Animal Nervous System (Earthworm & Fish)

7/30/2019 Animal Nervous System (Earthworm & Fish)

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Animal Physiology lab Group 2

4B Benson, Jonah

Date Performed: (leader) Catayas, Keth Laurel

Date Submitted: Legarda,Criscire

Activity 3

Animal Nervous System

I. Earthworm Dissection

Materials and MethodsMaterials: Reagents:

Earthworm Ringers Solution

Dissection tray - 6g NaClDissection pins - 0.12g KClForceps - 0.2g CaCl2

Scalpel - 0.1g NaHCO3Scissors - bring to 1 liter distilled waterProbeWater bath

WaterIce

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 ofthe worm was obtained using a ruler and an analytical balance, respectively. Photos were

taken for documentation.

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).

Dorsal blood vessel


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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).

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 2. Incision to the worms skin Figure 3. Incision towards the anus

Figure 5. Application of Ringers solutionFigure 4. Cutting of septa


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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.

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).

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

Figure 7. Removal of the thin film

Figure 8. Isolation of ventral nerve cord Figure 9. Nerve cord in Ringers solution

Figure 6. Removal of internal organs


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

(Figure10b).

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 hair-

like 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)

a b

Figure 10. Measurements obtained from the earthworm (a) length (b) weight


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.

Figure 11. Earthworm nervous system

Cerebral an lion

Circumpharyngeal

connective

Peri heral nerves

Segmental gangliaVentral nerve

cord

Subpharyngeal

ganglion


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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 Phosphate Buffered Solution:-Dissecting tray - 6.7g NaCl

-Scissors - 0.1g KCl-Pins - 100ml 0.1 M PO4, pH 7.3

-Forceps - 150ml Distilled H20-Scalpel

Spinal Cord

To start the dissection proper, the body weight and length of the fish was measuredfirst 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. Thefirst incision was at the belly of the fish from the front edge of the operculum through thetrunk to the anus (Figure 12).

Figure 12. Incision 1 Figure 13. Incision 2


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The second incision was done along the side of the fish near the caudal fin to thebackbone (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 trunkand other organs were removed except the head and the backbone (Figure 15). Thebackbone 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 tissuescovering 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 vertebraewere gently separated (Figure 18). The spinal cord was placed immediately in the PBS in

preparation for staining (Figure 19).


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Figure 18. Isolation of spinal cord Figure 19. Spinal cord in PBS

Brain

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 usingthe 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 brainusing the forceps. The tissue surrounding the forebrain was removed using the forceps

(Figure22). The brain was the gently isolated from the braincase and immediately placedon the PBS for staining purposes (Figure 23).

Figure 22. Isolation of brain Figure 23. Brain in PBS


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B. Results and Discussion

It is said that the any organisms body size is relatively 30-fold of that the size ofthe 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 (Figure24b).

a b

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 aspinal 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 identifiedwere the telencephalon, optic lobes and cerebellum. The telencephalon was small, oftenappeared 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 opticlobes 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 andlightly 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 theposterior 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 fulllength 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, sinceonly 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 wholesurfaces of the spinal cord and brain, respectively.


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a b

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 bonyfish'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 receiveand 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 telencephalonis 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 smelland 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 importantsense, have smaller olfactory lobes (Ramel, 2012).

Connecting the forebrain to the midbrain is the diencephalon which is locatedbelow the optic lobes and basically not visible. The diencephalon performs functions

coupled with hormones and homeostasis. The pineal body lies just above thediencephalon 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 oflearning). 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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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 thatcontrols 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 inmaintaining equilibrium. Fast-swimming bony fishes usually have an enlarged hindbrain.

Hagfish and lampreys have relatively small cerebellae, while the mormyrid cerebellum isbig 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 wastedisposal. 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 governsrespiration 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 thefull 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

Optic lobes

Telencephalon

Cerebellum

Posterior Part

Anterior Part


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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 thatexits 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 aneuronal pathway (Ramel, 2012)

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

Spinal Cord

Brain


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a

Figure 28. Isolated nervous systems wrapped in gauze (a) earthworm (b) fish

a

Figure 29. Wrapped nervous systems in Golgi-cox stain (a) earthworm (b) fish

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).

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).

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

b

b


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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 1st

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).

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 studyof 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

Sensory

neurons

Motor

neurons


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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 eachmidbody ganglion. The axons of these enter the CNS through the 1

stand 3

rdsegmental

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

a bFigure 32. Fish Neurons (a) Brain 100x (b) Spinal Cord 100x

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

Cell body

Axon

Cell body

Cell body

Axon

Dendrites

Dendrites


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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 ofLumbricus 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 NeuroscienceEducation, 10, 85-87.

Documents

Animal Nervous System (Earthworm & Fish)