BIO112 Laboratory Guide #7
DIVERSITY OF THE VERTEBRATES I
INTRODUCTION
Compared to the diversity of invertebrate phyla body plans, the vertebrates are a pretty homogeneous group. Vertebrates are properly a subphylum of Phylum Chordata. As such, all vertebrates are segmental deuterostomes which evidence each of the following defining characteristics of the chordates at some stage in their development:
1. A dorsal hollow nerve cord, which is a tube composed of neurons that runs beneath the dorsal surface of the animal. The anterior portion of the nerve cord is modified to form a brain.
2. A notochord, which is a flexible connective tissue rod that extends the length of the body between the gut and the nerve cord. In more primitive vertebrates this is maintained in the adult. In the more advanced vertebrates, the notochord is replaced by the segmental vertebral bodies.
3. Pharyngeal gills slits, which are openings from the upper portion of the digestive tract through the body wall. The fate of these slits during development varies in the different vertebrate classes.
4. A post-anal tail.
Unlike members of the other two chordate subphyla, the Urochordata and Cephalochordata, members of Subphylum Vertebrata have a 'backbone'; i.e., a column of vertebrae composed of bone or cartilage which surrounds and protects the nerve cord. Anteriorly, the vertebral column connects to the cranium, which surrounds the brain. Other defining characteristics for the vertebrates include the following:
1. A three-part brain: forebrain, midbrain, hindbrain.
2. A cartilaginous or bony cranium which surrounds the brain.
3. Replacement of the notochord in the adult with cartilaginous or bony segmental vertebrae.
4. Semicircular canals for sensing equilibrium and acceleration.
5. Tissues derived from neural crest cells.
6. The presence of nephritic kidneys which remove metabolic wastes.
7. A closed circulatory system.
In this exercise, you will see many of the above shared characteristics, as well as those shared derived characteristics which differ among the eight major vertebrate classes and represent evolutionary "steps". However, you should recognize three things about the taxonomy of vertebrates:
1. There is no universally accepted taxonomic scheme for the vertebrates. The taxonomy presented here is a widely used classification scheme, but not the only one in common use.
2. Traditional "phenetic" taxonomies are based almost exclusively on morphology. They do not reconcile very well with the actually phylogeny (evolutionary history) of the vertebrates. As an example, birds are phylogenetically a subset of the dinosaurs, which are themselves a subset of the reptiles. However, traditional taxonomy artificially elevates birds to the level of a class, while relegating dinosaurs to a mere order in Class Reptilia. Similarly, traditional taxonomies totally miss the rather close phylogenetic relationship between the birds and the crocodilians; crocodilians are in many ways anatomically and physiologically more similar to birds than to any other living reptiles.
3. The alternative "cladistic" taxonomies which rely strictly on monophyletic groups are prohibitively cumbersome to use because they are not strictly hierarchical and they define many logical modern vertebrate groups by exclusion; e.g. dinosaurs become "non-avian dinosaurs".
This lab will use an evolutionary taxonomy which respects phylogenetic relationships, but accepts paraphyletic Classes, because they more convenient, straightforward and familiar. We will also study only extant (living) groups of vertebrates, while recognizing that the vast majority of vertebrate species and the majority of higher taxa are, in fact, extinct.
In the first part of this lab you will study the diversity of the heterothermic vertebrates, that is Classes Myxini (hagfishes), Cephalaspidomorha (lampreys), Chondricthyes (cartilaginous fishes), Osteichthyes (bony fishes), Amphibia (amphibians), and Reptilia (reptiles). We will leave the two homeothermic Classes Aves (birds) and our own Mammalia for next week.
In the second part of the lab you will conduct an experiment in vertebrate neuromuscular physiology. This experiment will involve a very classical "preparation", electrical stimulation of the the sciatic nerve and mechanical response of the gastrocnemius muscle from the hind leg of a frog.
After completing this laboratory you should be able to:
1) describe the distinguishing characteristics of the living vertebrate Classes.
2) correctly name the major taxa within each Class.
3) classify representative animals from each taxon.
4) describe the major adaptive trends in vertebrate evolution, especially those accompanying the movement of some ancestral groups from water to land.
5) describe the methodology, results, and significance of electrically induced muscle twitches in the frog.
PART I. DIVERSITY OF THE VERTEBRATES
Materials
Representative live, preserved, skeletal, and/or fossil specimens
for most taxonomic groups.
Models for some taxonomic groups
Poster guides to the diversity within some groups.
Procedure
Specimens of each vertebrate class are on display. These specimens include skeletal remains, preserved animals, live animals, and some behavioral artifacts
1. Work through these displays.
a. A dichotomous key which outlines the major distinguishing features of each vertebrate class is provided below.
b. Following the key is additional information about each class, as well as a guide to lower taxa (subclasses, infraclasses, superorders, orders) within each class. On the sample specimens, identify and examine all of the external structures written in bold print in this guide.
c. Additional information will be on display with the specimens.
d. Your textbook has additional information on these vertebrate classes. The Biology Department has several additional vertebrate biology textbooks.
2. Be able to describe the distinguishing features of each class of vertebrates. Within each class, be able to recognize the major subdivisions.
3. Be able to use the distinguishing features to reliably classify these sample vertebrates. Pay particular attention to animals from different classes which look superficially similar. For example, think about how could you reliably distinguish a salamander (Amphibia) from a lizard (Reptilia), a shark (Chondrichtyes) from a sturgeon (Osteichthyes), or a lamprey (Cephalaspidomorphi) from an eel (Osteichtyes) or snake (Reptilia).
Study Suggestions
1. Make detailed sketches and notes on specimens. This will help you to look at the specimens more closely, as well as to help you study later.
2. Plan to come view the specimens once or twice more before the lab test. Test yourself by attempting to identify the specimens as accurately as possible by their common names, as well as to classify them without first looking at their labels.
3. The words in bold print in the extended guide below are words you should know and/or structures you should be able to identify or describe.
A Dichotomous Key to the Vertebrate Classes
Note: This key is based, in some cases, on secondarily derived characteristics of adult animals. This key roughly follows actual phylogenetic relationships. Notice that this produces an "unbalanced" key with lots of exceptions (e.g. snakes are reptilian tetrapods but do not have four legs) and omissions (e.g. non-avian dinosaurs).
1. Organisms are without jaws MYXINI, CEPHALASPIDOMORPHI[also: lack paired appendages (fins)]
1. Organisms have jaws
2. Organisms have fins
3. Organisms have cartilaginous skeletons CHONDRICHTHYES[Also: most have no operculum covering gills slits; have 5-7 gills with
separate openings; have non-overlapping, placoid (bony) scales with
projecting points; have subterminal mouth; tail is either heterocercal
or whip-like; have no swim bladder but use oil instead for flotation]
3. Organisms have partially bony skeletons OSTEICHTHYES
[Also: have an operculum over gill slits; have thin, overlapping dermalscales; most use swim bladder for flotation]
2. Organisms are tetrapods ('four-footed') as adults or embryologically
3. Organisms have moist skin AMPHIBIA
[Also: terrestrial but remain tied to aquatic habitats; usually haveexternal fertilization; eggs have jelly-like membrane coverings;
development includes metamorphosis from aquatic larval form
to lung breathing adult (usually); are ectothermic
3. Organisms have dry skin
4. Organisms are ectothermic or heterothermic REPTILIA[Also: have scales; have amniotic egg with leathery shells;
have internal fertilization; have homodont dentition or
are adontoid]
4. Organisms are endothermic or homeothermic[Also have well-developed brains with enlarged forebrains;
have an erect stance; have extended care of dependent young]
5. Organisms have feathers AVES [Week 8]
[Also: have front limbs modified for flight (usually); have scaleson feet; have an amniotic egg with calcareous shell;
have beak and are devoid of teeth]
5. Organisms have fur (hair) MAMMALIA [Week 8]
[Also: have mammary glands; have heterodont dentition(usually); have prehepatic diaphragms; have well-developed
forebrains]
SUPERCLASS AGNATHA (jawless fishes)
Members of superclass Agnatha are primitive, jawless fish. Their endoskeletons are composed almost exclusively of cartilage and fibrous tissue, with virtually no bone. They have no true lateral appendages. Extinct groups of agnathans were ancestral to all modern vertebrates. Modern agnathans include the hagfishes (class Myxini) and lampreys (class Cephalaspidomorphi). Both hagfishes and lampreys are eel-like in form and have a single dorsal nostril.
CLASS MYXINI (hagfishes)
Hagfishes are scavengers; they attach to the flesh of dead fish with their mouths and use their rough tongues to scrape away tissue. They retain a pattent notochord into adulthood, have no cranium, and have very little cartilage.
Observe the preserved hagfish on display. Find the single dorsal nostril and the lateral gill slits. Note the multiple sensory tentacles surrounding the jawless mouth.
Watch the videos of two unusual and novel hagfish behaviors. The first behavior is "sliming". When irritated, hagfish secrete a small amount of a proteoglycan from specialized slime glands. This reacts instantly with sea water to produce a simply astounding amount of thick slime; an effective deterrent to most would-be predators which gives then the name "slime-hag". The second behavior is "knotting". In this behavior the hagfish actually ties its entire body into a simple knot, then pulls the head through the center. This is an effective way to gain the leverage necessary to pull chunks of flesh from its dead meals.
CLASS CEPHALASPIDOMORHA (lampreys)
Most lampreys are parasites which attach with their circular mouths to living fish, rasp away enough tissue to maintain a blood flow, and then ingest the blood. Unlike the hagfish the lampreys have a partial cartilaginous cranium enclosing the brain.
Observe the preserved, sectioned lampreys, as well as the "plastomount" preparations. Identify the round "cyclostome" mouth and tongue lined with file-like teeth. Identify the small eyes and the multiple lateral gill slits. Find the anus and note the characteristic chordate post-anal tail. In the sagittally-sectioned lampreys identify the notochord and the dorsal hollow nerve cord running the length of the body. At the rostral end of the nerve cord find the tiny brain. Just deep to the single nostril and rostral to the brain find the blind-ended olfactory sac.
Watch the video of lampreys clinging to rock in fast-moving streams. The round mouth makes an excellent suction cup and accounts for their alternative class name of Petromyzontia - literally "the rock clingers".
SUPERCLASS GNATHOSTOMA (jawed vetebrates)
Members of superclass Gnathostoma have three common derived characters which distinguish them from the Gnathostomes. The first is the presence of upper and lower jaws derived from the anterior gill arches. The second is the presence of paired pectoral (anterior) and pelvic (posterior) appendages.
CLASS CHONDRICTHYES (cartilaginous fishes)
Chondrichthyans are the most primitive living form of gnathostomes. They are exclusively marine fish. Their endoskeletons are composed entirely of cartilage. They have placoid scales, composed of a bony dermal "teeth" which project through the epidermis, giving the skin its characteristic "sandpaper" feel. They are negatively buoyant and must actively swim to stay off the bottom. Chondrichthyans are generally divided into two subclasses: Subclass Elasmobranchii (sharks, rays, skates, and sawfishes) and Subclass Holocephali (chimeras and ratfishes). Some species of sharks have remained essentially unchanged since the Jurassic Period, more than 65 million years ago.
Subclass Elasmobranchii (literally "plate gills")The elasmobranchs have multiple exposed gill slits. The most anterior gill slit on each side is reduced into a small, dorsally located spiracle. Sharks are free-swimming, have exposed lateral gill slits and a heterocercal tail (dorsal lobe is larger than ventral lobe). Buoyancy is provided by the heterocercal tail, hydroplaning action of the flat pectoral (anterior) fins, and oil produced by the liver. Skates, rays, sawfish, and some sharks are compressed dorsoventrally, have ventrally located gill slits, and have pectoral fins flattened into broad, wing-like structures. They are predominantly bottom-feeders.
On the shark skeleton find the horizontally mounted pectoral and pelvic fins as well as the heterocercal tail. On the skull locate the chondrocranium surrounding the brain, the separate upper and lower jaws, and the multiple branchial gill arches which support the gill plates.
Look closely at the shark jaws on display . Notice that both the upper and lower jaws are only loosely attached to the skull. This allows sharks to extend both jaws during feeding to tear flesh from their prey. Notice also the multiple rows of surface-mounted teeth. As individual teeth are lost during the feeding process, new teeth move forward to replace them. The evolutionary longevity of the sharks and their habit of losing teeth accounts for the large numbers of fossil shark teeth.
Compare the preserved shark, skate, and ray specimens. When handing these watch out for the sharp dorsal spines! On each fish find the nostrils leading into blind-ended olfactory sacs, the prominent eyes, the dorsal spiracle (actually the most rostral gill opening), and the multiple exposed gill slits. Feel the placoid scales by rubbing the skin in several directions. How are these scales oriented? Pressing lightly on the skin of the rostrum will produce a light waxy discharge from the ampullae of Lorenzini, electrosensory organs of the skin. On the ventral surface of each animal find the mouth and the cloaca which serves as a common reproductive, digestive, and urinary opening.
Subclass Holocephali (literally "complete heads)
The holocephalans look a bit as if they were constructed from mismatched parts of other animals. This is why the group is alternatively called the "chimerae" and individual members are called either "ratfish" or "rabbitfish".
Examine the preserved ratfish and the ratfish skull. Find the three pairs of prominent plate-like teeth which give this fish its name. Note that its gill slits are covered with an single plate-like operculum on each side. Notice that the cranium is completely enclosed with cartilage (hence the group name "Holocephali"). How does this compare to the shark chondrocranium?
CLASS OSTEICHTHYES (bony fishes)
Osteichthyans remain the most speciose class of vertebrates; there are more species of these bony fish than of all of other vertebrate classes combined. They inhabit both marine and fresh-water environments. In osteichthyans the endoskeleton is composed partly to primarily of bone. They are mostly free-swimming, with buoyancy provided by a gas-filled swim-bladder (see the fish skeleton on display), derived as dorsal outpocketing of the digestive system. The gills are covered with a bony plate, called an operculum, which increases the efficiency of ventilation (movement of water across the gills). Most bony fish are suction-feeders, they eat by rapidly expanding the pharynx to suck water, food items, and/or prey into the mouth. The skin is generally covered by overlapping dermal scales which do not project through the epidermis.
The two main groups of extant bony fish are the Subclass Actinopterygii (ray-finned fish) and the much smaller Subclass Sarcopterygii (lobe-finned fish).
Subclass Actinopterygii (ray-finned fishes)Actinopterygians may be divided into three superorders. Order Chondrostei (sturgeons, paddlefish, bichirs) have skeletons composed mostly of cartilage. Living species are exclusively fresh-water. They have heterocercal (uneven lobes) or holocercal (single lobe) tails. Order Holostei (gars, and bowfins), have largely bony skeletons, but the cranium is cartilaginous. They are fresh-water fish with holocercal tails. Order Teleostei (all other ray-finned fish) is by far the most speciose and, in many ways, the most modern group. Teleost fish skeletons are composed almost exclusively of bone, and they generally have homocercal (same size lobes) tails.
Examine the skeletons and preserved specimens of ray-finned fish. Note the prominent operculm, which increases the efficiency of respiration in non-mowing fish. Note the swim-bladder preserved with the carp skeleton.
Subclass Sarcopterygii (lobe-finned fishes)
Sarcopterygians are "lobe-finned" fish, with a characteristic fleshy lobe at the base of each lateral fin. They tend to be bottom-dwelling or shallow water fish, in which the fins are used to maneuver along the substrate. There are two orders of living sarcopterygians. Order Actinista/Crossopterygii is currently represented by only a single marine species, the coelocanth, a so-called "living fossil". Order Dipnoi includes the fresh-water lungfishes and the ancestors of all of the tetrapod vertebrates. Lungfishes have some "primitive" features, includinga prominent notochord and a mostly cartilaginous skeleton. They also have some "modern" features similar to amphibians, such as paired atria in the heart, and paired ventral lungs, which allow them to breath air.
Examine the models of the coelocanth and lungfish. How are the fleshy appendages a preadaptation to moving on land?
CLASS AMPHIBIA (amphibians)
Amphibians (literally "dual life") are a group of terrestrial and aquatic (fresh-water) vertebrates. Modern amphibians all have moist, smooth, scale-less skins, with numerous mucous and poison glands. Terrestrial amphibians generally must return to the water to lay eggs. Terrestrial amphibians have paired lungs. Respiration in aquatic amphibians and larval forms is via either external gills or the skin (cutaneous respiration). Amphibians have a three-chambered heart with two atria and a single ventricle. Modern amphibians have kinetic skulls, meaning that there are multiple jaw hinges. Feeding is generally by suction in aquatic forms and by "lingual feeding" (capturing prey with an extended tongue) for terrestrial forms. Amphibians are "cold-blooded" ectotherms whose body temperature is largely determined by the surrounding environmental temperature.
Amphibians are generally divided into three orders - Order Urodella/Caudata (salamanders), Order Anura/Salientia (frogs and toads), and Order Apoda/Gymnophiona (caecilians)
Order Urodela/Caudata (salamanders)
Salamanders have four legs (usually) and a prominent tail. The legs do not support the trunk off the ground. Salamanders move by undulating the body, with the legs serving merely as "pivot-points". Salamanders all have aquatic larval forms. Some species remain aquatic throughout their lives, some species have a terrestrial "eft" stage then return to the water as adults, and some species metamorphose directly into fully terrestrial adults. Salamanders have internal fertilization; sperm are passed from male to female in a spermatophore sack.
Examine the salmander models and preserved specimens and the very generalized body structure relative to the frogs and toads.
Order Anura/Salientia (forgs and toads)Anuran eggs and sperm are shed into the water, where external fertilization takes place. These eggs hatch into tadpole larvae. Tadpoles grow and eventually metamorphose into tail-less adults. Additional diagnostic features of frogs and toads are the relatively long hind legs used for jumping and/or swimming, and a prominent external eardrum or tympanum associated with each ear.
Examine the skeletons and preserved specimens of the frog and toads. Note the highly modified pelvic structure, including the enlarged and fused pelvic girdle bones and the central urostyle. Note the tadpole larvae and the metamorphosis process that leads to adulthood.
Order Apoda/Gymnophiona (caecilians)Caecilians have no legs and no pelvic or pectoral girdles. They are burrowing animals of tropical rainforests.
CLASS REPTILIA (reptiles)
Reptiles are a diverse group of vertebrates. Primitive reptiles descended from primitive scaled amphibians, and in turn, gave rise to modern reptiles, birds, and mammals, as well as several extinct groups. Reptiles have a dry skin, covered with cornified epidermal scales which prevent desiccation. Reptiles and most of their descendants have amniotic eggs, with a delicate sac-like membrane which surrounds the embryo and maintains a fluid environment within the egg. Reptile eggs are also covered with a leathery egg shell which prevents desiccation (see display egg). This, and internal fertilization, frees many modern reptiles from a dependence on water. The reptilian heart has a ventricle which is partially or completely partitioned into two separate chambers for pulmonary vs. systemic circulation. Reptiles, like all of the groups discussed so far, are heterotherms, meaning that their body temperature fluctuates with the environmental temperature. However, most reptiles generate at least some internal heat by metabolic processes, making them at least partially endothermic.
Classical taxonomy divides modern reptiles into three major orders: Order Chelonia/Testudinata (turtles), Order Squamata (lizards and snakes), and Order Crocodilia (alligators, crocodyles, caimans.
Order Chelonia/Testudinata (turtles and tortoises)
Turtles and tortoises are characterized by their shell, which consists of a dorsal carapace and a ventral plastron. Each is constructed of elaborated dermal plates. The ribs and vertebrae are fused to the shell, and the pelvic and pectoral girdles are relocated inside the rib cage. Turtles have no teeth; feeding is by a combination of biting beak-like jaws, and suction-feeding in aquatic forms. Turtle cardiovascular anatomy is clearly adapted for diving.
less lizards.
Examine the turtle skeletons on display. Locate the fused vertebrae inside the dorsal carapace of the shell. Of what common skeletal elements is the bulk of the shell formed? Note the position of both the pelvic and pectoral "girdles" inside the shell? How do you suppose that this animal breathes, given that the ribs are not flexible?
Order Squamata (lizards and snakes)The squamates are traditionally divided into the lizards (Suborder Lacertilia), snakes (Suborder Serpentes), and amphibeans (Suborder Amphisbea). However, recent molecular evidence suggests that the snakes and venemous lizards for their own clade (Toxicofera) and that the legless amphibeans evolved within one of the remaining lizard groups.
Squamates have kinetic skulls and feed by grasping the prey and swallowing it whole. Most lizards are quadrupeds, but look closely at the display samples of the legless lizards or "glass-snakes". They may be distinguished from both salamanders and snakes by the presence of external ear openings and eyelids. Lizards also tend to hold the trunk slightly off of the ground, allowing somewhat more rapid and efficient terrestrial locomotion than in salamanders. Snakes are a group of legless descendants of lizards and move by undulating the body against the substrate. Most snakes are terrestrial, but some sea snakes are marine and return to the land only to breed. Snakes provide an extreme example of a kinetic skull with multiple jaw bones and hinges. A snake can generally swallow whole a prey which is larger than the apparent size of the snake's head. Most snakes are oviparous (lay eggs), but a few species are ovoviviparous (eggs hatch in the oviducts and live young are born). Amphisbeans are another group of essentially legless lizards.
Examine the skeletons and preserved specimens of lizards and snakes. Compare the lizard and salamander skeletons. What skeletal structures are more developed in the lizards? How do these relate to the more erect stance/posture of most lizards? How can you reliably distinguish between snakes and legless lizards, based on external anatomy?
Order Crocodilia includes crocodiles, caimans, alligators, and gavials. Crocodilians are distinguished from other reptiles by the elongated snout, the akinetic skull with a single pair of sturdy jaw hinges, and by the two postorbital temporal fenestrae (diapsid = two skull openings behind the eye socket). Crocodilians also have a true four-chambered heart, however there are vascular shunts between the systemic and pulmonary circulations. Crocodilians are also partially homeothermic and have extended care of their young.
Examine the crocodilian skeleton and skulls on display and compare them to those of the squamate lizards? What features of the crocodilian skull allow for a significantly stronger bite? How does crocodilian feeding differ from that of the lizards?
Locate the bony gastralia under the ventral abdomen. What is the function of this unusual structure?
CLASSES AVES (birds) and MAMMALIA (mammals)
You will explore the diversity of the birds and mammals in next week's lab.
PART II. VERTEBRATE NEUROMUSCULAR PHSYIOLOGY
The basic unit of skeletal muscle contraction is called a muscle twitch. You will use a muscle preparation dissected from the leg of a freshly-killed frog to study three aspects of skeletal muscle twitches: 1) the induction of a single twitch by electrical stimulation of the associated nerve, 2) the process of recruitment, where muscle contractile tension increases with increasing stimulus intensity, and 3) the process of temporal summation, where successive twitches closely spaced in time sum together to produce a stronger contraction.
The muscle "preparation" consists of the femur (of the thigh), gastrocnemius muscle and Achilles tendon (of the calf), and the distal part of the sciatic nerve (which runs from the spinal cord to the lower leg). The recording apparatus will consist of a force transducer (converts muscle tension into a graded electrical signal), connected to a bridge amplifier (amplifies the signal), connected to a MacLab A/D box (filters the analog signal and converts it to a digital signal data stream), connected to a Macintosh computer running MacLab software (processes the digital data stream and graphically displays the result). The stimulating apparatus consists of an electronic stimulator connected to a stimulator probe, which is in contact with the sciatic nerve. A connection between the stimulator and the MacLab box allows recording "sweeps" of the MacLab software to be synchronized with stimulator output pulses.
The instructor and lab assistant will steer you through the mechanics of this somewhat complicated experimental apparatus. You are not expected to learn all of the nuances of the equipment in the limited time available. You are, however, expected to understand the theory behind what you are doing, and what your results demonstrate about muscle physiology.
Materials
PowerLab/PC physiology recording stations
Recording "peripherals" set up for stimulating and recording from frog muscles
Pithed frogs
Procedure
a. Muscle Preparation:
1. Put on some gloves.
2. Obtain a decapitated or pithed frog from the instructor. Cut off the hind leg at the proximal (upper) end of the thigh and give the frog and remaining leg to another group. Strip the skin from the hind leg, being careful not to let the outside of the skin touch the muscle or nerves.
3. Locate the sciatic nerve in the thigh, free it between the knee and the hip using a glass hook, tie a thread around it near the hip, then cut it proximal to the thread. Do not touch the nerve with your fingers or anything metal. Lay the nerve along the gastrocnemius muscle of the calf. Now cut away all of the thigh muscles (but not the nerve!!) to expose the femur.
4. Tie a thread tightly around the Achilles tendon, then cut the tendon close to the ankle. Use this thread to gently lift the gastrocnemius muscle and sciatic nerve, and reflect them back over the femur. Now cut through the tibiofibula bone and the other lower leg muscles immediately below the knee. You should now have the gastrocnemius muscle attached distally to a thread at the Achilles tendon, and proximally to both the femur and a length of the sciatic nerve. Keep this preparation moistened with Ringer's solution (saline) at all times.
5. Mount the shaft of the femur in the femur clamp and securely tighten the jaws. Do not let the sciatic nerve touch the femur clamp or the ring stand. Tie the Achilles tendon thread to the force transducer and adjust the transducer to stretch the muscle to approximately its original resting length in the frog.
6. Your instructor will adjust the clamps, transducer position, and bridge amplifier offset to give you a zero volt baseline reading on the Macintosh display.
7. Position the silver stimulating electrodes as close to the knee joint as possible. Gently wrap the nerve around the stimulating electrodes, trying not to stretch it too much. Moisten the nerve and the muscle with Ringer's solution, then coat all of the exposed nerve with a generous layer of Vaseline. This will keep it from drying out.
b. Recording a Muscle Twitch:
1. We will be start by using a PowerLab application called "Scope" to monitor the muscle twitches. This application produces a display on the computer screen which emulates that of an oscilloscope. You instructor will set this up for you.
2. Adjust the stimulator to give a stimulus delay of 100 msec., a duration of 0.05 msec., and an intensity of 0.1 volts.
3. Click once on the START button at the lower right of the computer screen. This triggers both a recording sweep on the display and a stimulus pulse delivered from the stimulator to the sciatic nerve.
4. Continue to click the START button to deliver pulses repeatedly to the nerve, while watching both the computer display and the muscle itself. Slowly increase the stimulus intensity using the VOLTS knob and multiplier switch on the stimulator. Continue to increase the voltage until you get a distinct twitch from the muscle and a clear vertical deflection in the recorded trace.
5. The minimal stimulus duration and voltage required to elicit a distinct twitch is called the threshold. Record your threshold stimulator settings.
c. Recruitment:
1. Clear out the earlier traces by clicking on New under the File menu.
2. Now, starting from your threshold settings, record successive traces, gradually increasing the voltage for each recording sweep. Notice that the sweeps are accumulated as numbered pages on the screen. Make sure that you keep a clear written record of the voltage and duration settings for each sweep
3. As the stimulus amplitude increases more nerve fibers will be activated, hence more muscle fibers will be activated, and hence the amplitude of the muscle twitch will increase. This process is called recruitment. At a stimulus intensity which is above threshold for all of the motor fibers of the sciatic nerve, the twitch tension of the muscle will be maximal. Do not increase the stimulus amplitude too far beyond this point.
4. Save your Scope traces to a named file, using the Save command under the File menu.
d. Temporal Summation and Tetanus:
1. For the final part of this experiment you will record your muscle data with a PowerLab application called "Chart". This application produces a continuous scrolling record on the screen display, which emulates the output of a chart recorder. The stimulator will also need to reset for repetitive pulses. The instructor or lab assistant will reset the equipment for you and help you get started.
2. Set the stimulator to a voltage just sufficient to produce a maximal twitch for single stimulator pulses, based on the previous experiment. Set the stimulator frequency to 0.5 Hz (1 stimulus every 2 seconds).
3. Start the Chart display by clicking on the START button at the lower right of the screen.
Initiate repetitive stimulation by clicking the MODE switch to the REPEAT setting. At these settings you should get a distinct muscle twitch once every 2 seconds.
4. Allow the Chart display to run for about 5 seconds, then quickly double the stimulus frequency to 1.0 Hz. After 5 more seconds, double the stimulus frequency again to 2.0 Hz. Continue this process of intensity doubling until a sustained, continuous, maximal contraction is produced. Don't stimulate at this rate for more than 10 seconds. To terminate stimulation, click the MODE switch on the stimulator back to the OFF setting. Stop the Chart recording by clicking on the STOP button at the lower right of the screen.
5. Save this record to disk, using the Save option under the File menu.
6. Scroll back to the beginning of your Chart display, then scroll slowly forward. Notice that, as the individual twitches begin to overlap in time, the muscle tension starts to sum and the peak muscle tension increases. This process is called temporal summation and is related to the accumulation of calcium in the cytoplasm of individual muscle cells. At a critically high stimulation frequency a sustained maximal contraction called tetanus is produced. If you maintain this frequency or proceed past it for very long, tension may drop off as the muscle depletes available ATP and creatine phosphate supplies and becomes exhausted.
7. When you are finished, exit from Chart, remove and dispose of the frog leg preparation, and carefully clean your recording apparatus.
e. Printing out your results:
1. Open your Scope data file. Print out the superimposed traces using Print under the File menu. Exit from Scope when you are through. On each printout, locate and label traces corresponding to subthreshold, minimal, half-maximal, and maximal response amplitudes. Add to each label the stimulator duration (in milliseconds) and amplitude (in volts) settings.
2. Open your Chart data file. Print out a section of the record demonstrating distinct twitches, temporal summation, and tetanus. Exit from Chart when you are through.
On the printout, write the stimulus pulse duration and intensity settings and clearly label each change in stimulation frequency. Finally mark the point at which temporal summation begins and the point at which tetanic contraction begins.
f. Cleaning up:
1. Bag up your frog remains in a plastic bag.
2. Rinse off the surgical instruments and dissecting tray and spread them out to dry. Wipe any blood or fluid off of the frog femur clamp and transduced. Do not get the trasducer wet!
3. Turn off the Grass electronic stimulator. Turn off the PowerLab box (switch near floor level at the back of the cart).