Topic 2

Neurons and Glia

Lange

Biology 463 - Neurobiology

Camillo Gogi & Santiago Raymon y Cajal… research rivals that shared the 1906 Nobel Prize, but disagreed vehemently on the

organization of the microscopic level of the nervous system.

Introduction

• Cajal and Golgi were in disagreement about their ideas on how they the nervous system operated…. be it continuous (think of the Fluid Dynamic Model for example) or in a chain of individual cells.

• The Neuron Doctrine says that the neural network consisted of cells that interacted together by contact. This was the major idea of Cajal.

• Glia and Neurons– Glia: Insulates, supports, and nourishes neurons– Neurons

• Process information• Sense environmental changes • Communicate changes to other neurons• Command body response

Obstacles to the advancement of our understanding of building blocks of the nervous system:

•Small size requiring compound microscopy

•To examine brain TISSUE, with compound microscope, the tissue had to be extraordinarily thin to allow light to pass through.

•The consistency of brain, spinal cord, and other nervous system tissue was much less firm than most other tissues. It is often equated with having a consistency of “Jello”.

In fact, the stereotypical “Brain Jello” you can often find people making around Halloween, when used in a brain mold

is VERY realistic in texture and shape.

“Brain” Jello

Yield: 11 Servings

Ingredients:

2 (6 ounce) boxes gelatin, mix any flavor (peach or watermelon give the best color) 1 3/4 cups boiling water 3/4 cup cold water 9 ounces fat-free evaporated milk (must be fat-free or it will curdle)

To add a greyish cast, add this additional food coloring:

15 drops red food coloring 15 drops green food coloring 15 drops blue food coloring

1.Spray or the inside of the brain mold with a small amount of non-stick spray, then wipe out the excess. 2.Put the gelatin mix in a large bowl and add the boiling water. 3.Stir about two minutes until the mix is dissolved. 4.Stir in the cold water. 5.Stir in the evaporated milk and food coloring.6. Pour the mixture in the brain mold, stopping about 1/4 inch from the top

To overcome the issues of brain tissue texture, specialized techniques in histology were

developed.

The microtome (“wax embedded” on the left and “freezing” on the right) allows reserachers to slice soft tissues in very

thin sections (microns)

Yet, another major problem for studying the nervous system tissues involved its

lack of contrast.

The first advances to overcome this obstacle occurred in the late 1800s with the development and identification of a series of differentially staining dyes.

Franz Nissl – (1860 – 1919) founding father of the study of neuropathology . “Nissl” stain is named after him and was his own creation.

Uses for Nissl Stain:

This method is used for the detection of Nissl body in the cytoplasm of neurons on paraformaldehyde or formalin-fixed, paraffin embedded tissue sections. The Nissl body will be stained purple-blue. This stain is commonly used for identifying the basic neuronal structure in brain and spinal cord tissue.

A Nissl body is a large granular body found in neurons. These granules are rough endoplasmic reticulum (with free ribosomes) and are the site of protein synthesis.

This is a coronal section of a rat brain with a tissue thickness of 10 microns.

Camillo Golgi Developed a specialized stain, now named in his honor.

Uses for Golgi Stain:

This method is used for the detection of a number of novel facts about the organization of the nervous system, inspiring formation of the neuron doctrine:

The nervous system is made up of discrete individual cells.

•Golgi's method stains a limited number of cells at random in their entirety. •Dendrites, as well as the cell soma, are clearly stained in brown or black and can be followed in their entire length.•This allowed neuroanatomists to track connections between neurons and to visualize the complex networking structure of the brain and spinal cord.

Golgi's staining is achieved by impregnating fixed nervous system tissues with potassium dichromate and silver nitrate.

This is a section of a rat brain showing the Hippocampus impregnated by the Golgi stain.

• The soma or perikaryon is the more “bulbous” end of a neuron, containing the cell nucleus.

• The soma of a neuron is often called the "cell body". • A neurite is a generalized term that refers to any projection from the

cell body of a neuron.

Again, with Golgi staining, the entirety of a neuron could be visualized.

Cajal’s work uncovered some of the tremendous complexity of the range of cell types seen in the brain.

Cajal differed from Golgi in adamantly suggesting that:

Neurons communicate by contact and are not continuous.

–Cajal discerned the cellular nature using Golgi’s staining method.

–However, even with the best and most careful use of the Golgi Staining technique, it was still not definitive which theory (Cajal’s or Golgi’s) was most accurate.

–In the 1950s, the electron microscope would definitively provided definitive evidence for Cajal’s neuronal doctrine.

A stereotypical representation of the axon projecting from a neuron. The axon will function like a “telegraph” wire or “electrical wire” from the soma carrying impulses.

Structure of the plasma membrane according to the fluid mosaic model.

Integralproteins

Extracellular fluid(watery environment)

Cytoplasm(watery environment)

Polar head ofphospholipid molecule

Glycolipid

Cholesterol

Peripheralproteins

Bimolecularlipid layercontainingproteins

Inward-facinglayer ofphospholipids

Outward-facinglayer ofphospholipids

Carbohydrate of glycocalyx

Glycoprotein

Filament of cytoskeleton

Nonpolar tail of phospholipid molecule

Membrane proteins perform many tasks for general cell growth, development and survival…. but many of these tasks are also essential for neural function.

A protein (left) that spans the membrane may provide a hydrophilic channel across the membrane that is selective for a particular solute. Some transport proteins (right) hydrolyze ATP as an energy source to actively pump substances across the membrane.

(a) Transport

Signal Transduction

A membrane protein exposed to the outside of the cell may have a binding site with a specific shape that fits the shape of a chemical messenger, such as a hormone. The external signal may cause a change in shape in the protein that initiates a chain of chemical reactions in the cell.

(b) Receptors for signal transductionSignal

Receptor

Please note that we shall extensively examine PLB proteins used in this context throughout the semester.

Attachment via PLB Proteins

Elements of the cytoskeleton (cell’s internal supports) and the extracellular matrix (fibers and other substances outside the cell) may be anchored to membrane proteins, which help maintain cell shape and fix the location of certain membrane proteins. Others play a role in cell movement or bind adjacent cells together.

(c) Attachment to the cytoskeleton and extracellular matrix (ECM)

Emzymatic Activity

A protein built into the membrane may be an enzyme with its active site exposed to substances in the adjacent solution. In some cases, several enzymes in a membrane act as a team that catalyzes sequential steps of a metabolic pathway as indicated (left to right) here.

(d) Enzymatic activity

Enzymes

Intercellular joining

Membrane proteins of adjacent cells may be hooked together in various kinds of intercellular junctions. Some membrane proteins (CAMs) of this group provide temporary binding sites that guide cell migration and other cell-to-cell interactions.

CAMs

(e) Intercellular joining

Cell-to-Cell Recognition

Some glycoproteins (proteins bonded to short chains of sugars) serve as identification tags that are specifically recognized by other cells.

(f) Cell-cell recognition

Glycoprotein

Diffusion through the plasma membrane.

Extracellular fluid

Lipid-solublesolutes

Cytoplasm

Lipid-insoluble solutes (such as sugars or amino acids)

Small lipid-insoluble solutes

Watermolecules

Lipidbillayer

Aquaporin

(a) Simple diffusion of fat-soluble molecules directly through the phospholipid bilayer

(b) Carrier-mediated facilitated diffusion via a protein carrier specific for one chemical; binding of substrate causes shape change in transport protein

(c) Channel-mediated facilitated diffusion through a channel protein; mostly ions selected on basis of size and charge

(d) Osmosis, diffusion of a solvent such as water through a specific channel protein (aquaporin) or through the lipid bilayer

Primary Active Transport: The Na+-K+ Pump

Extracellular fluid

6 K+ is released from the pump proteinand Na+ sites are ready to bind Na+ again.The cycle repeats.

2Binding of Na+ promotes phosphorylation of the protein by ATP.

1Cytoplasmic Na+ binds to pump protein.

Na+

K+ released

ATP-binding site Na+ bound

Cytoplasm

ATPADP

P

K+

5 K+ binding triggers release of the phosphate. Pump protein returns to its original conformation.

3 Phosphorylation causes the protein to change shape, expelling Na+ to the outside.

4Extracellular K+ binds to pump protein.

Na+ released

P

K+

PPi

Na+–K+ pump

K+ bound

The use of the sodium – potassium pump PLB proteins will be shown to be crucial to the transmittance of the “electrical” signal that is the “message” carried within a neuron.

Copyright © 2010 Pearson Education, Inc.

Overview of Endocytosis & Exocytosis

Recycling ofmembrane andreceptors (if present)to plasma membrane

CytoplasmExtracellular fluid

Extracellularfluid

Plasmamembrane

Detachmentof clathrin-coatedvesicle

Clathrin-coatedvesicle

Uncoating

Uncoatedvesicle

Uncoatedvesiclefusing withendosome

To lysosomefor digestionand releaseof contents

Transcytosis

Endosome

Exocytosisof vesiclecontents

Clathrin-coatedpit

Plasmamembrane

Ingestedsubstance

Clathrinprotein

(c) Receptor-mediated endocytosis

Extracellularfluid

Cytoplasm

Bacteriumor otherparticle

Pseudopod

Clathrinprotein

(b) Phagocytosis

Clathrinprotein

Membranereceptor

(a) Clathrin-mediated endocytosis

1

3

2

Phagosome

(a) PhagocytosisThe cell engulfs a large particle by forming pro-jecting pseudopods (“false feet”) around it and en-closing it within a membrane sac called a phagosome. The phagosome is combined with a lysosome. Undigested contents remain in the vesicle (now called a residual body) or are ejected by exocytosis. Vesicle may or may not be protein-coated but has receptors capable of binding to microorganisms or solid particles.

Comparison of three types of endocytosis.

Comparison of three types of endocytosis.

Vesicle

(b) PinocytosisThe cell “gulps” drops of extracellular fluid containing solutes into tiny vesicles. No receptors are used, so the process is nonspecific. Most vesicles are protein-coated.

Comparison of three types of endocytosis.

Vesicle

Receptor recycledto plasma membrane

(c) Receptor-mediatedendocytosisExtracellular substances bind to specific receptor proteins in regions of coated pits, enabling the cell to ingest and concentrate specific substances (ligands) in protein-coated vesicles. Ligands may simply be released inside the cell, or combined with a lysosome to digest contents. Receptors are recycled to the plasma membrane in vesicles.

Exocytosis.

1 The membrane-bound vesicle migrates to the plasma membrane.

2 There, proteinsat the vesicle surface (v-SNAREs) bind with t-SNAREs (plasma membrane proteins).

(a) The process of exocytosisExtracellular

fluid

Plasma membraneSNARE (t-SNARE)

Secretoryvesicle

VesicleSNARE(v-SNARE)

Molecule tobe secreted

Cytoplasm

Fusedv- and

t-SNAREs

3 The vesicleand plasma membrane fuse and a pore opens up.

4 Vesiclecontents are released to the cell exterior.

Fusion pore formed

Exocytosis.

The endomembrane system.

Golgiapparatus

Transportvesicle

Plasmamembrane

Smooth ER

Rough ER

Nuclear envelope

Lysosome

Nucleus

The nucleus.

Chromatin (condensed)

Nuclear envelope Nucleus

Nuclear pores

Nucleolus

Cisternae of rough ER

(a)

The endoplasmic reticulum.

Nuclearenvelope

Ribosomes

Rough ER

Smooth ER

(a) Diagrammatic view of smooth and rough ER

(b) Electron micrograph of smooth and rough ER (10,000x)

Golgi apparatus.

Cis face—“receiving” side of Golgi apparatus

Secretoryvesicle

(a) Many vesicles in the process of pinching off from the membranous Golgi apparatus.

(b) Electron micrograph of the Golgi apparatus (90,000x)

Transport vesiclefrom the Golgi apparatus

Transportvesiclefromtrans face

Trans face—“shipping” side ofGolgi apparatus

New vesiclesforming

New vesicles forming

Cisternae

Transport vesiclefrom rough ER

Golgi apparatus

The sequence of events from protein synthesis on the rough ER to the final distribution of those proteins.

Protein-containing vesicles pinch off rough ERand migrate to fuse with membranes ofGolgi apparatus.

Proteins aremodified withinthe Golgi compartments.

Proteins arethen packagedwithin differentvesicle types, depending ontheir ultimatedestination.

Plasmamem-brane

Secretion byexocytosis

Vesicle becomeslysosome

Golgiapparatus

Rough ER ERmembrane

Phagosome

Proteins incisterna

Pathway B:Vesicle membraneto be incorporatedinto plasmamembrane

Pathway A:Vesicle contentsdestined for exocytosis Extracellular fluid

Secretoryvesicle

Pathway C:Lysosome containing acid hydrolaseenzymes

1

3

2

This process is crucial for neurotransmitter production (and release) from the axon terminal in neurons.

Classification of neurons can occur from a variety of standpoints.

The number of neurites can be used for classification:

– Single neurite

• Unipolar

– Two or more neurites

• Bipolar- two

• Multipolar- more than two

Classifying Neurons Based on Morphology

• Classification based on dendritic and somatic morphologies

– Stellate cells (star-shaped) and

– Pyramidal cells (pyramid-shaped)

Also note that spiny or aspinous processes may be seen on any type of dendtrite classification.

Further classification can be made:

– By connections within the CNS

• sensory neurons – carry sensory information to the CNS• motor neurons – carry “commands” out from the CNS to the PNS• Interneurons – carry signals within the CNS

– Based on axonal length

• Golgi Type I - a neuron having with a “long” axon that begins in the grey matter of the central nervous system and may extend considerably from there.

• Golgi Type II - a neuron having very short axons or no axon at all.

– Based on neurotransmitter type• Cholinergic (aka Acetycholine) a neurotransmitter group that uses acetylcholine as its primary

communication chemical released at the synapses between neurons.• Dopamenergic (aka Dopamine) a neurotransmitter group using primarily dopamine

Glia (Accessory Cells)– Cells That Support Neurons

1. Astrocytes

– Most numerous glia in the brain

– Fill spaces between neurons

– Influence neurite growth

– Regulate chemical content of extracellular space via functioning as part of the blood brain barrier

2. Microglial cells – immune response cells specialized for the brain and spinal cord. These are the resident macrophages of the CNS. Microglia are constantly scavenging the CNS for damaged

neurons, plaques, and infectious agents. The brain and spinal cord are considered "immune privileged" organs in that they are separated from the rest of the body by a series of endothelial cells known as the blood-brain barrier,

3. Ependymal Cells - cerebrospinal fluid producing cells (CSF) that line the ventricles. These cells are ciliated to promote circulation of the CSF throughout the CNS.

4. Oligodendrocytes - myelin producing cells.

5. Schwann Cells – myelin producing cells.

The role of the myelin produced in both the oligodendrocytes and in the Schwann Cells is to insulate neurons to promote a more rapid transmittance of a neural signal.

The key difference between oligodendrocytes and Schwann Cells is in their location. Oligodendrocytes are found in the CNS whereas the Schwann cells are found in the PNS.

• Myelinating Glia

• The production of the myelin sheath is called myelination.

• The production of myelin begins in the fourteenth week of fetal development, however only small amounts of myelin exist in the brain at the time of birth.

• During infancy myelination continues and growth occurs quickly and does not slow until adolescence .

• Because of rapid myelination during early life, it is essential that

children under the age of two receive a diet higher in fats than an adult.

Theodor Schwann – German physiologist who discovered the mylenating glia in the peripheral nervous system, the discovery and study of pepsin and the invention of the

term metabolism.

Rudolf Virchow –biologist, pathologist, and physician

Virchow discovered and described myelin in 1854 at the age of 33.

Oligodendrocytes can also be called “oligodendroglial cells”

– Nodes of Ranvier• Region where the axonal

membrane is exposed allowing the process of saltatory conduction

• Found associated with myelinated neurons in both the PNS and CNS (and therefore associated with oligodendrocytes and in Schwann cells.)

• The Axon & Axon Terminal

– The Axon Terminal

• Differences between the cytoplasm of axon terminal and axon

– No microtubules in terminal

– Presence of synaptic vesicles

– Abundance of membrane proteins

– Large number of mitochondria

• The Axon– Synapse

• Synaptic transmission• Electrical-to-chemical-

to-electrical transformation

• Synaptic transmission dysfunction

– Mental disorders

Differences in dendritic spine shape and size can exert significant effects on function. ( see Purpua, 1974 )

Notice the fewer number of spines and their thin, longer extensions in the affected group.

The theory is that the changes in the synaptic interactions that these dendritic spine differences lead to is the root cause of mental retardation in these individuals.

Structural characteristics of a neuron tell us about

its function

NEURONSSomaAxons

DendritesSynapse

Elaborate structure of

dendritic tree = receiver

e.g., Dense Nissl stain = protein;

suggests specialization

Structure Correlates with

Function

END.