C. elegans Neurobiology

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C. elegans Neurobiology Monica
Driscoll Rutgers, The Sate University of New
Jersey
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Humans make lousy experimental subjects
genetically heterogeneous environmental
differences slow reproduction, few
offspring live too long reluctant to give up
tissues or inject neurotoxins
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Genetic Model Systems are Powerful in Analyzing
Biology in Whole-Animal Context
Flies
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Advantages of Alternative Species

• EPA requiring multiple species in toxicological
  test
• Most agencies are encouraging the use of
  non-vertebrate species
• Fewer or no animal welfare concerns
• Genetics/transgenics
• Rapid assays
• Lower cost

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• C. elegans Anatomy 101
• Small, Short Life Cycle (1mm, 2.5 days)
• Inexpensive Maintenance
• Easily Stored Dauer long lived form, Freezing
• Hermaphrodites (self fertilization), Males
  (crosses)
• Many Mutant Strains, Available from a Strain
  Center

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Common tissues, easy to observe
Worms have many tissues shared with
humans Neurons/Nerve Ring Polarized intestinal
cells Muscle Kidney-like canal
cells Skin/Hypodermis Immune-like cells
Worms are transparent tissues can be
easily observed in living animals by microscopy
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C. elegans has a 2.5 day reproductive lifecycle
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C. elegans life cycle
Aging
Early development Reproductive adult
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Fast, Cheap and Productive Living-Genetic
Homogeneity!
2.5 day lifecycle Inexpensive maintenance Self
fertilization yields 300 genetically identical
progeny
2.5 days!
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Making a nematode embryonic development
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Molecular Biology Full Genome Sequence Fragments
Available on Cosmids
20,000 genes annotated in WORMBASE
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Easy, Rapid Genetic Engineering
Easy generation of transgenic lines -injection hi
gh copy number transgenes extragenic transgene
array) -microprojectile low copy number
transgene Integrated transgene Gene-based
fluorescent proteins (GFP) visible in living
animals
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or bombard in
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Easy, Rapid Genetic Engineering Easy
Generation of Transgenic Animals Several
Gene Knockout Methods Reporter GFP Fusion
in Live Animals
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Isolate Specific Deletions by Screening a Library
Detect Deletion by PCR
WT gene X Deletion in gene X
3 KB 1 KB
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RNAi Functional Knockout dsRNA Produced in vitro
Induces Degradation of Corresponding mRNA
Microinjection of dsRNA into the gonad or
intestine
in vitro produced dsRNA
Selectively targets only homologous mRNA
X
F1 generation will have specific RNAi-induced
phenocopy traits
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RNAi by Feedingthe Bacteria Produces dsRNA
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Genetic Analysis in C. elegans Can Be Used to
Identify Components of Molecular Pathways
Identify C. elegans Gene
Identify Human Homolog
Mutation Blocks Cell Death
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Basic Biological Mechanisms Are Conserved

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C. elegans nervous system

• Patterning and development of the nervous system
• Neuron and growth cone migration/axon guidance
• Synaptic transmission
• Synapse formation
• Neuron cell polarity and protein trafficking
• Synaptic plasticity (structural and behavioral)
• Mechanosensation
• Chemosensation
• Thermotaxis
• Developmental arrest in response to environment
• Physiology of cyclic/rhythmic patterns
• (e.g., feeding and defecation)
• Neurodegeneration
• Behavioral analysis

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C. elegans Neuroanatomy
Phil. Trans. Royal Soc. London. Series B, Biol
Scien. Vol.314, Issue 1165 (Nov 12, 1986), 1-340
The Mind of the Worm
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Serial section electron microscopy of an
entire animal
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The C. elegans Nervous System
Neuron Cell Bodies
Ventral Nerve Cord (Dendrites)

• Simple Nervous System.
• Transparent.
• Tolerates Nervous System Defects.
• Forward Genetics.

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Individual Neurons Can be Identified by Their
Position and Their Processes
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Some amphid and phasmid neurons are directly
exposed to the environment
ASH is a polymodal sensory neuron -high osmotic
strength -volatile repellents -detergents -alkaloi
ds -heavy metals -acid pH
Dye filling reveals proper development and
viability
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Full Wiring Diagram All Chemical Synapses Gap
Junctions are mapped
This is the only animal for which a complete
wiring diagram has been compiled.
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Types of C. elegans Neurons
Sensory neurons - neurons capable of sensing the
outside environment and transducing the
information to the CNS. Typically bipolar.
sensillum
nerve ring
sensory dendrite
cell body
axon
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Types of C. elegans Neurons
Interneurons - neurons that receive input and
send output to severa ldifferent neurons. Often
involved in integrating information. Often
unipolar.
nerve ring
cell body
ventral cord ganglia
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Types of C. elegans Neurons
Motorneurons - neurons that receive input from
the CNS and send output directly to muscles via
neuromuscular junctions (NMJs). Often complex
morphology.
dorsal cord ganglia
commissure
ventral cord ganglia
ventral cord ganglia
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DB
VD
DA
VB
DD
VA
Neurons killed Phenotype
No movement
DA,DB,VA,VB
Contracts both dorsally and ventrally
DD,VD
No movement
DA,DB,VA,VB,DD,VD
Moves forward only
DA,VA
Moves backward only
DB,VB
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Dorsal Muscle
DB
VB
Ventral Muscle
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Conservation Between C. elegans and Mammals

• Basic metabolic proteins
• Stress response
• Cell cycle control
• Signal transduction pathways
• Insulin
• Retinoic Acid
• MAPK/Ras
• Toll
• p53
• TGF
• WNT

• Neurotransmitters
• Dopamine
• Acetylcholine
• GABA
• glutamate
• serotonin
• nitric acid
• Diseases
• Cancer
• ALS
• Lysosomal storage disease
• Polycystic kidney disease
• Huntingtons disease
• Parkinson's disease

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Voltage-Gated Calcium Channels
Pore-forming ?1 core subunit--L type --egl-19
(muscle essential) --blocked by L type
inhibitors nifidipine, verapamil,
diltiazem non-L type (N, P/Q) --unc-2
(neurons) T type --cca-1 Two additional ones.
Both conserved orthologs and outliers..
Accessory
?2-? subunit--unc-36 also T24F1.6
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K Channels are abundant--80
TWIK-50!
Many loss of function mutations have no
effect --functional redundancy --known
mutations gain-of-function
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K Channel mutants can model disease Human KCNQ
channel in heart (long QT syndrome) Benign
familial neonatal convulsions Non-syndromic
autosomal deafness Worm version expressed in
pharynx- pumping organ, mutant analysis
providing novel insight into channel
function In vivo
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GABA signaling Human--inhibitory on CNS Worms
acts at NMJ to relax bodywall muscle
(inhibitory) -can also be excitatory for
enteric muscle contraction
26 neurons make GABA
Genetic analysis identified genes required for
biosynthesis, signalling, receptors, transporters
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Conserved Glu vesicle-packing
transporters Ionotrophic receptors Metabotrophic
receptors Glutamate transporters Receptor
associated proteins
Glutamate signaling
Pre
sGluTs
Ca2
Na
Glia
Post
PDZ
mGluRs
iGluRs
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Inhibitory Glu Receptors--specific to nematodes
drug targets
Inhibitory receptors-Glu-gated Cl-
channels avr-14--extrapharyngeal nervous
system --MNs, touch neurons--inhibits
locomotion avr-15 extrapharyngeal nervous
system, part of pharynx--inhibits pharyngeal
muscle contractions --convey sensitivity to
ivermectin
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sGluTs are Involved in Isothermal Tracking, a
Memory Paradigm
Worms Remember the Good Temp. and Move Along
Isotherm
Worms are Well Fed at 20c
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Disrupting glutamate signaling disrupts learning
behavior
Wild type
When synaptic Glu concentration is elevated..
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GLR-1 Is Localized to Clusters Along the Ventral
Cord Dendrite Bundle
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Many other neurosignalling strategies are
conserved Acetylcholine-- choline
acetyltransferase 42 acetylcholine
receptors Heterotrimeric G proteins and targets
Neurotransmitters 20 G? subunits dopamine
2G? serotonin 2 G? octopamine 12
RGS 3 adenylyl cyclases G coupled
receptors 18 class A amine receptors 50 class
A peptide receptors 4 class B peptide
receptors 3 GABA-B receptors gt700 G coupled
orphan receptors--odorant receptors TRP-related
channels DEG/ENaC channels
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Some things are missing
Voltage-gated Na channels --though do have 24
DEG/ENaC channel family members Rhodopsins
Some things are there but different
Gap junctions--innexins Olfactory receptors--G
protein coupled receptors
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Synaptic Function
Chalfie Jorgensen Trends Genet. 1998
Dec14(12)506-12.
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Molecules involved in synaptic signaling are
conserved
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Many behaviors can be measured..
swimming
Social behavior
chemotaxis
crawling
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Two Morphological Patterns of Cell Death
apoptosis compaction development and homeostasis
necrosis swelling cell injury
programmed cell death
necrotic-like cell death
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Apoptotic cell death in C. elegans
initiation
execution
Conserved death and corpse consumption programs
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Non-apoptotic caspase-independent death
mechanisms play important roles in injury and
neurodegenerative disease
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Molecular mechanisms of necrosis
Ca
Na
Protease activation
Calpains CLP-1 CLP-5/TRA-3 H25


calreticulin, ER Ca2 release channels
hyperactive ion channels
Cathepsins ASP-3 ASP-4
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Intracellular human AD Ab1-42 in C. elegans
muscle leads to progressive paralysis
Chris Link, U. Denver
Several transgenic models of human
neuroodegenerative disease
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Dopaminergic neurons are poisoned by 6-OH
dopamine uptake
Nass, Richard et al. (2002) Proc. Natl. Acad.
Sci. USA 99, 3264-3269
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Technical Approaches of Use in C.elegans
Neurobiology -Electrophysiology -Image-based
analysis of neuronal function -Cell culture
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Many C. elegans channels can be heterologously
assayed in Xenopus oocytes
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Neurons are tiny (1 um cell body) but can be
patched by experts
Goodman and Chalfie
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Cameleon a tool for measuring changes in
intracellular Ca2

• Cameleon is a genetically encoded fluorescent
  Ca2 binding molecule.
• YFP and CFP are far away from each other in low
  Ca2. NO FRET.
• When Ca2 binds CaM YFP and CFP come in close
  proximity to each other enabling FRET.

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Cameleon-based analysis of neuronal activity in
vivo
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Cameleon reports changes in touch neuron Ca2i
during touch stimulation dependent on mec-4 and
mec-2
intracellular stomatin
channel
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C. elegans Cell Culture
Embryonic cells differentiate in culture
pharmacology easier
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Culture -neurons differentiate --pharmacology
possible But! Embryonic cells only Some
neurons might not differentiate.
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The C. elegans Model Offers Many Advantages for
Neurotoxicology Studies a
Inexpensive Rapid In vivo criteria for neuronal
viability and function Capacity to rapidly
address mechanism (genetics, pharmacology) Track
record of providing huge insight into parallel
processes in mammals