MCDB 423 Lecture 38: Lecture 38

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Molecular, Cellular and Developmental Biology
MCDB 423
John Kuwada

Lecture 38 Slide 1 So fru expression is important for mating behaviors in flies There are subsets of neurons that express fru -These neurons mediate courtship behaviors This suggests that it must be the activity of the fru neurons that’s necessary for courtship behaviors in males This was tested -Utilized a mutation called shibire which is a geen required for synaptic vesicle recycling -So in a shi mutant fly, the recycling doesn’t occur -So eventually the terminals will run out of synaptic vesicles and now the synaptic transmission is eliminated There is a temperature sensitive shi mutation that is dominant -If you drive gal4 in fru+ neurons and you are uas-shi temperature sensitive, you can block synatpic transmission with temperature -At high temperature, the shi function is blocked -So you can induce inhibition of synaptic release by using genetics and temperature When you do this -Controls have high courtship index -In a transgenetic fly that’s fru-gal4 and uas-shi mutant, at high temperature the courtship index decreases -You are blocking the activity of fru + neurons in the males but at low temperatures that inhibition shouldn’t exist and you should get normal courtship index and this is what you get So synaptic activity by fru neurons is necessary for normal courtship behavior by males Slide 2 It gets more complicated FruM is expressed by males and expressed by a subset of neurons in the brain -It’s also expressed by the sensory neurons in the antenna What you see here is a fly that’s fru-gal4 uas-membrane bound gfp -What you see is that there are many of these neurons -All the sensory neurons in this particular fly are being driven by a all neuron gal-4 driver called elav -You can see that the antenna is full of sensory neurons -But if you have an antibody against fruM, you see that only a small portion of the sensory neurons actually express fruM (Only 15 percent express it in a male; 0 percent in a female) In the transgenetic flies now, because they’re expressing a membrane bound gfp, the gfp will diffuse laterally in the membrane and end up labeling the presynaptic terminals -What you see is that in the male, the da1 glomerous, the vl2a and vl1v are innervated by fru + sensory neurons and in a female -However the glomerous are larger than the females -This is dependent on fru slide 3 This is what we know about the neural circuit so far -There are sensory neurons that express fruM in a male that project synapses onto the da1 PN which also express fruM which then activate specific lateral horn targets -We know that the activity of fru neurons is required for normal courtship and mating behaviors by male There are two sets of neurons that are within this neural circuit -So can we distinguish between the activity of the fruM sensory neurons vs. the fruM da1 neurons slide 4 This is the genotype of the fly that was used -Has 3 different transgenes involved -One is eyeless flipase and recall that eyeless is a transcriptional factor expressed by all antenna cells including the sensory neurons -So since eyeless is controlling the expression of flipase, all the antenna cells are expressing flipase -It’s also transgenetic for uas-frt-stop-frt-shi temperature sensitive -It’s also fru-gal4 So what happens is that the antenna neurons will express eyeless and therefore express flipase -Those cells would exercise the transcriptional stop to give you the uas-shi(ts) -Only the eyeless cells that are neurons will express it because only those sensory neurons express fru This allows you to selectively manipulate the activity of the fruM + sensory neurons as opposed to other neurons that are fru + Slide 5 How does this affect the behavior? -The two high bars represent controls thus they have high courtship index -Looking at the transgenetic we were just talking about, it has a decreased courtship index -This suggests that synaptic transmission by fru + sensory neurons is required for courtship In the ven diagram, it’s only the intersection of all 3 that represents the fru+ sensory neurons What about the rest of the fru neurons in the brain? Slide 6 Answer: C So you’re fru-gal4 so all the fru + neurons are making gal4 You’re also ey-FLP so all the sensory neurons are expressing flipase So only in the sensory neurons would you exercise the shi-ts -So that means those neurons are no longer sensitive to temperature anymore in terms of being able to release NTs -However all the other neurons that are fru+ in the brain that don’t’ express flippase and not have this temperature sensitive allele of shi exercised, they will still have it -Therefore, they will be responsive to temperature to block synaptic transmission or not Blocking this transmission in the brain fru+ males does affect courtship index -These then have to be the da1 neurons since we know they express fruM slide 8 There are fruM + neurons including the or67D sensory neurons but also other sensory neurons -The or67D project to the da1 glomerous which are innervated by the da1 PN which also express fruM which project to the mushroom body and the lateral horn slide 9 The fru gene is also found in the mesothoracic ganglion (MG) -mcd8 is just a membrane protein What you see is that there are a lot of gfp + in the MG -This is the case in both males and females The MG are the very ganglion that control the wings of a fly -So it makes sense that the fru + in the MG may actually form neural circuits that generate the song by vibration by the wings The brain neurons that are fru+ project axons to the MG Slide 10 Goal was to figure out if those fru+ neurons in the MG do constitute a pattern generator for this song What he does: -Created a fly pod which are headless -This is how he eliminated the brain neurons -A fly can live for about half a day without a head The fly is fru-gal4, uas-p2x2 -p2x2 is an ATP gated channel that passes positively charged ions -So if they bind ATP, + ions flow though and activate that neuron -Drosophila lack endogenous ATP gated channels So all the fru positive neurons will express gal-4 and thereby activate p2x2 but you need ATP -He solved this by injecting a caged ATP into the thoracic region of the fly What is caged ATP? -It’s a version chemically modified version of ATP that almost looks like there’s a cage around it and prevents it from interacting with other substances -He made it a UV sensitive caging -You can shine UV light to caged ATP to uncage it and now the ATP can bind to p2x2 In this case, it’s a headless male -Once you do the UV flash, the wings start virbrating So the fru+ neurons in the MG are capable of when activated of emitting a song -When he did this with the WT female, the female also sang slide 11 If you take a closer look at the songs, they are different for male and female -The song we heard consist of two parts: a sine song and a pulse song -A fruM female that only makes fruM emits a song that’s the same as WT male song So given that the songs are different, do they have different affects on WT female? Slide 12 Both of these flies are WT but the male is wingless -So the male can’t sing -The female is WT virgin female He placed them in the chamber -Then he played songs (WT male songs, fruM female song, etc.) and checked to see if he got mating The left is pl
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