Lecture 3: Investigating Neural Circuits- Courtship and Mating in Drosophila Flashcards
What is courtship?
- A sexually dimorphic behaviour- male and female behaviour is different in flies
- Appropriate species need to be elicited by males (conspecific- of same species)
Describe the difference in appearance of the male and female Drosophila
- Male fly is smaller, more rounded abdomen
- Female is bigger than male, pointed abdomen (where eggs are laid, larger because contains eggs).
Discrete ordered behaviours in fly courtship
- Orientation- the male orients towards the female when in close proximity, if the female walks away he follows
- The male taps the female with his front legs
- Wing extension- one or another wing is extended, the wing is vibrating and this creates a courtship song/pulse- the female hears this and becomes receptive.
- Licking- Eventually, the female fly slows down from walking and the male licks her genitalia
- Copulation- The fly is trying to copulate in this time, it can take seconds to minutes to occur
Measurable courtship parameters
- Total time courting- during the observation period
- Latency to initiate courtship- the time it takes for the male to actually court from orientation to courtship (this is a measure of how well the male can perceive the female- see/smell her).
- Latency to copulation- the time it takes the male to copulate (could be due to how well the male courts, how quickly the female becomes receptive, how receptive she is is she too young or previously mated)?
- Courtship steps performed (order, latency and total duration).
- Courtship index: total time courting/ observation period
Is courtship and innate or modifiable behaviour
- It is innate, the fly can perform this behaviour from the egg.
- But, it can also be modified
Modification of fly courtship via learning and memory
- When a male meets a virgin female she produces attractive pheromones and is receptive.
- However, if she has already mated and received sex peptide from the male, then this causes a change in behaviour and the production of pheromones and inhibitory pheromones.
- The inhibitory behaviour of a female and the production of inhibitory pheromones will result in a reduction in courtship by the male (because the female has already mated).
- If a male then comes into contact with a virgin female again he will not mate with her because he has made an association with the attractive and the inhibitory pheromones.
- This is an example of learning and memory and highlights the complexity of behaviour
What are the courtship signals
- Visual
- Olfactory
- Auditory
- Gustatory
How was the visual courtship signal discovered?
Visual: measured under red light means flys cannot see the stimuli (flies can’t perceive red light). Courtship still occurs but takes longer to initiate as the male takes longer to start and locate the female
How was the olfactory courtship signal discovered?
Olfactory: Measure mutant flies lacking olfaction (involves sensory receptors, smell). The male is sensing volatile pheromones floating around, if you mutate the Ors the fly won’t court as much due to a delay in initiation of courtship.
How was the auditory courtship signal discovered?
Auditory: Measure mutant flies lacking audition (FEMALE cannot hear the fly courtship song). Copulation latency is longer in mutant female flies. So hearing speeds up receptiveness of the female.
How was the gustatory courtship signal discovered
Gustatory: Involved in non-volatile pheromones (taste) when a fly receives the pheromones on the cuticle of the female. Measure using gustation
Which stages of courtship involve which sensory modalities?
- Orientation- Olfaction and vision (males sees and smells the fly).
- Tapping- involves gustation (taste on cuticle of female)
- Wing vibration (audition for female and olfaction for male).
- Licking- taste when male licks female
when the male is not perceptive the male uses hearing too.
Which class of visual projection neurones are involved in courtship (orientation)?
- LC10 visual projection neurones are required for tracking a fly- sized object.
- These sense moving visual objects
- Activation of LC10 neurones elicits courtship (orientation and wing extension) that is enhanced by courtship arousal (pheromones).
- If you knock out LC10 neurones the fly can’t identify visual objects
What are the lobe like structures in the fly brain called?
What is there function?
Optic lobes
- These receive inputs from the command eye
Where do visual neurones project to?
Lateral region of the brain
- Important in information processing and the production of behaviour
Chemosensory signals secreted by females
Chemosensory (olfactory and gustatory) signals- Pheromones
Females:
7-11-HD and 7-11-ND secreted on cuticle
- These are attractive to males
- Allow males to identify females of the same species
Chemosensory signals secreted by males
Chemosensory (olfactory and gustatory) signals- pheromones
Males:
11-cVA, 7-T and 7-P on the surface of their cuticle
- Are attractive to females (and very young males)
- Inhibit or elicit aggression in other males
How does the female hear the courtship song?
- Through the ( feathery structure in-between the segments of the antennae).
- Vibration of arista causes movement of the joint between the two segments of the antenna (segment A3 and A2).
- Johnson’s orgaan is found within the joint of the antennae - this houses a transduction module.
- Transduction module: system of proteins that when it moves in response to arista moving triggers a signal in the neurones)
- Mechanosensory neurones that project to brain from Johnston’s organs
- These neurones project to mechanosensory and motor centre in the brain. So sound neurones project to this region
How does the female hear the courtship song?
- Through the ( feathery structure in-between the segments of the antennae).
- Vibration of arista causes movement of the joint between the two segments of the antenna (segment A3 and A2).
- Johnson’s orgaan is found within the joint of the antennae - this houses a transduction module.
- Transduction module: system of proteins that when it moves in response to arista moving triggers a signal in the neurones)
- Mechanosensory neurones that project to brain from Johnston’s organs
- These neurones project to mechanosensory and motor centre in the brain. So sound neurones project to this region
What is the arista?
- Feathery like structure between the segments of the antennae
- Vibration of arista causes movement of the joint between the two segments of the antenna (segments A2 and A3).
What is the Johnston’s organ?
- Found within the joint of the antennae
- This houses a transduction module ( a system of proteins that triggers a signal in the neurones when the arista moves).
- Mechanosensory neurones in the Johnston’s organ- these project to mechanosensory and motor centre in the brain.
Mating decisions in the male
- The nervous system has to make sense of the sensory inputs and make decisions about which behaviours to recruit
- Male has gustatory, visual and olfactory input. These inputs are integrated and a decision made
- The behaviours are recruited sequentially
- The NS integrates the inputs and if no mating occurs that is fed back and goes to integration centre again and the whole courtship steps are repeated (integration centre is the mushroom body).
Mating decision in the female
- Receives olfactory input (smell-pheromones) and auditory (song).
- She has to decide whether the male is the right species and if she has already mated and received sex peptide she will reject.
- If she hasn’t mated, her NS will reach thresholds of excitation due to sensory inputs and she will accept mating- will stop walking.
What are the genes involved in specifying and controlling male and female behaviours?
- Courtship behaviour is governed by a genetic hierarchy that directs all aspects of sexual differentiation.
- A branch of this hierarchy in which fruitless (fru) is the first regulatory gene is essential for male sexual behaviour.
- fru is essential for male sexual behaviour
- Female behaviour is governed by the fru and doubles (dsx) genes.
Describe how the sex determination pathway in the fly depends on X chromosomes
- In females with XX, there is a pre-mRNA of a gene called sex lethal (Sxl) that has regulated splicing.
- So the two X chromosomes result in regulated splicing of the sex lethal RNA to produce sex lethal protein.
- But, default splicing (without regulated splicing) leads to a different mRNA which has stop codons and no proteins are produced (so in males there are no Sxl).
- Sex lethal is an RNA binding protein that regulates splicing itself and the next gene in the hierarchy is called transformer
Transformer (TRA) protein and Transformer-2 (TRA-2)
- TRA is another RNA binding protein that directs splicing of two further proteins
MALE:
- In males with no Sxl, transformer is spliced by default splicing leading to no TRA production.
- In males the default splicing pattern does still produce a functional protein but it is a male specific fru and dsx.
FEMALE:
- In females TRA is present via regulated splicing
- TRA directs splicing of two downstream proteins dsx and fru
- dsx and fru are spliced in a female specific manner
Describe the phenotype of Dsx males
- Dsx mutant males can carry out most aspects of courtship except from one part of the courtship song
- Dsx in males is therefore not involved in courtship per se, but just in some of the courtship song.
EXPERIMENT: Expression of male dsx in females
What happened?
- Expression of male dsx in females resulted in transformation into males except they did not perform male courtship
- Shows that dsx is not involved in the courtship of males per se
EXPERIMENT: fru null mutant male flies
- Phenotypically male but show no courtship behaviour
- This showed that fru was important in male flies.
EXPERIMENT: less severe fru mutant alleles and allelic combinations
- Produce males that display defects in different stages of courtship
- showing fru importance in courtship
Describe the function of the fru gene
- Controls male courtship behaviour
Describe the function of dsx gene
- Controls one aspect of the courtship song and is involved in somatic differentiation into a male
Transcription and Splicing Patterns of the fru gene
- The fruitless transcript that is differentially spliced comes from the PI promoter.
- There are other promoters in the fru gene that are present in both sexes and produce RNA (they are required proteins, flies die if they have them).
- Mutants in fruitless would only knock out the P1 promoter.
- Flies to be viable have to have transcript from P2-4.
- So its only males lacking fruM (male fru) that have defects in courtship
Where is fruM (male) expressed
Regions of NS involved in sexually dimorphic courtship and behaviour
- In sensory neurones
- In gustatory neurones (proboscis and legs)
How can we identify other regions of the brain involved in controlling courtship behaviour? (that are not sexually dimorphic)
- Not all the neural circuitry involving courtship is sexually dimorphic so other experiments have been carried out to identify other regions of the brain involved in controlling behaviour:
1. Genetically modify a region of the brain
2. Measure the effects on behaviour - If a behaviour change is seen conclude that region is involved in the behaviour
- If no change in behaviour, then conclude that region is not involved in controlling behaviour
- Visualise/identify modified cells and neurones
- Repeat with multiple discrete and or overlapping patterns of brain modification to identify brain regions/cells involved in or required for a behaviour.
How were sexually dimorphic regions of the CNS involved in male courtship identified?
- Using gynandromorphs (male female genetic mosaics, Gynandromorphs are individuals that contains both male and female characteristics).
- This identified broad sexually dimorphic regions of the CNS involved in male courtship
How did we refine the location of sexually dimorphic brain circuitry involved in courtship?
- GAL4/UAS system to refine the location of sexually dimorphic brain using UAS-traF driven by GAL4 lines
- UAS-traF (UAS- transformer female)
- UAS-transgenes were used to alter the activity of neurones e.g., USA- toxin (Ttx).
Proposed circuitry controlling initiation of male courtship and male song in the CNS
- Olfactory input going to the antennal lobe and projecting to higher brain centres
- Lateral region- pheromone processing centre- projects to descending neurones that elicit motor behaviour
- For gustatory input you have subosephageal ganglion receiving input and projecting to gustatory processing centre in the lateral region.
- In terms of initiating courtship this information is integrated in the higher order processing centres (lateral region of brain) and we think of this as a command centre.
- The information integrated in the lateral regions of the brain projects to descending neurones that project a region of ventral ganglion that control flight muscles called the song generator (motor neurones controlling the wing).
- So olfactory, gustatory and visual information is processed and decisions are made in higher order processing/command centre whether to trigger courtship initiation or not.
How does the brain process multiple stimuli to elicit the appropriate behavioural output?
- this is not well understood
