Neuroscience Winter 2015

Lab # 1: Surface anatomy of the brain and

cellular neuroscience
Objectives of the lab:
- Become familiar/review the surface anatomy of a sheep brain
- Examine the cellular components of the brain and nervous system
- Culturing undifferentiated neuronal cells and induction of cellular
differentiation in vitro

Introduction: a Brief overview of the organization of the nervous system:

The nervous system is divided
into the central nervous
system (CNS), which include
the brain and spinal cord, and
the peripheral nervous
system (PNS), which is made
of all other nervous tissue,
such as all spinal ganglia and
nerves, cranial nerves and the
autonomic nervous system
(system that innervates the
internal organs).
The brain is defined as being
the part of the CNS contained
in the skull and consists of the
cerebrum (also called
telencephalon), cerebellum,
brain stem (made of
diencephalon, pons, midbrain
and medulla), and retinas. The
spinal cord is the part of the
CNS located in the vertebral
column.
1

Note that the CNS and PNS are tightly connected; an individual
nerve cell can indeed have components in both the CNS and the
PNS. The peripheral nervous system has motor components
called efferent neurons (because they arise in the CNS and head
away from it to the periphery), and sensory components called
afferent neurons (because the impulse is initiated in a
specialized receptor in the PNS and heads toward the CNS). A
special component of the peripheral nervous system is the
autonomic nervous system (ANS). This is the portion of the
PNS that conducts impulses to smooth muscle, cardiac muscle
and glandular epithelium. It is classified into three divisions: the
sympathetic, parasympathetic and enteric divisions.
In many instances nerve cell bodies are found together in
clusters. Clusters of nerve cell bodies in the CNS are called
nuclei (sing. nucleus, not to be confused with the nucleus of
every cell that houses its DNA etc.). Clusters of nerve cell
bodies in the peripheral nervous system are called ganglia
(sing. ganglion).

Cellular components of the nervous system:

Nervous tissue consists of nerve cells and associated supporting cells. All cells exhibit electrical
properties, but nerve cells, also called neurons, are specifically designed to transmit electrical
impulses from one site in the body to another, and to receive and process information.
Supporting cells are non-conducting cells that are in intimate physical contact with neurons.
They provide physical support, electrical insulation and metabolic exchange with the vascular
system.

Neurons:
Nerve cells are very variable in appearance, but all neurons have a cell body, also called soma or
perikarion, and processes.

The cell body is similar to other types of cells. It has a nucleus with at least one nucleolus

and contains many of the typical cytoplasmic organelles. It lacks centrioles, however.
Because centrioles function in cell division, the fact that neurons lack these organelles is
consistent with the amitotic nature of the cell.

The processes extend from the nerve cell to communicate with other cells. There are two types of
processes: dendrites that receive impulses and axons that transmit impulses.
All nerve cells have an axon (usually only one), which is generally the longest process that
extends from the cell. Most nerve cells have dendrites, usually many, and these are generally
shorter and thicker than the axon.
2

Nerve cells can also receive impulses right on the nerve cell body, and some nerve cells have no
dendrites. The junction where a nerve cell communicates with another nerve cell, or an effector cell (eg.
muscle fibre) is called a synapse. The terminal part of the axon releases a chemical called a
neurotransmitter which acts on the membrane of the other cell, therefore allowing the propagation of the
impulse.

Many axons are wrapped in a lipid-rich covering called myelin. This myelin sheath insulates the
axon from the surrounding extracellular component and increases the rate of electrical
conduction. The myelin sheath is discontinuous at intervals called the nodes of Ranvier. The
areas covered with myelin are called internodal areas. In myelinated axons, the voltage reversal
(that is, the impulse propagation) can occur only at the nodes, with the impulse "jumpping" from
node to node. This is called saltatory conduction. In unmyelinated axons, the impulse is
conducted more slowly, moving as a wave of voltage reversal along the axon.
In the CNS, nodes of Ranvier (between myelinated regions) are larger than those of the PNS, and
the larger amount of exposed axolemma makes saltatory conduction more efficient.

In the central nervous system, areas containing nerve cell bodies, their myelinated and
unmyelinated processes and supporting (glial) cells are called grey matter. Areas containing
predominantly myelinated axons as well as some unmyelinated axons and glial cells but no nerve
cell bodies are referred to as white matter. In the brain, the grey matter is exterior to the white
matter, but it is the reverse in of the spinal cord.

Classification of Neurons

Structural Classification - grouped according to the number of processes extending from

their cell body:
o

Anaxonic - axons and dendrites are indistinguishable; found in brain; functions poorly
understood.

Multipolar neurons - three or more processes (usually with a single axons); most
common type, major neuron in CNS.

Bipolar neurons - two processes (axon and dendrite) extend from opposite sides of
neuron; rare in adult but may be found in retina and olfactory mucosa.

Unipolar neurons - one process extending from cell body which forms both central and
peripheral processes

Central process associated with secretory region

Peripheral process associated with sensory region (receptor)

Unipolar also referred to as Pseudounipolar (originally bipolar but processes fuse

in development).

Functional Classification - according to direction in which nerve impulses travel relative to

the CNS
o Sensory (afferent) neurons - transmit impulses from sensory receptors toward CNS

Unipolar neurons - skin or internal organs to CNS for interpretation

Bipolar neurons - special sense organs, retina

o Motor (efferent) neurons - carry impulses away from CNS to organs

Multipolar neurons - cell body located within CNS and neurons form
neuromuscular junctions with effector cells

o Association neurons (interneurons) - transmit impulses within CNS (usually sensory to

motor); found in CNS only; mostly multipolar and 99% of neurons in body.
4

Support Cells
Support cells are essential to the function and survival of nerve cells. The CNS and PNS each
have their own specific types of support cells.
Support cells in the CNS:
The general term for support cells in the CNS is glia or neuroglia (glial cells, neuroglial cells). Glial

cells do not conduct nerve impulses, but, instead, support, nourish, and protect the neurons. Glial
cells are far more numerous than neurons and, unlike neurons, are capable of mitosis.
There are three types of neuroglial cells.
(1) Oligodendrocytes, the myelin-secreting cells of the CNS.
(2) Astrocytes, which provide physical and metabolic support for nerve cells.
(3) Microglia, or microglial cells (also called mesoglia), which are the phagocytes of the CNS.

Oligodendrocytes are often found in rows between axons. The myelin sheath around axons is
formed by concentric layers of oligodendrocytes plasma membrane about 1 m thick and is
made of 80% lipid and 20% protein. Each oligodendrocyte gives off several tongue-like
processes that wrap itself around a portion of the axon, forming an internodal segment of myelin.
One oligodendrocyte may myelinate one axon or several (up to 50). The nucleus-containing
region may be at some distance from the axon(s) it is myelinating. Unmyelinated axons in the
CNS are truly bare, that is they are not embedded in any glial cell process (in contrast to the
situation in the PNS, described below).

Astrocytes. Astrocytes are the largest of the neuroglial cells. They have elaborate processes that
extend between neurons and blood vessels. The ends of the processes expand to form end feet,
which cover large areas of the outer surface of the blood vessel. Astrocytes are believed to play a
role in the movement of metabolites and wastes to and from neurons, and in regulating ionic
concentrations within the neurons. They may be involved in regulating the tight junctions in the
capillaries that form the blood-brain barrier. Astrocytes also cover the bare areas of neurons, at
nodes of Ranvier and synapses. They may act to confine neurotransmitters to the synaptic cleft
and to remove excess neurotransmitters.
Two kinds of astrocytes have been identified,
protoplasmic and fibrous astrocytes. Both types
contain prominent bundles of intermediate
filaments, but the filaments are more numerous
in fibrous astrocytes. Fibrous astrocytes are
more prevalent in white matter, protoplasmic
ones in grey matter.

Microglia. These are the smallest of the glial cells, with short twisted processes. They are the
phagocytes of the CNS, considered part of the mononuclear phagocytic system. They are
believed to originate in the bone marrow and enter the CNS from the blood. In the adult CNS,
they are present only in small numbers, but proliferate and become actively phagocytic in disease
and injury. Their alternate name, mesoglia, reflects their embryonic origin from the mesoderm
(the rest of the nervous system, including the other glial cells, is of neuroectodermal or neural
crest origin).
Support cells in the PNS:
The support cells of the PNS are called satellite cells and Schwann cells.
Satellite cells. Satellite cells surround the cell bodies of the neurons in ganglia (ganglion cells).
These small cuboidal cells form a complete layer around the nerve cell body, but only their
nuclei are visible in routine preparations. They help maintain a controlled microenvironment
around the nerve cell body, providing electrical insulation and a pathway for metabolic
exchange. In paravertebral and peripheral ganglia, nerve cell processes must penetrate between
satellite cells to establish a synapse.

Schwann cells. Schwann cells are responsible for the myelination of axons in the PNS. A
Schwann cell wraps itself, in a spiral around a short segment of an axon. During the wrapping,
the cytoplasm is squeezed out of the Schwann cell and the leaflets of plasma membrane of the
concentric layers of the Schwann cell fuse, forming the layers of the myelin sheath. An axon's
myelin sheath is segmented because it is formed by numerous Schwann cells arrayed in sequence
along the axon. A Schwann cell can only myelinize one axon. The junction where two Schwann
cells meet has no myelin and is called the node of Ranvier; the areas covered by Schwann cells
being the internodal regions.
The lack of Schwann cell cytoplasm in the concentric rings of the myelin sheath is what makes it
lipid-rich. Schwann cell cytoplasm is however found in several locations. There is an inner collar
of Schwann cell cytoplasm between the axon and the myelin, and an outer collar around the
myelin. The outer collar is also called the sheath of Schwann or neurilemma, and contains the
nucleus and most of the organelles of the Schwann cell. The node of Ranvier is also covered with
7

Schwann cell cytoplasm, and this is the area where the plasma membranes of adjacent Schwann
cells meet. (These adjacent plasma membranes are not tightly apposed at the node, so that
extracellular fluid has free access to the neuronal plasma membrane.) Finally, small islands of
Schwann cell cytoplasm persist within successive layers of the myelin sheath, these islands are
called Schmidt-Lanterman clefts.
Not all nerve fibres in the PNS are covered in myelin, some axons are unmyelinated. In contrast
to the situation in the CNS, unmyelinated fibres in the PNS are not completely bare, but are
enveloped in Schwann cell cytoplasm. The Schwann cells are elongated in parallel to the long
axis of the axons, which fit into grooves on the surface of the Schwann cell. One axon or a group
of axons may be enclosed in a single groove. Schwann cells may have only one or up to twenty
grooves. Single grooves are more common in the autonomic nervous system.

(a) Unmyelinated axons and (b) Myelinated axon in PNS

Cell Culture:
Cell culture commonly refers to the growth of cells derived from multicellular eukaryotes in a
synthetic environment. All cell lines originate from cell cultures with a limited lifetime, but
occasionally some cells keep on multiplying due to a mutation. Culture conditions (such as
growth media, pH, and temperature) vary widely for each cell type, and variation of conditions
for a particular cell type can result in different phenotypes being expressed. Cells can be cultured
for a longer period of time if they are subcultured (passaged) regularly: growth medium is then
replaced and the cells are diluted.
The cells you will be using in this experiment are human neuroblastoma cell line SH-SY5Y
(ATCC # CRL 2266). This cell line is derived from SK-N-SH, and resembles immature
sympathetic neuroblasts in culture.
8

The SH-SY5Y cells will be cultured in DMEM/ F12 Hams growth media containing 10%
FBS (fetal bovine serum) and Gentamycin (antibiotics to prevent bacterial growth), and
maintained in a humidified atmosphere (95% air, 5% CO2) at 37oC.
These cells are typically locked in an early neuronal differentiation stage, characterized
biochemically by the low presence of neuronalmarkers. Human neuroblastoma SH-SY5Y cells
are, however, able to acquire neuron-like phenotypes with neurite outgrowth and branches by
adding retinoic acid (RA) to the growth media. RA is essential in embryonic development and
maintenance of growth and differentiation of epithelial, fibroblastic and myelomonocytic cells
Hence, RA controls cellular differentiation processes by modulating the expression of several
RA-responsive genes by the activation of retinoic acid/retinoid nuclear receptors. In vitro, RA
also plays a role in regulating transition from the proliferating precursor cell to post-mitotic
differentiated cell.

Observing culture cells:

In this experiment, you will use an inverted microscope to assess growth stages of different of
SH-SY5Y cell cultures as well as compared cells exposed and not exposed to retinoic acid.
Inverted microscopes are useful for observing living cells or organisms at the bottom of a large
container (e.g. a tissue culture flask) under more natural conditions than on the glass slides used
with a conventional microscope. As the name suggests, an inverted microscope is upside down
compared to a conventional microscope.
-

The light source and condenser are above the stage pointing down.

The objectives and turret are below the stage pointing up.

The only things that are "standard" are that (1) a specimen (as dictated by the laws of gravity) is
placed on top of the stage and (2) the binocular tube is not upside down but in the standard
position pointing at a conventional viewing angle. As a result, one is looking up through the
bottom of whatever is holding the specimen that is sitting on the stage rather than looking at the
specimen from the top as in conventional microscope.

Phase contrast: Phase contrast microscopy, first described in 1934 by Dutch physicist Frits
Zernike, is a contrast-enhancing optical technique that can be utilized to produce high-contrast
images of transparent specimens, such as living cells (usually in culture), microorganisms, thin
tissue slices, lithographic patterns, fibers, latex dispersions, glass fragments, and subcellular
particles (including nuclei and other organelles). One of the major advantages of phase contrast
microscopy is that living cells can be examined in their natural state without previously being
killed, fixed, and stained. As a result, the dynamics of ongoing biological processes can be
observed and recorded in high contrast with sharp clarity of minute specimen detail.
The phase contrast microscope uses the fact that the light passing through a transparent part of
the specimen travels slower and is therefore shifted compared to the uninfluenced light. This
difference in phase is not visible to the human eye. However, the change in phase can be
increased to half a wavelength by a transparent phase-plate in the microscope and thereby
causing a difference in brightness. This makes the transparent object shine out in contrast to its
surroundings.

The phase-plate increases the phase difference to half a

wavelength. Destructive interference between the two sorts of
light when the image is projected results in the specimen
appearing as a darker object.

Phase contrast pictures showing four

different stages during the cell cycle.
Notice the halo effect around objects,
which is a result of the phase contrast
technique.
Phase contrast is therefore a useful technique for high-contrast imaging of unstained specimens
and will be used when examining cells using the inverted microscope. To set up your microscope
for phase contrast, look for the Ph number written of the objective you are using and select the
corresponding phase ring on a slider located between the light source and the condenser.

10