Structure & Function of Neurons

Structure & Function of Neurons 10:00 on 9/15/2015

Dr. Lindsley MS-539, [email protected]

Lecture Objectives: Introduction to the histology and cell biology of neurons, emphasizing the special features of neurons that distinguish them from other cell types, esp. axon transport. Using the example of a simple spinal reflex circuit, students learn to integrate their understanding of neuronal structure and direction of information flow to the gross anatomy of spinal nerves.

Student Learning Objectives:

1. Describe important histological characteristics of neurons, nerves and nervous

tissue.

2. Describe key features of the 3 kinetic components of axon transport (fast anterograde, fast retrograde and slow anterograde).

3. Relate what you know about the structure of neurons and direction of information flow to the anatomical organization of the spinal nerve.

1

1. Highly polarized functional compartments The cell body, dendrites, and axon are distinct compartments that differ in

structure and function. Each compartment has subcompartments whose molecular characteristics

provide for specialized functions. Very high level of transcription and protein synthesis, which occur almost entirely in

the cell bodies and dendrites (nissl substance). Intracellular materials must be transported to regions far away from the site of

synthesis (axon transport). Axon transport is regulated by activity and myelination. 2. Excitability & Conductivity → Cell-to-Cell Communication (Dr. Shin’s lectures) All cells have different ion concentrations inside and outside the plasma membrane. Muscle cells and neurons use this differential ion concentration to generate

electrical signals; both cell types respond to physical or chemical stimuli with a change in the electrical potential across their plasma membrane.

The unique event in neurons occurs in axons: This electrical signal propagates over long distances as a self-regenerating, non-decremental and uni-directional wave (from the cell body to the synaptic terminals ) called an action potential.

What is special about neurons compared to other cell types?

2

What do you already know about the structure of neurons?

Q: How many cells are depicted in this drawing? Add Labels: dendrite, neuronal cell body, nucleus, axon, Schwann cell nucleus, myelin sheath or internode, Node of Ranvier, axon terminals. Q: What is the direction of information flow in neurons? 3

Modified from Kandel et al. 4th Edition. Fig 2.8

There is a large diversity of neurons in the nervous system – more than any other organ system or cell type.

They all have regions for input, integration, conduction & output.

*

NO dendrites NO synapses on soma

Input from special sensory organs

4

Classification of Neurons

There are several hundred different types of neurons that may be classified by one or more of the following characteristics:

• shape; multipolar, bipolar, unipolar, pseudounipolar, spiny, pyramidal, etc.

• “modality”; ex. sensory or motor

• whether they are connected to local targets (interneuron) or distant targets (projection neuron)

• neurotransmitter they release; ex. GABAergic, glutamatergic, acetylcholinergic, etc.

• Electrophysiological firing patterns; ex. bursting, regular spiking, silent, etc.

• various other characteristics

Details at 10am 9/17

5

Ultrastructure of Neurons

Cell body – input and integration • Postsynaptic regions (axo-somatic synapses) • Nissl substance • Axon hillock

Dendrites – input and integration • Mixed polarity of microtubules • Postsynaptic regions (axo-dendritic synapses) • Dendritic spines

Axons – conduction and output • Uniform polarity of microtubules • Initial segment • Node of Ranvier • Presynaptic regions (with output to neurons and other effector targets)

6

Histology of Dendrites • Input region - Primary target for synaptic input from other neurons. Electrochemical signals travel along dendrites to the soma.

• They extend from the soma and taper in diameter.

• Length, thickness and pattern of branching is highly variable.

• Shape influences electrical properties and surface area correlates with # of synapses.

• Cytoplasm composition is similar to soma (next slide), except that the golgi apparatus and Nissl bodies are only in the proximal part of the dendrites.

Unique features Dendritic spines are tiny mushroom-shaped protrusions on the dendrites of some neurons. They increase the receptive surface area for synapses, esp. excitatory input. Thought to be important in neuronal plasticity & learning.

“Somatodendritic Region” = combined input region; dendrites and soma of the cell. 7

K. Harris 2004

Spines on a dendrite. Reconstructed from serial EM

Dendritic Spines: Ultrastructure

• Most spines are mushroom-shaped; spine neck and head. • Spines receive synaptic input, primarily on their head region

(i.e. spines are postsynaptic). • Spine shape changes can occur rapidly in response to

synaptic activity and other conditions. • Spine shape influences electrophysiological properties.

8

The structure of axo-spinous synapses in the CNS: LM and EM

A. Multipolar neuron with spiny dendrites. B. Dendritic shaft (orange) and spines (green).

C. and D. abbreviations: pre presynaptic ax axon s.v. synaptic vesicle PSD postsynaptic density dend dendrite

9

Dendritic Spines: Plasticity

Spine number and shape is modified by synaptic activity. - The spine neck is rich in actin (shape change modifies electrical properties) - local mRNA translation (activity-dependent changes in protein expression)

Spine number and shape may correlate with neural function and can be modulated by a variety of conditions:

- Spine number is lower in conditions marked by cognitive impairment, ex. Autism spectrum disorders, Down’s syndrome, depression, chronic stress, normal aging.

- Spine number is increased by environmental enrichment and estrogen replacement.

More about spines in learning & memory in Nervous System I theme.

Before (left) and after (right) repetitive synaptic stimulation at a synapse onto the spine indicated by the arrow.

10

Histology of the cell body (aka soma, perikaryon)

• Input and integration; receives and summates synaptic input.

• metabolic and genetic center of the cell • nucleus (often has prominent nucleoli)

o post-mitotic o lots of euchromatin (active transcription)

• lots of sER, mitochondria, golgi Unique features • Nissl substance – a neuron-specific

arrangement of rER and polysomes that is distributed throughout the cell body cytoplasm (except in the axon hillock).

• Axon hillock – a region of cytoplasm in the cell body that is adjacent to the axon and is devoid of Nissl. Thought to be the “trigger zone” for the action potential.

11

Nissl substance is a unique arrangement of cytoplasmic, free polysomal rosettes (one rosette is boxed) interspersed between rows of rough endoplasmic reticulum (RER) studded with membrane-bound ribosomes (arrow).

Nissl “bodies” refers to the clumped appearance of nissl substance in stained sections observed by light microscopy (next slide)

Squire et al., 2nd Edition, Fundamental Neuroscience

Nissl substance is unique to neurons. This is an EM of Nissl.

12

The axon hillock is located within the cell body adjacent to where the axon emerges. Axons have NO Nissl substance.

LM showing Nissl stained sections of spinal cord grey matter. Nissl bodies are the dark purple clumps in the cytoplasm. Recognize the axon hillock region of the cell body cytoplasm by the absence of Nissl.

Axon hillock

Neuronal cell bodies

13

Histology of Axons • The conducting and secretory output regions. • Thinner than the dendrites; do not taper • Length and branching vary greatly.

Unique features • Axons have no Nissl (low protein synthesis). • Initial segment of the axon is adjacent to the

axon hillock in the soma; it has a high density of Na2+ channels for initiating the action potential.

• May be myelinated (shown) or unmyelinated. Myelin is a multilayered wrapping of membrane produced by glial cells. (Dr. Mongin’s lecture)

• Internodal regions –segments wrapped in myelin • Nodes of Ranvier – unmyelinated regions

between adjacent internodal regions; nodes have high density of Na2+ channels.

• Axons have presynaptic specializations (at terminals or nodal regions) where chemical neurotransmitters are released; these “terminals” or “boutons” are the output region (Dr. Keller).

• All the microtubules have their plus-ends pointing away from the cell body.

+

+

+

+ +

+ -

- +

14

Histology of Synapses

15

What do you already know about the structure of synapses? dendrite

nucleus

cell body (soma)

Node of Ranvier

16

Neurons form synapses with other neurons. Types of synapses can be classified by their locations.

Axospinous

17

Histology of chemical signal transmission between neurons. The axo-dendritic synapse occurs between the axon on one neuron (presynaptic) and the dendrite of another neuron (postsynaptic). Notice the many synaptic vesicles and mitochondria in the presynaptic terminal. Notice the synaptic cleft (thickened pre- and post-synaptic membrane). Notice the thickened and complex structure of the postsynaptic density. Many synapses are surrounded by glial cells (“tripartite synapse”) – Dr. Mongin More about the cellular physiology of synaptic transmission from Dr. Keller.

18

Neurons also form synapses with muscle. The neuromuscular junction (NMJ) is a synapse between the axon of a multipolar motor neuron (presynaptic) and a muscle cell (postsynaptic). (More about NMJs from Dr. Mazurkiewicz).

axon of motor neuron

muscle cell

presynaptic terminal

muscle

LM EM

19

Mechanisms of Axon Transport

20

General Features of Axon Transport

Fast transport of membranous organelles – both directions: anterograde (away from the cell body) and retrograde (toward the cell body) Slow transport of cytosolic proteins and cytoskeletal proteins - anterograde direction only

Three Kinetic Components of Axon Transport

1. Fast anterograde 2. Fast retrograde 3. Slow anterograde (component a, component b)

21

1. axon transport requires microtubules and motor proteins.

2. axon transport depends on oxidative metabolism (requires ATP) 3. axon transport does NOT require protein synthesis

4. axon transport is independent of the cell body

All axon transport components share the following biochemical and molecular characteristics.

22

Review: MT in non-neuronal cells

Microtubules are hollow tubes with walls formed by 12–14 protofilaments. Each protofilament consists of a series of α- and β-tubulin dimers organized in a polar fashion, giving the microtubule a plus (fast growing) end and a minus (slow growing) end. Microtubules are approximately 24 nm in diameter and may be more than 100 μm in length. Various polypeptides called microtubule-associated proteins (MAPs) are typically associated with the surface of the microtubule. They regulate assembly, stiffness, spacing and stability of the microtubules. Microtubles in most cell types in interphase are dynamic and unstable.

Properties of microtubules (MT) in non-neuronal and neuronal cells

Squire et al., 2nd Edition, Fundamental Neuroscience

Microtubules in neurons - Lots of tubulin ~ 10% of brain protein! - are stabilized by microtubule-associated

proteins (MAPs). 2 important groups of neuronal MAPs: Taus: axonal, developmentally-regulated High MW MAPs: MAP-2 is dendritic, MAP-1c

is a cytoplasmic motor protein

- phosphorylated b-tubulin is neuron-specific

v In axons, microtubules have their plus ends distal (pointing away) from the cell body

v In dendrites, microtubules have ‘mixed polarity’; proximal dendritic microtubules have either plus end or minus end distal; only the most distal dendritic microtubules have plus ends distal (like axons).

β tubulin + end (fast growing)

α tubulin - end (slow growing)

23

Kinesins - 2 globular heads (motor domain) contain ATPase and microtubule-binding domains - Binding to microtubules (MTs) activates the ATPase. - tail domain (or stalk) interacts with cargo (e.g. membrane-bound organelles, MBO.) - The mammalian brain contains a neuron-specific form of kinesin.

Squire et al., 2nd Edition, Fundamental Neuroscience

Motor proteins involved in axon transport: Kinesins and Dynein

Motor domain: -ATPase -MT-binding

Tail domain: - MBO Neuron-specific

Kinesin

Dynein

Dynein - 2 globular heads (motor domain) contain ATPase and microtubule-binding domains - Binding to microtubules (MTs) activates the ATPase. - huge protein complex of ~1.5 megadaltons - associated with dynactin (multiple proteins), regulates dynein cargo binding and activity.

24

Axon transport provides a mechanism for transporting material between compartments in the axon.

1. Fast anterograde: - moves material away from the cell body, toward the + end of microtubules - moves membranous organelles e.g. synaptic vesicles, mitochondria - motors are kinesins - rate of ~200-400 mm/day 2. Fast retrograde: - moves material toward the cell body, toward the – end of microtubules - moves larger membrane-bound vesicles e.g. prelysosomal, NGF and NGF-R - motors are cytoplasmic dyneins - rate of ~100-200 mm/day

+ ends

- ends

25

3. Slow axonal transport (anterograde only; ave. 0.2 µm/sec) - moves material away from cell body component a - moves MT, NF and associated proteins transported in assembled form component b – moves variety of cytoplasmic proteins, including actin, mRNA, and enzymes (e.g.

for neurotransmitter synthesis) - motor may be a cytoplasmic dynein* - two components:

*Q: How can dynein that only carries cargo along MTs in the direction of their minus ends, be a motor for both retrograde AND anterograde transport?

- ends

+ ends

The MTs being moved are NOT bound to the cargo domain. Instead, the dynein is bound to actin near the plasma membrane and unable to move. The motor domain “walking” toward the (-)end displaces the MTs toward the terminal (anterograde direction).

26

Axon transport is important in the pathogenesis of neurologic infectious and neurodegenerative diseases. A few examples…

Rabies virus: Viral particles gain access to the nervous system by traveling in motor neurons by retrograde transport from the muscle to the cell body in the spinal cord. Recent studies suggest that an interaction between the rabies virus and the dynein light chain links the virus RNP to the host cell retrograde transport system, thereby facilitating axonal virus transport into the CNS. (Jacob et al., J. Virology 2000; Raux et al., J. Virology 2000.)

Charcot-Marie-Tooth type 2 (CMT2): Charcot-Marie-Tooth disease is a group of progressive disorders that affect the peripheral nerves, those connecting the brain and spinal cord to muscles as well as those that detect sensations such as touch, pain, heat, and sound. It is the most common inherited disorder that involves the peripheral nerves, and affects an estimated 1 in 3,300 people worldwide. Although the pathogenic mechanisms are still under investigation, evidence suggests that abnormal mitochondrial transport may contribute to the selective susceptibility of the longest axons, in which proper localization of mitochondria is critical for axonal and synaptic function (Baloh et al., J. Neurosci. 2007).

Familial Alzheimer’s Disease (FAD): In a transgenic mouse model of FAD, defects in anterograde fast axonal transport may contribute to reduction in the supply of neurotrophin receptors and other components of the presynaptic specialization, resulting in compromised neuron function. (Lazarov et al., J Neurosci. 2007)

27

Anterograde tracing: Injection of tracer* into the region containing neuronal cell bodies is followed by a period of time to allow anterograde transport of the label to distal axons. Labeled tissue is then fixed and sectioned for histochemistry to visualize the location of the labeling. * BDA (biotinylated dextran amine) or PHA-L (phytohemaglutinin-L)

Tract tracing methods take advantage of axon transport to trace connections in live, intact cells.

Retrograde tracing: Injection of tracer# into the region containing synaptic terminals is followed by a period of time to allow retrograde transport of the label to the cell body. # HRP (horseradish peroxidase) or FluoroGold

Retrograde axonal transport

28

Transneuronal labeling. Anterograde tracing may lead to the release of the label at terminals and it’s uptake into synaptic target neurons. This permits labeling of pathways involving multiple neurons.

Retrograde axonal transport

29

Neurons in Spinal Nerves

30

Neural CELLS are various types or classes of:

• neurons (this lecture) – specialized for intercellular communication • glia (Dr. Mongin) – wide variety of nervous system functions

Nervous TISSUE refers to structures that contain neural cells along with other cells and constituents. For example, nerves in PNS, including cranial nerves Dorsal root ganglia (aka sensory ganglia) in PNS Autonomic ganglia in PNS Grey and/or white matter of brain and spinal cord in CNS (aka neuropil)

What is the difference between neurons, neural cells, & nervous tissue?

31

http://education.vetmed.vt.edu/Curriculum/VM8054/Labs/Lab9/Lab9.htm

Know the difference between a “neuron” and a “nerve”. A neuron is a single cell. A nerve is a type of nervous tissue in the PNS. Nerves contain a bundle of axons (red) with variable amounts of myelin (yellow), the cell bodies of other cell types (ex. Schwann cells, connective tissue cells, the cells of blood vessels, and extracellular connective tissue material. There are no neuronal cell bodies in nerves.

32

Begin using correct terms for nervous tissue in the PNS and CNS. In the PNS (nerves and peripheral ganglia): A bundle of axons is a nerve, root, trunk or ramus.

A cluster of neuronal cell bodies in the PNS is a “ganglion”. In the CNS (brain, spinal cord and neural part of retina): A bundle of axons is “white matter” and may be called a tract, column, brachium, fasiculus, funiculus, or lemniscus.

A cluster of neuronal cell bodies is “grey matter” called a nucleus. A layer or sheet of neuronal cell bodies is “grey matter” called a lamina or cortex.

There are no nerves in the CNS. There are no collagen fibers between axons in white matter.

33

Formative Assessment Questions

34

From Noback et al., The Human Nervous System 6th Ed 2012, Humana.

Recall the anatomy of Dr. Martino’s typical “Spinal Nerves”

Locate: Spinal cord in cross section Gray matter (dorsal & ventral horns) Dorsal and Ventral Roots (Nerves) Dorsal Root Ganglia Nerves - typical spinal nerves - rami - nerves What is the direction of information flow? 35

Flow of information between PNS and CNS: A simple neural circuit -- To other regions of the CNS via white matter tract.

3 neurons participate in the circuit shown: sensory afferent neuron (red), interneuron (blue) and motor efferent neuron (purple).

What is the location of the cell body of each type of neuron? Where are the synapses? What part(s) of each neuron is in the CNS and what part(s) is in the PNS? Locate the ventral roots, dorsal roots and dorsal root ganglia. Locate the spinal nerves. What is the cellular composition of these nerves? 36

List of textbook references: Cui, Atlas of Histology, 1st Edition, Lippincott, Williams & Wilkins (Chapter 7; pgs 115-133) Kandel, Schwartz & Jessell, Principles of Neural Science, 4th Edition. Elsevier Kierszenbaum, Histology and Cell Biology: An Introduction to Pathology (2nd Edition). Elsevier Paulina textbook Purves, et al., Neuroscience, 4th Edition. Sinauer Squire, et al., Fundamental Neuroscience, 2nd Edition. Recommended for review: Henrikson, Megargel & Murphy, Histology Laboratory: An Interactive Review, CD Version 2.0

Just for Fun An animation of axonal transport called, “The inner life of the cell” http://www.studiodaily.com/main/searchlist/6850.html And with narration… http://multimedia.mcb.harvard.edu/media.html Harvard’s Department of Molecular & Cellular Biology

37