The loss of tßh in adult flies leads to decreased walking speed and an increase in stripe fixation. Rescuing the gene by a heat shock construct (flies from Henrike Scholz, Cologne) increases walking speed back to wild type level but cannot change stripe fixation.

It is possible that the phenotype in stripe fixation is not exclusively tßh-dependent but more due to other problems the tßh-mutants have, e.g. developmental defects. That would ask for another heat shock timing.

Results:

–          1^st cross

–          2^nd cross

Flies:

–          1^st cross (with heterozygous controls):

                  males                         females                                          females

w1118,tßh/y ;; HStßh  x  w+,tßh//FM7   ->    w1118,tßh//w+,tßh ;; HStßh//+
w1118,tßh/y                    x  w+,tßh//FM7   ->    w1118,tßh//w+,tßh
w1118,tßh/y ;; HStßh  x  w+                        ->   w1118,tßh//w+ ;; HStßh//+
w1118,tßh/y                    x  w+                        ->    w1118,tßh//w+

– 2^st cross (only mutants):

w1118,tßh/y ;; HStßh  x  w+,tßh//FM7    ->    w1118,tßh//w+,tßh ;; HStßh//+
w1118,tßh/y                    x  w+,tßh//FM7    ->    w1118,tßh//w+,tßh

Heat shock timing:

Draft of protocol

Material:

0.5 µl capillary with adequate pump, 0.5ml and 1.5ml Eppis, ice, peaked stylus, razor blade, 2 forceps (or scissors)

Procedure:

sting 3 holes in a small eppi (size is important, not to small so that enough hemolymph can go through, not to big so that no fat or other dirt is going through), put into big eppi.

20 adult flies on ice, cut their wings (not sure about the reason)

spear the fly’s thorax with the peaked stylus

store flies in the small eppi with holes

centrifuge the small eppi within the big one (1 min, 5000 rpm)

throw away small eppi, cut big eppi (to access the pellet easier), soak the pellet with a capillary

record the amount of soaked hemolymph with a ruler (e.g. by photographing under disscetion scope)

pump the hemolymph out of the capillary into a fresh small eppi and freeze it.

Remarks:

Everything has to be done on ice, cooling chain should as poosible not be interrupted

(5000 rpm can be differ with different centrifuges…)

Chrisi’s day…

1 Collected virgins for our ill post-doc, made some crosses:

d42-GAL4 males x tubGAL80-UAS-PKCi and x CS

d42-chaGAL80 males x tubGAL80-UAS-PKCi and x CS

2 Buridan: Prepared colored food (10mg/ml, 2.5ml total in the very little vials, ~ 5 flies in each vial). Denise tested in the afternoon females in the Buridan. Data (4n now) will be shown in the lab meeting.

3 Imaging Made a new cross for the Imaging-project:

TDC2-GAL4 males x UAS-GCaMP3(2)

replaced Neloy’s flies by flies from stock

Florian’s larval preparation (good!) showed that GCaMP-expression is much less than GFP-expression. They hope for the homozygous line.

4 Flight: Performed flight experiments with the tbh,TDC,UAStbh flies. Raw data, see below. Thinking about analysis (probably tomorrow)

Protocol: glued the day before, sugar in wheel chambers, little EVIAN-water.

5s filter on the legs before starting, aspirated, stopped when no response 3 times in a row, stopped time when 30min, stopped after 15 aspiration trials

5 Trehalose: Found time to try out Gérard’s idea (no pigment in the probes to not disturb the NanoDrop measurement). 4 groups: 10 flies with or without heads, 50 flies with or without heads. Hilde showed me how to remove heads with liquid nitrogen and vortex. The other flies (with heads) were frozen at -20° as usual. Trehalase incubation over night. Tomorrow glucose oxidase and measurement

Hi all, I forgot about the lab book email, because it reached me in New Orleans… My day:

1 Collected virgins for our ill post-doc

2 Prepared colored food (10mg/ml, 2.5ml total in the very little vials, ~ 7 flies in each vial). Vicky tested in the afternoon females in the Buridan. The the duration of drug application was increased to 3h  because there was no effect after 1 hour. Data will be shown in the lab meeting.

3 Answered to Dennis Pauls to make an appointment for the Trehalose measurement in Würzburg

4 Glued hooks for a flight experiment I did not do because I did not know, how… (idea was to test whether the UAS-tbh cross is ok)

5 Checked my TDCxGCaMP flies for expression in the fluorescence dissection scope (very cool!) in Marco’s room (borrowed from Konstantin). Then realized that there are pupa in the virgin vials :(

6 Temperature-dependent experiments in Buridan: wanted to test the infrared lamp with the dimmer (measure the temperature change). Was impossible because the thermometer itself heated up. So, I finally looked for an infrared thermometer (you can point on the area you want to measure).

7 boring stuff… Dienstreiseabrechnung was wrong… Email to the Chinese guys again about the cylinder for Buridan… Explained Florian (Marco’s master student) some fly stuff…

A lot of things I learned at the neurofly. First my old friend Benjamin is doing a great postdoc in Bruno van Swinderen lab. It was great to see his data on isoflurane effects and the correlation between sleep disorder and sensitivity to anesthesia.

More related to our interests, I learned that Paul Tchénio has now a prototype to visualize neuronal activity in about half of the fly brain at a decent frequency. It may be interesting to get in touch with him again.

Andre Fiala is doing/has already done the TDC-Flp construct and is using his own UAS-stop-TRPA1 line to get a subset of octopaminergic neurons. Christine will contact him soon to have more information and maybe start a collaboration with him. The student was not at his/her poster when we went there, and we could not talk directly to him/her.

I nice talk by F. Mohammad (Singapore) showed that centrophobism can be associated with anxiety: the majority of treatments used with mices (for instance immobilisation in a pipet tip) also induce more (or less for some treatments) centrophobisms in flies. I told him about the CeTrAn, he said he will look at it. (his flies were in a squared box, with their wings untouched).

Jhl21 (receptor for JH) change of larval behavior at the  wandering stage (go slower and turn more): it acts at the NMJ to change the clustering of glutamate receptors. Similar thing may happen during the first hour of life of the fly, when they become phototactic ?

A nice poster from the Heisenberg’s lab show that flies do have a preferred 0 torque. Even when giving some closed loop drift, the histogram of torque suggest that the preference for the 0 torque is still there (non-uniform distribution around the +1 torque which stabilize the drift. It seems the distribution around the 0 torque is seen only if you have long enough data, I told them I would ask Satish to look at his distributions. This emphasizes why we need to be very careful in preparing the fly for our experiments…

I may have to look at the OK6 Gal4 line for motorneurons, and new RNAi lines seem to be available for PKC53e and FoxP…

I also talked to Flybase people, David was there. It seems the Buridan results should lead to 2 different entries: one linking each genotype to a dichotomic description of the phenotype (“mutant1” – “has larger”- “median_speed”) and one to the raw data and analysis. This will not be easy to automatize, but looks interesting.

I am probably forgetting a couple of things in addition to my discussion with Anette Schenk and her student Anna about FoxP..

Octopamine is a biogenic amine involved in insect physiology and behavioral control. In Drosophila, it was suggested to be necessary for appetitive olfactory learning (Schwaerzel et al., 2003). The available fly mutant tßh cannot convert tyramine into octopamine. We have found that the preference of these mutant flies for sugar (tested in a T-maze) as well as the responsiveness to a serial dilution of sugar (tested in a proboscis extension assay) is decreased compared to their genetic control “w+” (unpublished). We wondered whether the flies have different physiological state and hence different motivation, or if octopamine is involved in the neuronal coding of motivation per se. We therefore want to examine the physiological state of these flies.

In a first approach, we starved flies to death in bottles containing only a cotton pad moisturized with water. Dead flies were counted periodically and kept in the bottles. The survival curve shows that the tßh mutant flies die later (Fig.1a): The time point for 50 % of the flies to die is significantly higher in tßh mutant flies than the one of the control w+. Gender had no effect (ANOVA, gender: p=0.13, genotype: p<0.04). Data from male and female were thus pooled in the figures. These results show that starvation affects differently control and mutant flies. Obviously tßh mutants are more resistant to starvation.

The different physiological state could be due to different activity levels and consequently less energy requirements in tßh flies. To test for this hypothesis, we will use a second approach and measure trehalose content in the hemolymph of the flies. It has been shown that trehalose level decreases with starvation (Meunier et al., 2007). Furthermore there is a correlation found between trehalose level and survival (Isabel et al., 2004). A different metabolic rate in tßh mutants may explain a different trehalose content and the higher survival rate after the same starvation time.

In the future, we hope to be able to titrate the physiological state of the fly using the measure of the trehalose content in their hemolymph, in order to test the sugar responsiveness of mutant and control flies with adapted physiological state. This will allow us to separate the role of octopamine on the fly physiology and its role on the fly motivation.

Fig. 1a: Survival curve.

Fig. 1b: LD50.

after one week, the pattern of my work here start to emerge: flies are ordered, crossings are planned, and a beautiful side project (waiting for the new flight simulator) is planned:
establish appetitive olfactory learning in Berlin.

My last post-doc dealt with appetitive learning, I would love to do one last control experiment in Berlin with a new protocol. The use of a H-maze will be more “hand-made” paradigm, but if it works well, it may well be developed further…

nice time to come…

Monday, I will present my project(s)… have to work on it!