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Systems Neuroscience > Neural Control Circuits > Flashcards

Neural Control Circuits Flashcards

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
Q

Temporal regulation of foraging behaviour

A

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
Q

Questions to be addressed

A

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
Q

Circuits

A

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
Q

Circuit for simple context dependent behaviour - foraging

A

Neurones that regulate locomotor behaviour
Neurones that sense presence/absence of food
Neurones integrate and process this info

29
Q

C elegans NS

A

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
Q

Celegans muscle

A

Adult transgenic worm
Cell type specific reporter
YFP go myosin promoter, micro injected and extrachromosomal expression
YFP fluorescent marker expressed in muscle cells

31
Q

Neuromuscular signalling

A

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
Q

Normal locomotor behaviour

A

S shape
Muscles on either side so almost like crawling on it’s side
Sinussoidal wave form

33
Q

Muscle activity and motility

A

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
Q

Backward vs forwards locomotion

A

Forwards: b- motoneurones (VB, DB)
Backwards: a motorneurones (VA, DA)

Function assigned using laser microsurgery to ablate them and observing behaviour
Calcium imaging

35
Q

Backward and forwards motor neurones

A

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
Q

Experimental approaches: calcium signalling censors

A

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
Q

Motor neurone function

A

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
Q

Chemosensory neurones: amphids

A

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
Q

Experimental approaches: gCAMP

A

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
Q

Removal of odour is detected by chemosensory neurones

A

Fluid in different chamber
4 chambers
Can control smell release essentially

41
Q

gCAMP signalling

A

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
Q

Calcium signalling in AIY

A

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
Q

Calcium signalling in AIB

A

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
Q

Circuit for detecting absence of food

A

Absence of food AWC activated and realeases glutamate
Activated GLC3 and inhibits AIY
Glutamate actives GLR1 in AIB

45
Q

Circuit for detecting food

A

Presence of food AWC inactive
AIY released from negative regulation and so active
AIB inactive

46
Q

Circuit for simple context dependent behaviour

A

Sensory circuit
Command circuit
Motor circuit

47
Q

Circuit for connecting sensory signals to motor outputs

A

VA and DA (backwards)
VB/DB (forward)
Connected by command interneurons

48
Q

Command interneurons regulate pattern of locomotor behaviour

A

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
Q

Genetic screening in Celegans using foraging behaviours

A

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
Q

Mutants lacking glutamate signalling investigation

A

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
Q

Mutants lacking glutamate signalling is defective in local area search

A

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
Q

Bioinformatics identified glu receptors in Celegans

A

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
Q

Determine where receptors are expressed

A

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
Q

GLR1 AMPA Receptor

A

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
Q

Gain of function GLR1 increases reversal behaviour and high angle turns

A

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
Q

Suppressor screens

A

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
Q

Lurcher mutation

A

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
Q

GLR1 regulation of command circuit output

A

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
Q

Circuit for simple context dependent behaviour (altogether)

A

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
Q

What are signalling mechanisms that control the temporal aspects of circuit adaptation and underlie the transition from LARS to dispersal

A

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
Q

Temporal regulation of a circuit for simple context dependent behaviour

A

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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