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Neural Control Circuits Flashcards

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1
Q

C elegans difference from earth worms

A

Nematode
Smaller
Non segmented body
Zigmacoidal movement

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2
Q

Why c elegans?

A

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)

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3
Q

Conserved features

A

Synapses - Plasticity
Worm has 302 neurones vs billion in human brain
Fundamentals of how neurones communicate and synaptic communication are conserved

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4
Q

What can we investigate in c. Elegans (neural circuits)

A

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

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5
Q

Sydney brenner (1960s)

A

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

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6
Q

Growing c elegans

A

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

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7
Q

Novel prize

A

Genetic regulation of organ development and programmed cell death
RNA interference - gene silencing by double stranded RNA
Development of green fluorescent protein GFP

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8
Q

Advantages of c elegans

A

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

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9
Q

Mutagenesis

A

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

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10
Q

Genetic screening

A

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

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11
Q

C. Elegans nomeclature

A

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

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12
Q

Micro injection

A

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

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13
Q

CRISPR-CAS9/RNAi

A

CRISPR (cas9) allows genomic DNA to be edited (single nucleotide resolution)

RNAi allows knockdown of gene expression

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14
Q

Conserved tissues

A

Differentiated tissues that make up its body
Epidermis
Muscle
Nervous system

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15
Q

Conserved neurotranitters

A

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

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16
Q

Difference in neurotransmitters

A

Worms use octopamine instead of noradrenaline
They have glutamate gated chloride channels (allows hyperpol)

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17
Q

Conserved mechanisms of synaptic release

A

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

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18
Q

Neurotransmitter receptors in c elegans

A

Glutamate
42 ACH
GABA
4 glutametropic receptors (g coupled receptors activated by glu)

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19
Q

Model for behavioural plasticity

A

Defined genome
Mutagenesis/train genesis
Defined connective
So can model behavioural plasticity in c elegans
Modulation of neural circuits modifies behaviour

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20
Q

What does a worm do when you take away food

A

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?

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21
Q

C elegans exhibit context dependent behavioural plasticity

A

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

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22
Q

Rationale for food approach

A

Sensory cue
Previous experience
Execution of motor programme
change pattern of movement based on learning

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23
Q

Microcircuits and behaviour human and worm

A

Input from internal and external cues
Integration from sensory neurones
Interneurons cause output
Motor neurone cause behaviour

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24
Q

C elegans locomotion sub behaviours

A

Reversals
Turning
Head movement
Pausing

Show behavioural states ie foraging

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25
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
26
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?
27
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
28
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
29
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)
30
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
31
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
32
Normal locomotor behaviour
S shape Muscles on either side so almost like crawling on it’s side Sinussoidal wave form
33
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
34
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
35
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
36
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
37
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
38
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
39
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
40
Removal of odour is detected by chemosensory neurones
Fluid in different chamber 4 chambers Can control smell release essentially
41
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
42
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
43
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
44
Circuit for detecting absence of food
Absence of food AWC activated and realeases glutamate Activated GLC3 and inhibits AIY Glutamate actives GLR1 in AIB
45
Circuit for detecting food
Presence of food AWC inactive AIY released from negative regulation and so active AIB inactive
46
Circuit for simple context dependent behaviour
Sensory circuit Command circuit Motor circuit
47
Circuit for connecting sensory signals to motor outputs
VA and DA (backwards) VB/DB (forward) Connected by command interneurons
48
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
49
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)
50
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
51
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
52
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
53
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
54
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
55
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
56
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)
57
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
58
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
59
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
60
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
61
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

Systems Neuroscience (5 decks)

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