Neural Control Circuits Flashcards
C elegans difference from earth worms
Nematode
Smaller
Non segmented body
Zigmacoidal movement
Why c elegans?
Simple nervous Sydney but fundamental level key features conserved
Used to investigate - how does a nervous system adapt behaviour in a context dependent manner
Worms have conserved feature an
Inform on neural underpinnings
Adaptive behaviour
Easy to study
Human brain has multiple regions, circuits (imaging, genomic approach but missing molecular, synapse etc) etc worms easier
Genes, molecules, synapses, circuits, systems, behaviour (can go through all of these)
Conserved features
Synapses - Plasticity
Worm has 302 neurones vs billion in human brain
Fundamentals of how neurones communicate and synaptic communication are conserved
What can we investigate in c. Elegans (neural circuits)
How neural circuits function
How neural circuits respond to environment to coordinate behaviour
Study all levels of biological organisation
Simple behaviours eg locomotion and foraging
Sydney brenner (1960s)
Introduced c elegans as model organism
Unicellular to multicellular
How do cells function to develop systems
Fulfilled this need, hermaphrodites (cloned lines) (genome identical easy for mutants), translucent (can easily count eg pharynx movement and see cell types)
Have connectome for it
Growing c elegans
Transfer between culture plates
1 hermaphrodite on plate, leave for couple days, ~300 progeny
Synchronise worms on developmental stage
Increase heat to 30 Celsius, hermaphrodite will produce males (only 1% naturally males)
Genetic crosses (double and triple mutants)
Knock outs to figure out signalling pathways
Life cycle - 3 days
Novel prize
Genetic regulation of organ development and programmed cell death
RNA interference - gene silencing by double stranded RNA
Development of green fluorescent protein GFP
Advantages of c elegans
Small, easy, cheap and maintain
Translucent
Simple behaviours for complex mammalian behaviour
Genome sequenced
>40% if predicted proteins have homologues in mammals
Mutants available for majority of genes
Highly genetically tractable
Mutagenesis
Experimental approach
Adult with mutagen eg through uv, chemical
Random mutations to progeny
F2 - wild type and rare phenotype and screen genes to find mutation responsible
Genetic screening
Forward screening (unbiased)
Mutagenises if 1000s if worms
Identify mutants
Discover gene for phenotype
Reverse screening
Mutate a known gene
Look at phenotype (need design of assay)
Good for GWAS, good for neuroscience eg increased risk of genes but shown how through this approach
C. Elegans nomeclature
Gene names (function or molecular features) eg unc30 and mgl -1
Allele name: eat-4(ky5)
Strain name: MT6308 for eat4 (ky5)
Transgenic nomenclature: extrachromosomal or integrated
Micro injection
Makes transgenic lines
Make peices of DNA plasmids
Inject into gonad (reproductive)
Forms extra chromosomal array
See if progeny carry (transformed vs not)
Can make different transgenic lines
CRISPR-CAS9/RNAi
CRISPR (cas9) allows genomic DNA to be edited (single nucleotide resolution)
RNAi allows knockdown of gene expression
Conserved tissues
Differentiated tissues that make up its body
Epidermis
Muscle
Nervous system
Conserved neurotranitters
Conserved bioamones and aa NTs
Sign of the signal can be different eg glutamate
Enzymes also conserved
Conserved use of neuro peptides as neuro modulators
Difference in neurotransmitters
Worms use octopamine instead of noradrenaline
They have glutamate gated chloride channels (allows hyperpol)
Conserved mechanisms of synaptic release
Motor neurone releseaing 5HT (egg laying behaviour)
Cholinergic synapse release too
Unc18 (associated with vesicle) and syntaxin (pre synaptic membrane) forms SNARE complex important for synaptic release
Unc (motorloco - uncoordinated)
Munc in mouse
Neurotransmitter receptors in c elegans
Glutamate
42 ACH
GABA
4 glutametropic receptors (g coupled receptors activated by glu)
Model for behavioural plasticity
Defined genome
Mutagenesis/train genesis
Defined connective
So can model behavioural plasticity in c elegans
Modulation of neural circuits modifies behaviour
What does a worm do when you take away food
Agar plate with ip50 (food) and worm
Move to plate with no food (cleaning plate)
Move to another no food plate and observe (movement, feeding, egg laying, nutritional status, different time scales)
Fluorescent dyes for nutrition
Worms NS encoded how long there’s been abcence of food and cause temporal changes in behaviour. But how?
C elegans exhibit context dependent behavioural plasticity
Food
Fast pharyngeal pumping, egg laying, motor “dwelling” , slow rate of movement (enhanced slowing if previously starved)
No food
Initial decrease in pumping, reduced egg laying, progressive change in movement from local area search to dispersal, fast rate of movement
Food = powerful environmental sensory cue
Coordinate behaviour based on how long food has been absence and experience register
Rationale for food approach
Sensory cue
Previous experience
Execution of motor programme
change pattern of movement based on learning
Microcircuits and behaviour human and worm
Input from internal and external cues
Integration from sensory neurones
Interneurons cause output
Motor neurone cause behaviour
C elegans locomotion sub behaviours
Reversals
Turning
Head movement
Pausing
Show behavioural states ie foraging
Temporal regulation of foraging behaviour
We’ll fed - high angled turns, omega turns, long reversals (local search)
Starved - decrease angled turns, decreased omega turns, decreased reversals (dispersal)
Can encode info of how long ago they were in contact with food
Questions to be addressed
Which neurones are involved in the circuitry and how are they connected?
Which genes encode proteins that regulate behaviour?
Where do these proteins function in the circuit?
How does this circuit adapt to coordinate behavioural plasticity?
Circuits
Celegans has same bunker of synapses as single mammalian hippocampal neurone
Circuits are well defined, amendable to function and analysis
Amphid neurones - sensory neurones
Nerve ring - like brain and contains interneurons
Motor neurone - dorsal and ventral cords
Output to muscle cells
Circuit for simple context dependent behaviour - foraging
Neurones that regulate locomotor behaviour
Neurones that sense presence/absence of food
Neurones integrate and process this info
C elegans NS
Cuticle worm
Hyperdermis
Musculature (body wall and longitudanal)
Dorsal and ventral nerve cords
Muscle cells send projections to nerve cord (we send projections to muscles)
Celegans muscle
Adult transgenic worm
Cell type specific reporter
YFP go myosin promoter, micro injected and extrachromosomal expression
YFP fluorescent marker expressed in muscle cells
Neuromuscular signalling
Motorneurones
75 - 7 district classes Innervate body wall muscle
3 Innervate ventral body wall (VA, VB, VD)
4 Innervate dorsal muscles (DA, DB, DD, AS)
VD and DD are gaba others are ACh
Normal locomotor behaviour
S shape
Muscles on either side so almost like crawling on it’s side
Sinussoidal wave form
Muscle activity and motility
Activate and inhibit muscles coordinated for movement
Contracted = shorter
Relaxed = longer
Simultaneous excitation and inhibition
VA/VB release ACh exciting to contract
DD activate and releases glutamate causing extension
Backward vs forwards locomotion
Forwards: b- motoneurones (VB, DB)
Backwards: a motorneurones (VA, DA)
Function assigned using laser microsurgery to ablate them and observing behaviour
Calcium imaging
Backward and forwards motor neurones
Calcium is main ion driving depol in c elegans unlike sodium in us
So calcium reporter to see neurone activity and see how the neurone contributes to behaviour
Blue reacts, restrains worm, backwards and forwards but not out of view
Calcium censor eg chameleon - introduced into neurones using transgenics, cell specific expression downstream of promoter in plasmid and micro injection and observe progeny
Experimental approaches: calcium signalling censors
Blue and yellow fluorescent proteins linked by cal Mosul in and m13
No calcium, will fluoresce blue if calcium then yellow as conformational chain as ca2 binds to calmodulin and m13. CFP excited but transfer via FRET to YFP
Motor neurone function
VA motoneurones active during backwards movement
DB and VB motoneurones are active during forwards
Confirms laser ablation studies
Local area search involves backwards and forwards
In dispersal, backwards suppressed and mistily forwards
Chemosensory neurones: amphids
Amphibian neurones detect external cues
12 amphid neurones that are bilaterally pairs
Ciliates ending exposed to external environment
Amphid neurone body found in nerve ring, dendrite to sensory openings at the head of work. Axons project around nerve ring important for synapsing onto other neurones
AWCL (left r = right) olfactory and gustatory system
Experimental approaches: gCAMP
gCAMP genetically encoded calcium sensor
Used to determine role of AWC in sensing food/no food
GFP (Cam and M13 domains) so based on calcium levels, flourensce with ca2
Cell specific expession
Chemosensory neurones and analysis with microfluidic chambers (immobilisie, signal in controlled way and image)
Present olfactory signals
Removal of odour is detected by chemosensory neurones
Fluid in different chamber
4 chambers
Can control smell release essentially
gCAMP signalling
Neurone changes activity in response to olfactory cues
Odour on = less activity
Odour off = more activity
Increased custom of ca in AWC neurone when odour is removed
Same when bacterial odours removed
Calcium signalling in AIY
AIY activated by odour
Response in AIY depends on AWC (based on AWC ablations)
Food present = AWC inactive, AIY active
Food not present = AWC active, AIT not active
So AWC negatively regulates AIY activity
Calcium signalling in AIB
Activated by removal of odour
Depends on AWC
Food present = AWC inactive, AIB inactive
Food not present = AWC active, AIB active
AWC positively regulates AIB activity
Circuit for detecting absence of food
Absence of food AWC activated and realeases glutamate
Activated GLC3 and inhibits AIY
Glutamate actives GLR1 in AIB
Circuit for detecting food
Presence of food AWC inactive
AIY released from negative regulation and so active
AIB inactive
Circuit for simple context dependent behaviour
Sensory circuit
Command circuit
Motor circuit
Circuit for connecting sensory signals to motor outputs
VA and DA (backwards)
VB/DB (forward)
Connected by command interneurons
Command interneurons regulate pattern of locomotor behaviour
Laser ablation of AVE, AVA and AVD abolishes backwards
Laser ablation of AVB, PVC abolishes forward movement
Cross talk between the command circuits makes decision too
Genetic screening in Celegans using foraging behaviours
Worms on food
Cleaning plate
Observation plate
Utilising tracking analysis to record worm locomotion
5 mins off food (high turns, omega turns, during local search), 30-35 mins off (less turn, dispersal search)
Signal in the worms NS tells more high angle turns when placed off food (ARSi)
ARSi = rotio of high angled turns of 5 mins vs 35
0 seconds (near food) (dispersal)
2 seconds (on food) (ARSi reset)
Mutants lacking glutamate signalling investigation
Used ARSi to quantify behaviour of different mutants
Glu known to be key in command circuits
Eat-4 (vesicular glu transporter) knockout
Cannot load and release glu at synapse
MgluR2 (neuromidulators at synapses) exist
Mutants lacking glutamate signalling is defective in local area search
Glu needed for foraging
Cannot transition into foraging behaviour and can’t do high angle terms and reverse
Eat4 mutant worm goes straight into dispersal behaviour when placed off food
Glr1 rescue improvement around half way
Glr2 rescue better almost fully
May be due to not being reincorporated properly
Bioinformatics identified glu receptors in Celegans
Iontropic teceptors
NMDA like receptors encoded by nmr1 and 2
AMPA/KAINATE like receptors glr1 and 8
Fast excitatory signalling
Metabotropic receptors
8 subtypes in mammals
Group 1,2,3
3 subtypes in Celegans
MGL1,2,3
Determine where receptors are expressed
Access genome sequencing allows constructs to identify gene expression patterns
Regulatory sequence and reporter gene
Inject DNA into worm
Allow injected worm to lay eggs
Inspect progeny for expression of fluorescence
(Extrachromosomal array so not all will have)

GLR1 is expressed in command interneurons that coordinate backwards and forward movement (AVA, AVB, PVC, AVD, AVE)
Expression pattern is consistent with behavioural deficits in GLR1 knockout
GLR1 AMPA Receptor
Lurcher mouse - abnormal pattern of locomotion
Homozygous lethal
Cerebellum is absent of purkinje cells
Molecular basis a to y mutations causing GoF (AA)
Mutation in TMIII of mammalian receptor subunit
Electrophysiology reccirdings from xenopus oocyts expressing mutant ion channel showed ion channel always opens, so no longer glu gated
Gain of function GLR1 increases reversal behaviour and high angle turns
Receptor always open in heterologous expression system
Neurons expressing GLR1 GOF viable
Enabled worms to be used to investigate GoF on system function and behaviour
Predict they would show greater foraging phenotype
GOF GLR1 command circuit may cause hyper foraging behaviour
GLR1 GOF worms have increased reversal
Not really foraging, disrupted control of backwards and forward locomotion
Suppressor screens
Mutant worm (GLR1 GOF) exposed to mutagen
Progeny
Mutation in gene required for AMPA function
Lurcher worms and wild type movement (Lurcher phenotype suppressed so identify accessory genes required for AMPA receptor function eg auxiliary subunits)
Lurcher mutation
Duration forward and backwards movement in worms is very short
Reversals key behaviour in local area search and suppressed dispersal
Glu signalling through GLR1 expressed in command interneurons performs important role in regulating pattern reversals
GLR1 - equivantly short forwards and backwards
GLR1 regulation of command circuit output
GLR1 acts in command circuitry gate the frequency of reversals
AVA command interneurone
Forwards activity increases, reaches threshold, goes backwards
3 reversals but GoF and activates more reversals as thresholds reached quicker due to ion channel
Circuit for simple context dependent behaviour (altogether)
Coordinates reversals and high angled turns in response to removal of food and driven by AWC
Parts of circuit promote LARS others dispersal
GLR1 functions within circuit and coordinates early response of local area search
What are signalling mechanisms that control the temporal aspects of circuit adaptation and underlie the transition from LARS to dispersal
Things to look for
Neurone with sustained response to being moved off food could encode temporal aspects ie how long off food (AWC)
Signalling molecule that has longer time course of signalling eg neuropeptide. Neuro modulators, signal through GPCR pathways and known to have longer time course of both signalling and neuronal responses
Temporal regulation of a circuit for simple context dependent behaviour
One theory
AWC activation leads to GLR1 and GLC3 activation (fast)
Neurone downstream of AWC (AIA) release neuropeptides (insulin like)
Feedback inhibition of AWC
Inhibition of local area search and promotes dispersal behaviour
