ventral nerve cord Segmental ganglia

Figure 11. Earthworm nervous system

The peripheral nervous system, on the other hand, consists of the lateral segmental nerves that project along the ventral nerve cord. Typically, each ventral ganglion posterior to the subpharyngeal ganglion gives rise to three nerve pairs of lateral nerves, in which the 2nd and 3rd segments are attached to each other via nerve to septum. These lateral nerves supply filaments to the septa and muscular bands (longitudinal, oblique, and circular muscles). In addition, a stomatogastric nervous system arises from the circumpharyngeal connectives, which innervates the pharynx and the anterior region of the intestine (Mill, 1982). The prostomial and prestomial nerves are distributed to the muscular bands of the mouth, which after supplying the muscles of the anterior segments, terminates in the integument of the lower lip (Clarke, 1856). II. Fish Dissection A. Materials and Methods -Fish -Dissecting tray -Scissors -Pins -Forceps -Scalpel Spinal Cord To start the dissection proper, the body weight and length of the fish was measured first using an analytical balance and a ruler, respectively. Photos were taken for documentation purposes. The fish was placed on the dissecting tray with the head on the left and using the scalpel, an incision was made through the body wall of the fish. The first incision was at the belly of the fish from the front edge of the operculum through the trunk to the anus (Figure 12). Phosphate Buffered Solution: - 6.7g NaCl - 0.1g KCl - 100ml 0.1 M PO4, pH 7.3 - 150ml Distilled H20

Figure 12. Incision 1

Figure 13. Incision 2

The second incision was done along the side of the fish near the caudal fin to the backbone (Figure 13). Along the dorsal side of the fish near the lateral line, the last incision was completed through the ribs along the backbone to the tip of the operculum (Figure 14).

Figure 14. Incision 3

Figure 15. Removal of trunk flesh

The incisions were repeated at the adjacent side of the fish. The portion of the trunk and other organs were removed except the head and the backbone (Figure 15). The backbone was isolated from the head for easier isolation of the spinal cord (Figure 16).

Figure 16. Separation of the head

Figure 17. Removal of spines

Before isolating the spinal cord, the spines of the vertebrae and other soft tissues covering the vertebrae were removed using the scissors (Figure 17). The spinal cord was then isolated gently by using freehand method, in which each segments of the vertebrae were gently separated (Figure 18). The spinal cord was placed immediately in the PBS in preparation for staining (Figure 19).

Figure 18. Isolation of spinal cord Brain

Figure 19. Spinal cord in PBS

Using the scissors, the soft tissue along the ventral side of the skull was removed (Figure 20). The skin and the tissue on the dorsal side of the skull were removed by using the scalpel (Figure 21).

Figure 20. Removal of ventral tissue

Figure 21. Removal of dorsal tissue

The skull was carefully opened by removing the skull bone on dorsal side of the brain using the forceps. The tissue surrounding the forebrain was removed using the forceps (Figure22). The brain was the gently isolated from the braincase and immediately placed on the PBS for staining purposes (Figure 23).

Figure 22. Isolation of brain

Figure 23. Brain in PBS

B. Results and Discussion It is said that the any organisms body size is relatively 30-fold of that the size of the brain. With regards to this information, the weight and length of the fish were taken. The organism measured 112.90 g in weight (Figure 24a) and 21.5 cm in length (Figure 24b).

Figure 24. Measurements obtained from the fish (a) weight (b) length After the dissection, the nervous system was easily recognized because of its distinct morphological characteristics. The nervous system of the fish was composed of a spinal cord and a brain (Figure 27). The brain was reddish-white in color, measuring up to 1.3 cm long (Figure 25a) and 1.2 cm wide (Figure 25b). The brain was protectively encased within the skull and had several clearly visible parts. The only parts identified were the telencephalon, optic lobes and cerebellum. The telencephalon was small, often appeared to be as separate two lobes and located at the anterior part of the two optic lobes. The optic lobes were the most prominent among the parts observed. The optic lobes occurred also in pairs and situated between the telencephalon and cerebellum. The other optic lobe (at the left side facing the telencephalon) appeared to be indefinite and lightly distorted, possibly due to improper handling the skull bone during brain isolation. Lastly, the cerebellum showed as a single-lobed structure, which was positioned at the posterior part of the brain. Furthermore, it was divided by various lobes; however, folds were not present (Figure 26). The spinal cord appeared to be as a thick, white, and cylindrical nervous material that was attached from the base of the brain (posterior part) and extends along the full length of the fish's body. The spinal cord was thin, soft, and delicate, and was protected by the spinal column or vertebrae. It was decided not to measure the spinal cord, since only half of the spinal cord of the fish was isolated and the isolated parts were too fragmentary (Figure 27). The spinal and cranial nerves were not evident along the whole surfaces of the spinal cord and brain, respectively.

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Figure 25. Measurements obtained from the brain (a) length (b) width Fish typically have quite small brains relative to body size compared with other vertebrates, typically one-fifteenth the brain mass of a similarly sized bird or mammal, and is poorly developed compared to other vertebrates (Helfman et al., 1997). Fish have highly developed nervous systems organized around a brain. A bony fish's brain is divided into three sections: the forebrain, the midbrain, and the hindbrain. The forebrain of fish is dominated by the olfactory lobes, a pair of structures that receive and process signals from the nostrils via the two olfactory nerves that are responsible for the bony fish's ability to smell. Behind the olfactory lobes is the two-lobed telencephalon, the structural equivalent to the cerebrum in higher vertebrates. In fish, the telencephalon is concerned mostly with olfaction. It also seems to control behaviors such as taking care of the young and exploring the environment. Fishes that have an especial good of smell and primarily hunt by smell, such as eels, catfish, hagfish, and sharks have an enlarged forebrain (Helfman et al., 1997). The teleosts, for which sight is often the most important sense, have smaller olfactory lobes (Ramel, 2012). Connecting the forebrain to the midbrain is the diencephalon which is located below the optic lobes and basically not visible. The diencephalon performs functions coupled with hormones and homeostasis. The pineal body lies just above the diencephalon which involved in detecting light, maintaining circadian rhythms, and controlling color changes (Helfman et al., 1997). The midbrain or mesencephalon of a fish consists mostly of the two optic lobes, which vary greatly in size between species in accordance with their dependence on sight. In fish, the mid-brain is important in sorting out incoming information and it is also the main centre of learning (whereas in mammals it is the forebrain that is the main centre of learning). The optic lobes may be so large that they completely cover the forebrain such as those species that hunt by sight, like rainbow trout and cichlids. Blind bony fishes, such as blind cavefishes in the family Amblyopsidae, have a reduced midbrain (Helfman et al., 1997; Ramel, 2012).

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Anterior Part

Telencephalon Optic lobes

Cerebellum Posterior Part

Figure 26. Earthworm brain The hindbrain or metencephalon (medulla and cerebellum) is particularly involved in swimming and balance. The cerebellum is a single-lobed structure that controls motor coordination. This means that it controls the timing and interaction of muscles once a muscular action has been initiated. The cerebellum is also important in maintaining equilibrium. Fast-swimming bony fishes usually have an enlarged hindbrain. Hagfish and lampreys have relatively small cerebellae, while the mormyrid cerebellum is big and involved in their electrical senses. The medulla on the other hand, controls the operations of the inner organs such as heart rate, blood pressure, digestion and waste disposal. It is also a relay centre for many nerves sending messages to and from the mid and forebrain. The brain stem or myelencephalon is the brain's posterior. As well as controlling some muscles and body organs, in bony fish at least, the brain stem governs respiration and osmoregulation (Helfman et al., 1997; Ramel, 2012). Posterior to the brain is the spinal cord, which is the hollow dorsal nerve cord that is characterized by chordates. It is a thick sheath of nervous material that extends to the full length of the fish body, and protected by the neural canal of the vertebral column (Monterey Bay Salmon and Trout Project, 2009). It serves as the basis of many simple

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responses and act as the major link to the brain for sensory input and brain responses (Ramel, 2012). Apart from the brain and the spinal cord is a vast network of spinal nerves that exits from the cord and connects to the internal organs and muscles. Nerves are built of numerous neurons that travel the message to or from the brain or the spinal cord along a neuronal pathway (Ramel, 2012)

Brain

Spinal Cord

Figure 27. Fish nervous system

III. Staining of Nervous System A. Materials and Methods First, the Golgi-Cox stain was prepared by making three solutions; (solution A) 5% potassium bichromate solution, (solution B) 5% mercuric chloride solution, and 5 % potassium chromate solution. In preparing the solutions, 500 g of potassium bichromate, mercuric chloride, and potassium chromate were weighed individually using an analytical balance. Five hundred grams of potassium bichromate was stirred into warm 1000 ml

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distilled water until dissolved. Five hundred grams of mercuric chloride was also stirred into warm 1000 ml distilled water until dissolved. Five hundred grams of potassium chromate was added into cold 1000 ml distilled water while being continuously stirred until dissolved (Wright et al., 2011). Then, 100 ml of solution A was added to solution B. Separately, 120 ml of distilled water was added to 80 ml of solution C. The A/B solution was then slowly added to the diluted solution C, while continuously stirring. An orange color was observed in the final solution. The Golgi-cox solution was stored in the dark when not in use (Wright et al., 2011).

a b Figure 28. Isolated nervous systems wrapped in gauze (a) earthworm (b) fish Next, the newly isolated nervous systems of earthworm and fish were individually wrapped in gauze and placed in separate vials (Figure 28a and 28b). The gauze was completely immersed in the Golgi-cox stain (Figure 29a & 29b). Vials were sealed and also kept in the dark room for 14 days (Wright et al., 2011).

a b Figure 29. Wrapped nervous systems in Golgi-cox stain (a) earthworm (b) fish After 14 days, the stained nervous systems were harvested. The parts were removed from the gauze and placed in two separate petri dishes, one for the earthworm and the other for the fish, containing distilled water. Fractions or slices of the nervous system were immersed in diluted ammonium hydroxide (100ml of 20% ammonium

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hydroxide diluted with 400ml distilled water) for ten minutes. Then, the fixed slices were mounted and immediately viewed under the microscope. B. Results and Discussion Earthworm Figure 30 shows the neurons and a segmental nerve found in one segmental ganglion of the earthworm nervous system. The neurons were identified because of the dark-pigmented cell bodies and web-like axons. The cell bodies were grouped in a distinct band lying along the side of ganglion. Some axons were easily distinguished because of their dark and thick filaments while others were very thin and light in color. In addition, one intact segmental nerve was observed during the first viewing. Then, the ganglion was squashed and the nerve fibers were found from the segmental nerve. The fibers were transparent, however, the filaments were very distinct (Figure 30).

Segmental nerve

Axons

Neurons

Cell bodies

Segmental nerve fibers

100x Figure 30. Earthworm neurons located in one segmental ganglion

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In Lumbricus terrestris, the segmental nerves are very important because they serve as input centers from different sensory neurons. The tactile sensory axons enter the central nervous system via the 1 st and 3rd segmental nerves. Chemoreceptive inputs, on the other hand, pass primarily through 1st and 3rd segmental nerves, but also to some extent via the 2nd segmental nerves. Lastly, proprioceptive inputs (awareness of body position) enter the CNS via all three segmental nerves (Mill, 1982).

Motor neurons

Sensory neurons

Figure 31. Motor and sensory neurons (a) schematic diagram (b) actual ganglion stained with nickel chloride (Mill, 1982) The neurons were identified using the figures (Figure 31a and 31b) from the study of Mill (1982). In which, the schematic diagram and actual stained ganglion showed the distinct dark-pigmented cell bodies of both motor and sensory neurons lying on the sides of the ganglion. In Lumbricus, the sense organs are concentrated in two bands encircling the animal in each segment. Each sense organ contains great number sensory cells. There are

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as many as 50 sensory neurons in each sense organ associated to supporting cells to which they are attached by peripheral zonula adherens and desmosomes (Mill, 1982). Furthermore, motor neurons are mostly found on the sides of the segmental ganglia. There are 26 pairs of motor neurons with contralateral cell bodies in each midbody ganglion. The axons of these enter the CNS through the 1st and 3rd segmental nerves. There are also four pairs of giant motor neurons in each ganglion (Mil, 1982). Unfortunately, these giant neurons were not located and identified. Fish

Cell body Dendrites Axon

Axon

Cell body Dendrites

Cell body a Figure 32. Fish Neurons (a) Brain 100x (b) Spinal Cord 100x b

After almost 3 weeks of staining, the neurons were examined and their structures were identified under the microscope. The neurons from the brain and spinal cord were easily identified from others because of their similarities in branching characteristics. Only few cell bodies were evident and commonly distinguished because of their dark pigment-like structures, with few (two to three) or lightly dense set of dendrites arising from all over the surface. The cell bodies were almost asymmetrical and some were

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relatively circular in shape. The lightly visible dendrites were numerous, thin, short, and highly branched from their origin (cell bodies). Axons were few and structurally thin and long and frequently extend away from the soma or cell body. The axons appeared to be as thin fragments of thread that were aligned in linear fashion. Each of these axons was found connected with other cell bodies. Some dendrites and axons were superficially similar in structure under the microscope which somehow made the identification of the parts very complicated (Figure 32a & b). The neurons of humans are similar to the neurons of other animals like the fish. A typical neuron has an enlarged area called the cell body, which contains the nucleus. Neurons have a large number of extensions called dendrites. They often look like branches extending out from the cell body. The dendrites receive the information from the neurons. Each neuron usually also has a longer tail-like structure called an axon, which transmits information to other cells. This structure can easily be distinguished from the dendrites because of its length. Longer axons are usually covered with a fatty, segmented covering called the myelin sheath. This covering acts as an insulator which improves the ability of axons to carry nervous system signals rapidly. At the very end of the axon is the axon terminal also known as terminal button or synaptic knob which is separated from the next cell by a tiny gap called a synapse (Moreno & Tharp, 2007).

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REFERENCES Helfman, D., Collette, B., & Facey, A. (1997). The Diversity of Fishes. Blackwell Publishing, pp. 4849, 191. Lockhart, C. 1856. On the nervous system of Lumbricus terrestris. Mill, P. J. 1982. Recent Developments in Earthworm Neurobiology. Comprehensive Biochemistry and Physiology,73, 641-661. Moreno, N. P. & Tharp, B. Z. 2007. What Are Neurons?. Baylor College of Medicine. Monterey Bay Salmon and Trout Project. 2009. Fish Nervous System. Retrieved from http://mbstp.org/Fish/thenervoussystem.html Ramel, G. 2012. Fish Nervous System. Retrieved from http://www.earthlife.net/ fish/nerves.html Wright, K. A., Zimerman, E. L. & Harrington, M. E. 2007. A modified golgi-cox procedure in undergraduate courses. The Journal of Undergraduate Neuroscience Education, 10, 85-87.