Biol 3060- Lab # 1-Introduction-W2015

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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 dividedinto 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 theautonomic nervous system

(system that innervates the

internal organs).

The brain is defined as beingthe 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 theCNS located in the vertebral

column.


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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 componentscalled efferentneurons (because they arise in the CNS and head

away from it to the periphery), and sensory components called

afferentneurons (because the impulse is initiated in aspecialized receptor in the PNS and heads toward the CNS). Aspecial 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 muscleand 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 cellbodies 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 electricalproperties, 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 vascularsystem.

Neurons:

Nerve cells are very variable in appearance, but all neurons have a cell body, also called soma orperikarion, 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: dendritesthat receive impulses and axonsthat 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 generallyshorter and thicker than the axon.


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Nerve cells can also receive impulses right on the nerve cell body, and some nerve cells have nodendrites. 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 aneurotransmitterwhich acts on the membrane of the other cell, therefore allowing the propagation of theimpulse.

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. Theareas 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, andthe larger amount of exposed axolemma makes saltatory conduction more efficient.


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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 nervecell 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.

o Multipolar neurons- three or more processes (usually with a single axons); most

common type, major neuron in CNS.

o 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.

o 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 tothe 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 tomotor); found in CNS only; mostly multipolar and 99% of neurons in body.


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Support Cells

Support cells are essential to the function and survival of nerve cells. The CNS and PNS eachhave their own specific types of support cells.

Support cells in the CNS:

The general term for support cells in the CNS is gliaor neuroglia(glial cells, neuroglial cells). Glial

cells do not conduct nerve impulses, but, instead, support, nourish, and protect the neurons. Glialcells 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.


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Oligodendrocytesare 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-likeprocesses 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 theCNS are truly bare, that is they are not embedded in any glial cell process (in contrast to thesituation 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, atnodes 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 typescontain prominent bundles of intermediatefilaments, but the filaments are more numerous

in fibrous astrocytes. Fibrous astrocytes are

more prevalent in white matter, protoplasmic

ones in grey matter.


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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 neuralcrest 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 microenvironmentaround the nerve cell body, providing electrical insulation and a pathway for metabolic

exchange. In paravertebral and peripheral ganglia, nerve cell processes must penetrate betweensatellite cells to establish a synapse.

Schwann cells. Schwann cells are responsible for the myelination of axons in the PNS. ASchwann 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'smyelin 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 collarof 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


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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 ofSchwann 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 longaxis 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 conditionsfor 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 immaturesympathetic neuroblasts in culture.


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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 cellsare, however, able to acquire neuron-like phenotypes with neurite outgrowth and branches byadding 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 severalRA-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 downcompared 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) isplaced on top of the stage and (2) the binocular tube is not upside down but in the standardposition 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.


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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, thintissue 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 beingkilled, fixed, and stained. As a result, the dynamics of ongoing biological processes can beobserved 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. Thisdifference 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 fourdifferent stages during the cell cycle.

Notice the halo effect around objects,

which is a result of the phase contrasttechnique.

Phase contrast is therefore a useful technique for high-contrast imaging of unstained specimensand 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.

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Biol 3060- Lab # 1-Introduction-W2015