Histology

MUSCLE TISSUE
Smooth and Skeletal Muscle
Muscle is one of the four "basic" tissues. "Muscle" is a broad term that can be applied to several sub-categories based on
appearance and structure, but the fundamentals of muscle function are the same for all types. The two major
classifications are smooth muscle (sometimes called "visceral muscle" because of its common occurrence in internal
organs) and striated muscle. The striated category is subdivided further into skeletal and cardiac muscle.

Skeletal muscle is sometimes called "voluntary" muscle, because it's subject to conscious nervous system control. It's the
principal form in terms of mass as most of the body's bulk is skeletal muscle, used for locomotion and other conscious
activities; another word for it is "meat," and skeletal muscle is what you cook on the barbecue.

Cardiac muscle is a special form of striated muscle, found only in the heart.

Excitability
All muscle cells are "excitable cells," i.e., they are capable of responding to appropriate stimulation. The response to this
is contraction. The stimulus can vary. It may be an electrical signal or depolarization produced at the surface of the
muscle cell by the activity of neuron (which themselves "excitable" cells). Some muscle cells (particularly smooth
muscle) can respond not only to neuronal signaling, but to direct chemical or mechanical stimulation. Hormonal signals
can cause contraction in some situations.

Blood & Nervous Supply

Muscle is a metabolically dynamic tissue, so it is very well supplied with blood vessels -- many capillaries and small
blood vessels in among muscle fibers of all types. In keeping with the functional considerations of muscle as an "effector
organ" for the nervous system, you will of course also encounter profiles of nerves and other neural elements. To keep the
whole thing structurally intact, there is a good deal of collagenous CT present, as well.

Smooth Muscle
Smooth muscle is found in virtually all organ systems to some extent. It's present in large amounts

A. In some organs of the reproductive tract:

1. the uterus (especially)
2. the walls of the uterine tubes
3. the wall of the epididymis, and in
4. the ejaculatory apparatus).
B. Smooth muscle forms the bulk of the wall in the gut
C. Smooth muscle is present to some degree in all blood vessels larger than capillaries.

Individual smooth muscle cells are "fusiform" or "spindle shaped," that is, they are longer than they are wide, tapered at
each end and widest in the middle.
The images at left show smooth muscle in x-section and longitudinal section, diagrammatically and in an actual specimen.
The x-sections are at the top of the field in each case.

In cross sections, each smooth muscle cell is individually visible as a roughly circular profile, and the size of each profile
varies. Some are large, some are small: it depends upon the point in the double-tapered cell that the plane of the section
has passed through. Only the largest ones will have nuclei visible in them, because the nucleus occupies the central
"fattest" portion of the cell. The size of nuclear profiles is related to the size of the cell profile.

The boundaries of adjacent cells are not very distinct in the longitudinal section as they are in x-section. In x-section, the
separation between fibers is the space occupied by the endomysium around each fiber. It's there in the longitudinal
sections, but not easily made out because the cells overlap each other to form a continuous sheet. Furthermore, they're
firmly attached to each other for purposes of communication and tissue cohesiveness.

This drawing should make the lateral relationship between two smooth muscle cells a little more understandable. They're
represented as they would be seen in a 3-dimensional reconstruction from an EM. The "fat" part of one lies against the
tapered end of the adjacent cell. A gap junction is shown: since these cells have to contract in a coordinated manner
and don't have the precise neural control that skeletal muscle cells do, they need to interact with each other: that's the
function of the gap junction.

Smooth muscle have neural input but it lacks the elaborate neuromuscular junction of skeletal fibers. Neurotransmitters
released from the ends of nerve fibers that lie adjacent to the cells can be taken into the cells by pinocytosis. Pinocytotic
vesicles are a common feature of smooth muscle cells.

The images above shows smooth muscle in longitudinal section on one side and in x-section on the other; examine the x-
sections first. Right off, notice the varying diameters of the cell profiles, and the fact that only about one in ten shows a
nucleus. The different sizes result from the tapering shape of the cells; if cut near the tip the profile will be very small,
and if near the middle, much larger. If cut in the portion of the cell which contains the nucleus, a nuclear profile will be
seen; and since the nucleus is also slightly tapered, the same variability of size results.

Look at the longitudinally sectioned area. The cells lie neatly against each other, with the wide portion of one nested
against the narrow parts of its neighbors.

The "wrinkled" look to the nuclei in the longitudinal section is due to contraction of the muscle sheet. This is partly from
its natural tonus and partly a result of fixation, i.e., it's an artifact of preparation. Nevertheless, it's a pretty reliable one
and it can serve as an identifying feature of smooth muscle in section. Contraction of the cell and compression of the
nucleus into a shorter length causes this. In extension, the nuclei are often elongated, and in the relaxed but not contracted
state they may have a "cigar shape."

Typical smooth muscle cells are ca 15-20 microns wide, but they may be up to half a mm in length in some cases. This is
exceptional; 0.2 mm is more common. These cells have a single nucleus, located at the widest point. Since the nucleus'
length is much less than that of the cell itself, a sectioning plane that passes through the cell at right angles to its long axis
usually misses the nucleus.
Smooth muscle cells don't show a banding pattern like those of striated muscle. They do have internal actin and
myosin filaments, but their contractile units are not in register. The relationship of smooth muscle with the surrounding
CT is essentially the same as that of skeletal muscle, except that the epimysium doesn't continue into tendons.

You can also see these features in the smooth muscle of the intestinal wall. The outer portions of this section are
composed of two layers of smooth muscle oriented at right angles, so that one is cut in x-section and one longitudinally.

"Look Alikes" for Smooth Muscle

Smooth muscle can be confused with other tissues, especially regular CT, and one of the easiest traps to fall into is
mistaking one for the other. Note that the nuclei in smooth muscle are inside the cells, and you should have no problems
telling it from possible look alikes. It's always best to try to find areas of the tissue cut in x- section; this affords better
identification than the longitudinally cut areas.

Skeletal Muscle
By definition, skeletal muscle is that form which is attached to the skeleton (in common English usage, the word "brawn"
has come to mean brute muscle power). Remember that skeletal muscle is only one of the two forms of striated muscle,
and "skeletal" and "striated" are not synonymous terms. Cardiac muscle, which is restricted to the heart, is also striated.

Nomenclature
The myofiber is the individual skeletal muscle cell, and myofibers are bundled together into groups, or fascicles. These
fascicles are in turn "tied" together by CT to form anatomic muscles. The myofiber has internal structure, and each
myofiber contains hundreds of myofibrils. The myofibrils in turn are composed of smaller structures, the myofilaments.
These are the actual contractile parts of the cell, and are principally composed of the two specialized muscle proteins
myosin and actin.

Skeletal Muscle in Sections

At LPO, one can make out individual myofibers, or muscle cells. Skeletal myofibers are cylindrical in shape, about 100
microns or so in diameter. The really amazing dimension is their length. Most of them are at least several centimeters
long, and it's possible for individual myofibers in large anatomic muscles to be half a meter or more from end to end! In
the longitudinally section (below), you should be able to verify the elongated shapes of the myofibers. The banding
pattern of skeletal muscle should be quite obvious. It's a dead giveaway for the nature of this tissue; only the two types of
striated muscle show it.

This field shows the banding pattern

characteristic of skeletal muscle very
nicely. The alternating dark and light
bands so obvious in this image make this
tissue hard to mistake for anything else.

The dark bands are the A and the light ones

are the I bands. The I band shows a thin
darker Z-line within it; this actually is the
demarcation of the limit of the contractile
unit of skeletal muscle, the sarcomere.

An A band contains both actin and myosin

filaments; an I band contains only actin
filaments. The striated appearance comes
about because each of these myofibers
contains millions of myofibrils whose A
and I bands are in register with each other.
Each myofibril is composed of millions of
sarcomeres in series. A useful mechanical
analogy is the arrangements of railroad
trains in a switchyard on parallel tracks.

The Mechanism of Contraction: The "Sliding Filament Hypothesis"

The basic contractile unit of muscle is the sarcomere. In a sense, sarcomeres are subunits of myofibrils. They are
composed of myofilaments (actin and myosin) arranged in a very specific fashion. Within each myofiber, sarcomeres are
joined end to end, thousands of them in innumerable parallel myofibrils. Each one is anchored to the end of the anatomic
muscle. Find an area which the muscle is cut longitudinally, and you'll be able to make out the different bands quite well.
I bands are the portions of the sarcomeres in which only actin filaments are located, seen as light bands; the darker A
bands contain both actin and myosin fibers. The I bands you see here are actually two "half-I bands" back to back
between adjacent sarcomeres.

When a striated muscle contracts, the ends of all the sarcomeres approach each other, and the overall sarcomere length is
reduced. Since there are thousands of sarcomeres in a myofibril, and all of them shorten, the myofibril shortens quite a
bit. Since there are hundreds or thousands of myofibrils, all lined up in register, and they all contract together and all
contract the same amount, in the LM you will see the I bands diminish in length, while the A bands do not. This is caused
by the "telescoping" of the actin and myosin filaments past each other. The myosin filaments comprising most of the A
bands don't move, but the anchor points of the I bands do, and bring the tips of the actin filaments closer together. This
mechanical model of contraction is the sliding filament hypothesis.

Anatomic muscles can't expand; they can only contract. That's why they're paired: when one is contracted the other is
passively extended to "reset" the sarcomeres to their normal extended length (about 2.2 µm) from the contracted state
(about 1.6 to 1.8 µm). In contraction, the ends of the sarcomeres move closer to each other. The I-bands shorten or
disappear; the A bands don't change their length. If you have tens of thousands of sarcomeres arrayed end to end, and
each of them shortens by a few tenths of a micron, the overall effect on the anatomic muscle is that it shortens by quite a
lot. Since the myofibrils are laid side by side, and they all contract simultaneously, the summed force of their contraction
is very strong.

In transverse sections the arrangement of cells in skeletal muscle is even more apparent. Each myofiber is visualized as a
separate unit. The large size of the cells and the positions of the various nuclei present (see below) can be determined
easily. If a myofiber nucleus happens to be present in the plane of the section, it's seen inside the sarcolemma, right at the
periphery of the cell. The satellite cell nuclei are outside the sarcolemma, but within the basal lamina (which isn't visible
without special staining).

Nuclei of Skeletal Muscle

Notice that there are two kinds of nuclei directly associated with the myofiber. The first type is the myofiber nucleus
itself, which is clearly located within the fiber. Skeletal muscle cells are multinucleated (in fact, they are syncytial) and
may have anywhere from ten to hundreds of nuclei. These are all located right up under the plasma membrane
(conventionally called the sarcolemma in all types of muscle cells), because the internal volume of the cell is packed solid
with the contractile myofibrils. One can make this out pretty easily in the two x-sections shown below. One rarely sees
more than two or three nuclei in any x-section because the fiber is so long, that even if there are hundreds of nuclei in it,
they're strung out along its length so that the odds of getting a section plane through any given nucleus is small.
The second group of nuclei are those of supporting cells, found between the fibers and in some cases lying in shallow
depressions in the surface of the myofiber. Because fibroblasts are so numerous (they make the intercellular collagen
network) you'll see them in the CT that surrounds each fiber and bundles of fibers; and of course there will also be nuclei
of blood vessel cells, some wandering CT cells, etc., expected in any CT location. Note the large numbers of capillaries
between fibers. Skeletal muscle is very well vascularized, because it has such a high oxygen demand, so every myofiber
is laid up against a blood vessel somewhere.

There are also reserve cells, incompletely formed myofibers, called satellite cells. These are small cells closely associated
with myofibers. They rest in shallow depressions on the surface, and when a need for repair or hypertophy is present, can
divide and produce new muscle fibers or add to existing ones.

Connective Tissue Relationships

All muscle depends on CT to transmit the force of contraction. This "intercellular collagen network" is hierarchical in
arrangement, and it's diagrammed here. Each myofiber is invested by a delicate CT covering, the endomysium. These
collagenous fibers in turn are woven together to demarcate "bundles" of myofibers. The "bundles" are said to be joined
together by the perimysium, which is the next higher level of CT. The muscle as a whole is covered with CT, which is of
course continuous with the perimysium, and which represents the epimysium. The epimysium is attached to the end
points at which the muscle originates or inserts on a bone. Thus the CT provides something for the contracting muscle to
"pull on" so that work can be done.

 In this scanning EM image (approximately 400x) one can see the surface of a skeletal muscle fiber. The "fuzz" that's
obvious on the specimen is the lowest level of the intercellular collagen network (ICN) that surrounds all muscle fibers,
and binds the mass into a functional whole. This level, closely associated with and anchored to the surface of the muscle
fiber is the endomysium (Greek: "endo" = within; "mys" = muscle).

The endomysium of the ICN surrounds every fiber and grades without sharp demarcation into the next level, the
perimysium, which in turn is physically linked to the epimysium, the outermost covering. At the ends of an anatomic
muscle, the epimysium grades into the tendons that anchor it to the bones it moves.
In these transmission EM (approximately 25,000x and 12,000x), the very intimate relationship of the collagen fibers of the
ICN and the surface of the muscle cell is clear. These fibers run individually (small arrows) representing the
endomysium; some larger bundles of perimysium (C) are also visible. The collagen is in the space defined on one side by
one muscle cell's plasma membrane and on the other side by that of a second muscle cell. The amorphous material
surrounding them is essentially the glycocalyx of the cells. Various adhesion molecules and Type IV collagen are
involved in keeping these high-strength fibrils in place.

The ICN is crucial to muscle action. If it's not present for some reason, or if the collagen of which it's made is not capable
of handling the load imposed on it by muscle contraction, no force can be transmitted through the muscle mass and no
work can be accomplished.

Neural Relationships in Skeletal Muscle: Motor End Plates and Neuromuscular Spindles
Skeletal muscle is under nervous system voluntary control. Skeletal muscle must have neural innervation to function. If
the axons to a myofiber fail to develop, or are damaged in some way (e.g., by disease or by surgical severing) the muscle
cells will wither in the process known as neurogenic atrophy (see below). It's important to understand the structural
basis for the interface between a neuron's axon and the myofiber it serves; and the mechanical structures by which the
contraction signal is modulated and controlled.

Neuronal processes have to end somewhere. In the case of somatic motor fibers, the terminus is on a skeletal muscle cell
at the structure called the motor end plate or neuromuscular junction. This is a plaque-like synapse between the end of
the axon and the plasma membrane of a muscle cell. The motor end plate represents the final synapse in the motor
pathway, between a nerve cell and the myofiber it controls. As with any synapse, transmission of the signal is chemical.
In skeletal muscle the neurotransmitter acetylcholine (ACh) is released into the gap between the terminus of the neuron
and the surface of the muscle; this brings about a depolarization of the plasma membrane of the myofiber, internal
chemical changes in the cell, and contraction.

At left is a whole mount preparation of "teased"

muscle fiber. (In other words, it's not a sliced
section, but individual myofibers pulled apart, placed
on a slide, and stained.)

The "bump" on the myofiber in the center of this

field is a motor end plate or neuromuscular
junction. This is the site of interaction between the
nervous system and muscle fiber. One can easily
make out the axon that comes in to form the motor
end plate on this fiber. In vertebrates, each skeletal
muscle fiber — no matter how long it may be — has
only one such neuromuscular junction.

When ACh is released at this site, the muscle fiber

(the post-synaptic cell) undergoes membrane
depolarization and a change in ion permeability.
The result is a change in electrical charge on the
plasma membrane that spreads to cover the entire
surface. This in turn results in the release of calcium
ions from the myofiber's sarcoplasmic reticulum, triggering the "ratcheting" of actin and myosin filaments past each
other, and the overall shortening of that specific myofiber. As with neurons, depolarization and the subsequent
contraction, it induces an all or nothing response; when depolarization takes place, all the myofibrils inside that myofiber
contract simultaneously and maximally.
The detailed architecture of the neuromuscular junction really requires an EM to see clearly, but some features can be
made out in the LM. What you see above is a slide showing a group of motor end plates: they appear as raised plaques
on the surface of myofibers. Each end plate has large numbers of nuclei associated with it; some are nuclei of the
Schwann cells around the end of the neuron, and others are nuclei of the underlying muscle fiber. An end plate is only
associated with one myofiber, and you can see this when you look at them from the top. The oval object at the upper left
in this field isn't a motor end plate, it's a muscle spindle.

The myelin sheath of the axon is lost just before the end plate is formed (you can probably see where this happens). The
terminal end of the axon collateral arborizes, and the splayed out ends rest in shallow grooves on the surface of the
myofibers. These grooves are actually a complex set of folds in the sarcolemma, the synaptic clefts. When the neuron
fires, ACh is released into the space between the plasma membrane of the neuron and that of the muscle fiber; it diffuses
across the space, and its release triggers the series of intracytoplasmic events which cause contraction of the sarcomeres.

Motor Units
Take a good look at the image above and the one below at left: notice that the individual axons usually send out collateral
branches. A single axon may therefore innervate several muscle fibers via its axon collaterals. When the neuron fires, all
of these muscle fibers will contract simultaneously. This aggregation of muscle fibers, all controlled by one neuron, is a
motor unit. Any given anatomic muscle contains
thousands of such motor units, each with anywhere
from 10 to 100 myofibers. All of the motor end plates
in this teased preparation are served by collateral
branches of the same axon. Consequently, when that
axon fires, all of those myofibers (with one each motor
end plate) are also going to fire, simultaneously and
maximally.

The amount of force generated in an anatomic muscle

for a given task is dependent on how many motor units
in it are "recruited" into use. There may be hundreds
or even thousands of motor units in any given
anatomic muscle. Decisions on how many to use are
made continuously at the CNS level.

Not all motor units are "'on line" at any given time; if
the task is one that requires little force, only a few may
be "recruited" by the brain and as these fatigue, they
are shifted smoothly out of service and new motor
units switched into use, to maintain the steady level of
contraction that muscle has to have. If there's a
demand for greater force generation, more and more
units can be recruited and the total force the anatomic
muscle generates is increased. Eventually, if all the
motor units are in use, so that there's no reserve of rested and ready ones, the muscle shows complete fatigue and force
generation has to be halted.

Feedback from proprioceptors in among the myofibers and at the joints, tells the brain how much force is being exerted,
how much more or less is needed, and when to stop generating force. Coordinating thousands of inputs and outputs is in
most species a learned behavior (and it also depends on the degree of development of the cerebellum). Newborn puppies
(and human babies) are clumsy because they haven't integrated their nervous system control over skeletal muscle motor
units yet. Once the process of final maturation of the CNS is completed, and the "trick" of controlling skeletal muscle
precisely is learned, they're able to walk and run.

Muscle Spindles
Anatomic muscles can only contract, not relax; that's why they are paired, so that when one contracts the other is passively
extended. By regulating the relative degree of contraction, and the number of motor units involved, any desired level of
force can be generated and sustained. Obviously, in order for this system to work, there has to be a great deal of
informational input into the central nervous system. The CNS has to know the degree of contraction tension on any given
muscle, information that's obtained from a special sensor located deep in the muscle. This sensor is the muscle spindle, a
small tapered structure lying in among the myofibers. This information is not only necessary for voluntary functions
(such as throwing a baseball or writing a lab handout) but for "automated" ones (i.e., those learned in infancy) such as
standing up straight without falling down.

The sense of position and movement is proprioception. Proprioceptive information comes from many sources, of which
the skeletal muscle spindle is only one example. One of these can be seen on slide below (which also has several motor
end plates on it). The muscle spindle on this slide is a wedge shaped collection of nuclei, surrounded by a capsule. A
fiber leads from it to join a nearby nerve. The spindle is responsive to mechanical forces stretching its capsule, and sends
information on muscular tension to the CNS, which then can adjust the tension generated in the muscle accordingly.

Spindle input is vital to continuous monitoring of body position and movement, and all of this is fed through the
"automatic pilot" circuits in the cerebellum. Thus, you need not be consciously concerned about standing up straight,
balancing while walking, or not falling off a lab stool. The sensory/motor loops involving the proprioceptors and skeletal
muscle make the automatic fine adjustments of muscle tension that are required.

Above are two examples of muscle spindles. The left image is stained with H&E, the right with a Golgi stain. Both are
about 200x in magnification. The muscle spindle is an oval to wedge shaped collection of nuclei, surrounded by a
capsule. Nerve fibers to and from it provide both sensory and motor functionality.

The spindle contains highly modified skeletal muscle fibers. Since it has both sensory and motor supply, it's responsive to
mechanical forces that stretch the capsule. Pulling on it sends information on muscular tension to the CNS; the brain can
use this input to re-adjust the tension generated in the muscle appropriately for the task. The spindle works like a
thermostat: the brain can "set" the spindle to any degree of tension desired, and when the tension varies from the setpoint,
use the information it provides to send contraction signals to motor units.

Spindle input is vital to continuous monitoring of body position and movement. All of this information is fed through the
"automatic pilot" circuits in the cerebellum. Normal postural control (standing up straight, balancing while walking, or
not falling off a lab stool) is a matter of switching motor units in and out of service to maintain normal muscle tensions.
The sensory/motor loops involving the proprioceptors and skeletal muscle make the automatic fine adjustments needed.

As has been mentioned, much of this control learned behavior: in "precocious" animals, such as cattle, the cerebellum is
almost completely functional at birth, so that a calf can get up and walk most immediately. But in "altricial" species, such
as cats and dogs, the newborn is helpless and uncoordinated until the cerebellum completes post-natal development and
the circuitry is established.

Differentiation and Functional Considerations in Skeletal Muscle

All muscle is extremely differentiated, and specialized for its function of contraction. As a general rule, very highly
specialized cells have little or no capacity to divide, and skeletal muscle is no exception. Once a myofiber has
differentiated, that's it: it will never divide again. But the somewhat limited regeneration capacity of skeletal muscle
doesn't mean it can't change at all in response to environment or to stresses. It can and does undergo hypertrophy very
readily. Hypertrophy, defined as an increase in cell size without a corresponding increase in cell numbers, is the result of
the activity of proliferating satellite cells that fuse with existing myofibers to increase cytoplasmic volume, and also
increased synthesis of contractile units. The highly developed muscles of a bodybuilder or a ballet dancer represent the
increased amount of new contractile material within the cytoplasm, but not the production of new myocytes.

Conversely, in atrophy (which can result from disuse or malnutrition, among other causes) the volume of contractile
cytoplasm is diminished, but not the number of cells. There are, of course, diseases in which actual cell death occurs, and
when this happens, the loss can't be replaced. Loss of muscle cells to necrosis or trauma is made up by the formation of a
scar which is mainly collagenous in nature and has no contractile properties.

Since we've been considering the relationships of neurons and muscle fibers, it seems appropriate at this point to look at
what happens when the nerve supply is interrupted. This is really pathology, but it illustrates nicely the importance of the
neuro-muscular interplay and the structures that control it.

If you've ever broken a leg, and had it in a cast for a few weeks, you've seen how the muscles shrink; this is atrophy of
disuse, the counterpart of the hypertrophy of use seen in body builders. Atrophy of disuse is reversible: exercise causes
the re-building of lost contractile units. But what's shown here is not reversible. This is permanent neurogenic atrophy,
caused by mechanical injury to a nerve during surgery. The muscle cells no longer get any signals to contract. They start
to scavenge the components of their contractile apparatus, and get smaller and smaller, retaining their numerous nuclei.
They don't die: they remain metabolically capable of maintaining themselves, in "suspended animation" waiting for the
nerve signal that never comes. It's often the result of trauma: depending on the degree of injury, this can happen to a
single motor unit or to an entire anatomic muscle.