wrong 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 right 91 90 89 88 87 86 85 84 83 82 81 80 79 78 77 76 75 74 73 72 71 70 69 68 67 66 65 64 63 62 61 score 100 98.9 97.8 96.7 95.6 94.5 93.4 92.3 91.2 90.1 89 87.9 86.8 85.7 84.6 83.5 82.4 81.3 80.2 79.1 78 76.9 75.8 74.7 73.6 72.5 71.4 70.3 69.2 68.1 67 Animalia: Frog Name Today we are studying the circulation physiology of the African clawed frog, Xenopus laevis, as an example vertebrate animal. Frogs are hopping amphibians and they appear in virtually their current form in the fossil record of 190 million years ago! The earliest amphibian, Ichthyostega, appears in a fossil from Greenland dated at 370 million years ago in the Devonian Period. Today frogs are found in just about every freshwater ecosystem from desert to arctic winters, and on every continent except Antarctica. Frogs are poikilotherms, meaning that they are coldblooded. Their body temperature is not maintained homeostatically, but rather approximates the environmental temperature. In extreme heat of a desert, the Australian water-holding frog buries itself in the soil, sheds its skin to form a water-holding cocoon, and hibernates up to seven years awaiting a rainy season to emerge and mate. Wood frogs can live above the Arctic Circle and load up their internal organs with extra glucose as winter approaches as a kind of antifreeze. The peripheral parts of the body freeze solid through the winter! Sadly, amphibians and frogs in particular are sensitive to environmental factors that have combined to put many species into endangered status. In previous years this exercise involved the use of two dozen Rana pipiens grass frogs per semester. These were supplied to us by large companies that collect frogs from the wild for use in schools and universities. This continues to apply pressure to the decline of frog populations. We have chosen to modify this exercise to use Xenopus laevis because we have found suppliers that raise them in a commercial setting for laboratory and medical use. Thus our class is no longer contributing to the world-wide decline in wild frog populations. The posterior appendages (legs) are specially evolved to permit frogs to leap 20 times their body length for a tall human that would be a 40-meter leap. Tree frogs have specially-adapted toe tips for climbing and clinging to vertical surfaces. The Costa Rican flying tree frog uses its foot webbing like a hang-glider to soar from tree to tree. Xenopus is adapted as a mostly aquatic frog, and not surprisingly its posterior legs are mostly for swimming. Their posture is somewhat different from that of a common grass frog. Frogs are carnivores and feed mostly upon invertebrates, but the ornate horned frog of Argentina can swallow a mouse whole! The frog mouth lacks slashing or grinding teeth, so all the prey are swallowed whole. More-terrestrial frogs use a sticky tongue that extends a great distance out of the mouth to capture insects on the wing. The tongue can flash out a distance longer than the frog body and so quickly that just about any insect can be easily captured by a hungry frog. Xenopus lacks much of a tongue; it feeds by pushing food into its mouth with its rather small forelegs. More-terrestrial frogs have excellent vision and the position of the eye permits a frog to see predators approaching from just about any direction. The bulging eyes and nostrils (nares) at the anterior tip of the body allows the frog to rest with very little of its body extending above the water surface. Xenopus has comparatively small eyes as it spends most of its day in dark sediments on pond bottoms. Frog skin is a major site of gas exchange, but the skin must be kept moist for oxygen to exchange across it. Sure the frog has lungs, but frogs rely upon their skin for much of their gas exchange. About once a week the frog sheds its skin, pulling it over its head like a stocking cap and generally the frog eats the shed skin. This document Ross E. Koning 1994. Permission granted for non-commercial instruction. Koning, Ross E. 1994. Animalia: Frog. Plant Physiology Information Website. http://plantphys.info/organismal/labhtml/frog.html. (8-23-2013). Page 1
Page 2 Xenopus has comparatively large lungs that extend behind the diaphragm down most of the length of the body! It surfaces to fill those lungs and then can use the air for a long time while resting on the pond bottom. Of course it uses its skin for gas exchange, but sediments are notoriously anoxic. Moreover they are filled with decay organisms (bacteria, fungi, etc.), so Xenopus skin sheds rapidly and glands secrete tremendous quantities of mucus to isolate the tender frog skin from the environment. The slippery skin makes these frogs very hard to handle! Frogs are often cryptically colored, but there are poisonous frogs with aposematic (wildly colored) skin to warn would-be predators about their toxic properties. Of course there are non-toxic species that also have aposematic coloration, which probably helps them avoid predation by stealth! Many frogs can change their coloration to match their environment, or because of their temperament (lighter when normal and darker when aggressive), or because of the time of day (lighter in daytime and darker at night). The skin possesses chromatophores to assist in these color changes, some of which are under hormonal control. Frogs have vocal cords and vocal sacs that help produce characteristic calls, mostly by males. Frogs use these sounds to establish territory and to attract mates. Frogs have a large tympanic membrane just behind the eyes to hear each other's calls. If a male successfully attracts a female, he mounts on her back and wraps his forelimbs around the female's ribcage, interlocking his thumbs. One way to distinguish male from female frogs is the condition of the forelimb thumbs. The clasping behavior is called amplexus and it may last hours, days, or even a week or more. Most frogs are sexually dimorphic; females are generally larger than males. When the female is fully ready hormonally to shed eggs, she releases hundreds to thousands of eggs into the water. When this happens, the male on her back sheds clouds of sperm over them. The syngamy event occurs in the water (external syngamy). In most frogs the zygotes pass through the tadpole (larval) stage initially as herbivores with very little parental care. However there are other species that keep the larval frogs in their mouths, stomachs, vocal sacs, or embedded in the skin on their backs and assist in their feeding, early development, and survival in the face of predation. Because Xenopus is so slippery, we will not observe them while still fully alive as they would easily be maimed or injured unintentionally by falls from the bench, etc. Euthanasia In biology we treasure and value the life of an organism as we study it. This will require us to provide our animal with some level of comfort as we open its body to observe circulation physiology. Most of you have heard of "pithing" or "double-pithing" a frog, which means inserting a dissecting needle into the space between the skull and first vertebra, severing the spinal cord, and then driving the needle into the brain and disrupting it and also running the needle down the channel for the spinal cord to disrupt most of the frog's peripheral reflexes. For smaller frogs (such as Rana pipiens) decapitation with a pair of bone scissors is a faster, more reliable procedure to render the frog paralyzed and insensitive to our operations. For Xenopus, the skull and vertebral column is extensively calcified and the foramen magnum and spinal cord canal are very small. It also has very strong jaws. These features make both direct pithing and decapitation inhumane approaches. Therefore, the instructor will be euthanizing the frogs using methods embraced by wide range of agencies for humane euthanasia of Xenopus. The frog will be instantly stunned by a compression concussion with braincase fracture, immediately followed up by decapitation and double-pithing. There will be considerable bleeding; but this should not impact our project. External Anatomy Your frog will arrive ventral surface up on a dissecting tray. Carefully but quickly observe the morphology of the frog and make a sketch in the space provided below.
Page 3 On the edge of the head you will find the mouth. The frog has two anterior pectoral appendages (forelimbs) and two posterior pelvic appendages (hindlegs). Obviously the frog is a tetrapod vertebrate animal. The forelimbs have a hand with four digits and a rudimentary/vestigial thumb. The hindlegs have a foot with five digits and a rudimentary and vestigial sixth digit. The digits of the hindlegs are webbed for swimming. You might notice the size difference between the forelimb and the hindleg! Since Xenopus is more a swimming than a jumping frog, this dimorphism might be expected to be of less magnitude; its forelimb is used mostly for swimming and so is smaller than in jumping frogs. The organization of the proximal and distal parts of each limb are very similar to their counterparts in humans. You should be able to locate the wrist, forearm, elbow, shoulder and note that the range of motion and direction of folding match your own. Internally the bone structure and the musculature that operates the forelimb are very similar to those of your arms. The hind limb is also similar in composition to yours. The thigh is attached with a ball-joint at the pelvis, there is a knee hinging just like yours, then a shin ending at the heel of the foot. The foot of even this aquatic frog is disproportionately larger than yours, relative to the rest of the hindleg. The adult frog lacks a post-anal tail and so is called an anuran; the body axis ends with the cloacal opening. Make a sketch of your frog's external structure from the perspective of looking down at its ventral surface. Orient the frog's head to the left in your sketch. Draw lines from the labels given below to your sketch. Be sure your lines actually touch the structure named. Plan before you sketch, so you do not have any crossing lines. Use a { or [ to show longer parts. Pay close attention to correctly show how the legs are articulated ("fold"), your sketch must be biologically accurate! Try to avoid the "Gumby" look. mouth heel digits } foot forelimb: cloacal opening shoulder hindleg: hand{ digits wrist forearm elbow thigh knee shin /14
Page 4 Cardiac Physiology The main physiology project for today is to observe the frog cardiac muscle directly. The reason for using a frog heart is that it is rather thin-walled, allowing for rapid diffusion of chemicals that we can treat it with, and oxygen diffuses easily into the exposed heart. Our frog is no longer breathing. Moreover, as is true of most poikilotherms, the heart muscle operates over a broad range of temperatures. So the frog heart will still be beating and will continue to beat for a few hours after euthanasia. Assuming lecture has not covered the function of the chemical treatments, let us simply say here that the poikilotherm heart has a built-in pacemaker (a group of neural cells sending out regular impulses) allowing the heart to beat for hours even after the brain is no longer connected to the heart! The nerves that control the heart rate obviously must balance stimulation and inhibition of that pacemaker to maintain a pulse appropriate for the current conditions. These neurons that connect to the heart, influence the pacemaker by one of two nerve systems. The parasympathetic nerves slow down and weaken the contractions by stimulating the pacemaker with acetylcholine secretions the sympathetic nerves speed and (especially) strengthen contractions by stimulating the pacemaker with nor-epinephrine. Acetylcholine and norepinephrine are called neural transmitter substances. The responding cells must have receptors to receive these chemicals and respond accordingly. Atropine blocks acetylcholine receptors. Caffeine is a stimulant that also disorganizes the pacemaker's signals. We can now proceed to observing the still-beating heart. Turn the frog in the dissecting tray so that the ventral (belly) side is up. Using forceps, lift the abdominal skin near the pelvis and snip it open with the scissors. Remember we want only to cut the skin, so this cut needs to be shallow! Cut the skin up to the edge of the frog's jaw. Then cut the skin laterally on each side of the cut so that you can flap the skin open to see the abdominal muscles. (Your cuts will be in the form of the letter I). Repeat your cuts just going through the abdominal muscles from pelvis to the ribs. As you flap the muscle layers open and pin them, you should be able to see the abdominal organs inside the abdominal cavity. Dividing the abdominal cavity from the thoracic cavity is a very thin sheet of muscle (looks more like a "membrane"), the diaphragm. This attaches along the body wall at the level of the lowest rib. Use the point of the scissors to penetrate the diaphragm just under the rib cage and carefully cut through the ribs along the sternum (breastbone) up toward the throat but BE CAREFUL to keep the scissor points just under the ribs so that you do not cut into thoracic organs. After you have opened the thoracic cavity and pinned it open a bit, you should be able to locate the beating heart. The heart is surrounded by a pericardial membrane; in most Xenopus individuals, this has a speckled silvery-appearance. Again carefully lift this membrane with forceps and slit it open with the scissors to expose the dark pink heart. This is a three-chambered heart with one ventricle and two darker atria. You might quickly observed the contraction sequence between the two dark-looking atria (right and left) and the lighter pink ventricle. You should also notice the color change that happens as the ventricle contracts, and the degree of movement of the ventricle with each contraction. From this point onwards the heart needs to be kept moist. Apply isotonic room-temperature Frog Ringer's solution (6.5 g NaCl + 0.14 g KCl + 0.12 g CaCl 2 + 0.2 g NaHCO 3 + 0.01 g NaH 2 PO 4 H 2 O per liter) to the heart muscle and other internal organs to keep them from drying out. All of the drugs you will be using in this project are dissolved in Frog Ringer's solution. You should never use distilled water to moisten internal tissues.
Page 5 Make ventricle pulse observations over one full minute for each trial for our "control" setting. Add one drop of Ringer's solution to the heart muscle between the five trials. Ringer's control. weak normal strong After the fifth trial, blot out the excess Ringer's solution with a wad of dry paper towel. Immediately apply a few drops of acetylcholine dissolved in Ringer's solution. Allow one full minute for the chemical to diffuse into the heart muscle. Then repeat your pulse observations. 0.3% acetylcholine. weak normal strong If acetylcholine stops the heart, blot out the acetylcholine immediately, apply the atropine (dissolved in Ringer's) solution to counteract it and to soon restore the heartbeat (then enter 0 for the rest of the trials for acetylcholine). Allow one full minute for the atropine to diffuse into the heart muscle. Is a heart massage needed? Then repeat your pulse observations. 0.5% atropine. restored not restored Blot out the atropine solution and apply a few drops of the nor-epinephrine (dissolved in Ringer's) solution to the heart. Allow one full minute for the nor-epinephrine to diffuse into the heart muscle. Then repeat your pulse observations. In determining the relative strength of the contraction, compare what you have seen in terms of the amount of heart movement and the relative color change between the relaxed and contracted ventricle. 0.3% norepinephrine. weak normal strong Blot out the nor-epinephrine solution and apply a few drops of the caffeine (dissolved in Ringer's) solution to the heart. Allow one full minute for the caffeine to diffuse into the heart muscle. Then repeat your pulse observations. In determining quality of contraction, compare the normal sequence of the atrial contractions and ventricular contractions to anything that the caffeine may induce. 1% caffeine. regular irregular Now that your chemical treatments and timed intervals are over, you can proceed in a more relaxed way. Blot out the caffeine solution, and add a few drops of plain Ringer's solution. Complete your examination of the chest cavity by noting that blood from three dorsal veins including the posterior vena cava join together beneath the heart to form the sinus venosus. This feeds blood from the body into the right atrium (on your left!). The left atrium (on your right) receives oxygenated blood from the lungs. The ventricle pumps blood into the conus anteriosus passing ventrally over the right atrium. It then divides into branches including the dorsal aorta. /40
Page 6 Since you have been looking at it for such a long time, we should sketch this anatomy to commit your experimental environment to your notes. Make a diagram of the chest cavity of your frog (use your cranial zoom!) and label it by linking the structures sketched to the words in the word bank. Be sure your lines actually touch the structure named. Plan before you sketch, so you do not have any crossing lines. rib cage pericardium atrium ventricle Mouth Anatomy. Turn your frog over, and try to examine the inside of the mouth. The mouth of Xenopus is a bit simpler than that of a terrestrial frog. This frog does not have the long tongue for capturing insects as prey. This frog is more of an ambush predator. It lives in sediments, detects the vibration caused by the presence of prey using its lateral-line system. It grasps the prey in its mouth and swallows it whole. So the tongue is poorly developed and vomerine teeth are basically missing too. Slide your dissecting needle along the margins of the upper jaw (the maxilla) being sure to be inside the frog's lips. You should be able to feel the texture of the maxillary teeth. The teeth are equal unequal in size and point back to the throat straight up from jaw. In the floor of the mouth (lower jaw = mandible) there will be a small dome of soft tissue called the glottis with a slit in the middle of it. Just below this slit are the vocal cords. The slit leads into the trachea (breathing tube) that conducts air into and out of the two lungs on either side of the midline. Posterior in the mouth, the esophagus passes from the mouth, through the diaphragm, and connects into the stomach. Flip your frog back over to see if the lungs have become more visible (they lie deeper than the heart and are under the internal organs). Eye Anatomy. Using the frog's severed head and your dissection microscope and illuminator, get a closer look at the small eye of Xenopus. Terrestrial frogs generally have much larger eyes, but Xenopus lives in dark sediments in pond bottoms, so its eyes are less useful for predation and protection from prey than its lateral line system. In the space below, make a sketch of the eye and label it clearly as for previous sketches. You may need to use a dissecting needle to lift the nictitating membrane to find it! pupil iris nictitating membrane /9
Page 7 Abdominal Cavity. Posterior to the chest cavity you have watched so closely, there is a larger abdominal cavity. Most of our frogs will be female, so this area is dominated by olive-khaki eggs and light pink oviducts. There may also be a fair bit of yellow to orange adipose (fat) tissue. Carefully pull out and snip the eggs and oviduct tissues being careful not to disburb the digestive and other systems that are internal to all of this reproductive system. Just anterior to the eggs and oviducts, you will find the large and very dark brown liver. As you trace its edges you will see that it is lobed. In fact you should find that it has two separated sections (left and right). As you lift the right section of the liver (on your left) slightly you will find the dark-greenish gall bladder attached into its lower surface. The bile enters the digestive system at the level of the intestine. Beneath the left section of the liver (on your right) you should find a whitish j-shaped muscular stomach connecting anteriorly to the esophagus and posteriorly to the intestine (small in diameter, but long in length!). Between the stomach and intestine, you may find the gray-brown pancreas. The pancreatic duct leads digestive enzymes into the beginning of the intestine where enzymatic digestion and nutrient absorption occur. The later part of the intestine is where water absorption occurs. The intestine empties into the cloaca where the adjacent collapsed bladder also empties. Beneath the pubic bone the cloaca exits the body at the anus. The cloaca receives contributions from the reproductive, urinary, and digestive systems. Cut the digestive system at the esophagus well above the stomach and again at the cloaca near the anus, and remove the entire digestive system to a paper towel in one piece. Do not disarticulate the sections of the digestive system! This is a shortcoming of all those PC-Frog on-line dissection software programs. We want you to see the system as it is connected together and functioning as a whole sequenced system. Stretch out the digestive system to straighten it. You may need to cut mesenteries (the connectives that keep the conformation of the intestine in proper folded orientation and have many hepatic-portal blood vessels to extract nutrients from the intestine). Make a sketch of this system in its stretched-out-straight condition (esophagus to left, cloaca to right!); be sure to use your "cranial zoom" to fit it into the space below. Try to get relative sizes, diameters, and lengths right. Label your sketch as before. Shade in the organs to show relative colors, and label the colors in the blank above each label etc. esophagus stomach pancreas liver gall bladder spleen intestine cloaca Snip out the gall bladder. Blot it with paper towel. Place it on a fresh piece of white paper towel and pierce it with the dissecting needle. What color is bile? Looking deeper in the abdominal cavity you will find two bilateral brownish kidneys with a pink-white adrenal gland at the anterior end of each. These lie close to each other just above the backbone and are surrounded by a dense capillary system. The ureters conduct urine to the urinary bladder that empties into the cloaca. /17
Page 8 Make a sketch at this level and label it, as always avoiding crossing lines and touching the object demonstrated with the line you draw to it. adrenal gland kidneys bladder cloaca Muscular Anatomy. Using the forceps and scissors to get started, pull the skin from your frog. Each leg can be skinned easily by cutting the skin around the appendage and pulling the skin off like a latex glove. This will expose the muscles of the frog. You might notice that the frog's musculature is virtually identical to yours. The pectoral appendage has biceps muscles to bring the foreleg closer to the body (flex it), and triceps muscles to return it nearer to straight (extend it) at the elbow. The shoulder is extended by the deltoid muscles and flexed with the latissimus muscles. The hind leg has quadriceps muscles to extend the foreleg and hamstring muscles to flex it. The hindleg has a gastrocnemius muscle to extend the foot. This "calf" muscle and the quadriceps muscles are particularly well developed in a frog obviously an adaptation for jumping even though Xenopus mostly uses these for swimming. On the other hand, the hamstring muscles are weakly developed because frogs do not stand upright on two legs (contrary to some frog images you see around our town!). Make a sketch of your skinned frog below (head to top and frog's dorsal surface up) and label completely the appendicular muscles mentioned above in bold type. deltoid latissimus biceps triceps quadriceps hamstrings gastrocnemius /11