During my meeting Dr. Eichler mentioned about a new VNC single-cell RNAseq dataset to be more confident about my results on b1 & b3 motor neurons. Good part about this dataset is it can be accessed via web tool but it only focuses on pupae on different developmental points. It is also mapped to MANC connectome with cell types but this feature is not working that correctly.

Here are the top expressed receptor genes if I access the cells with MANC ids:

Same data but filtered according to percentage of cells express that receptor in the selected neuron group:

List of receptors if I filter them via transcription factors:

b1 top expression:

b1 filtered according to percentage of cells express that receptor in the selected neuron group:

b3 top expression:

b3 filtered according to percentage of cells express that receptor in the selected neuron group:

Same dataset but results for rutabaga and radish:

With connectome ids:

Filtered via transcription factors b1:

Filtered via transcription factors b3:

Experimental Group: mb247-Gal4>UAS-ChR2-XXL, Control 1: mb247-Gal4>w1118, Control 2: w1118>UAS-ChR2-XXL, Tests are performed blind, 1.5M Salt

Following the previous experiments investigating HSP70 expression in response to heat shock and optogenetic stimulation, this experiment intended to find out if cold stress could also induce HSP70 expression. Two types of cold stress were tested: long exposure to moderate cold (often referred to als ‘chilling temperature’) and short exposure to more extreme cold.

To ensure a larger sample size, two Western blots with the same experimental setup were performed. As a positive control, the same protein extract used in previous experiments was included, which had been obtained from mixed-gender flies exposed to 37 °C for 1 h. In addition, unstressed flies, flies exposed to 0 °C for 20 h, and flies exposed to -4 °C for 1 h were tested. For each of these conditions, male and female flies were analyzed separately.

Following the cold shock treatment, the flies were allowed to recover for 1 h at 25 °C before being frozen in liquid nitrogen. This recovery period was included because low temperatures are expected to reduce metabolic activity, potentially limiting the cellular processes required for HSP70 expression during cold exposure. Recovery at 25 °C may allow HSP70 expression to increase after the stress has ended.

Western blot analysis was performed using the same antibodies and methods as in the previous experiments. During the prolonged cold exposure, the temperature inside the cooling box fluctuated by approximately ±1 °C over time, which should be considered when interpreting the results.

The quantification revealed a clear, nearly 18-fold increase of HSP70 expression in females exposed to prelonged cold stress compared with unstressed females. In males, HSP70 levels increased approximately 4-fold under the same conditions compared with unstressed males. In contrast, the short exposure to – 4 °C for 1 h did not result in a remarkable increase in HSP70 levels in either sex. No increase was observed in males, while females exhibited only a minimal change, which is probably negligible due to the small sample size.

Overall, these results suggest that prelonged exposure to moderate cold can induce HSP70 expression, whereas the short, acute cold shock did not show a comparable response. The difference between males and females following prelonged cold exposure is not significant yet, but further experiments could determine whether this difference is reproducible.

The experiment is currently being repeated using different exposure times and temperatures based on the reported LT50 values. LT50 refers to the time required for 50% of the exposed flies to die under the given conditions.

Experimental Group: mb247-Gal4>UAS-ChR2-XXL, Control 1: mb247-Gal4>w1118, Control 2: w1118>UAS-ChR2-XXL, Tests are performed blind

As N1-8 were perfomed within one week and differences in the size/developmental stage of the larvae within the same crossings/genotype in different vials were observed, the results of the measurements from the first week (N1-8) and of the measurements of the second week (N9-12) are analyzed separately and were additionally pooled.

Trying to illustrate the potential physiological changes happening in motor neurons such as b1 during torque learning:

Top row: when attempting left turns, either spontaneously, or due to counter-clockwise rotating optomotor stimuli, the fly decreases its wing beat amplitude (blue lines) of the left wing and increases its wing beat amplitude of its right wing. Schematic recordings from left and right b1 motor neurons (lb1 and rb1, black lines) show different phase relations to the overall wing beat cycle (bottom, blue): lb1 is delayed compared to rb1, leading to a smaller wing beat amplitude on the left side vs. the right side (blue lines) and thus resulting in left-turning yaw torque. If the fly has been trained to avoid the left turning yaw-torque domain (red flash), then the amplitude of the torque response to counter-clockwise optomotor stimuli is reduced. It is not known if this decrease in optomotor response amplitude is due to an increase in wing beat amplitude on the left side of the animal (red line), a decrease of wing beat amplitude on the right side of the animal (red line), or both. Presumably, this change in optomotor response amplitude is due to changes in the phase relationship of b1 spike activity (red action potentials).

Middle row: when attempting right turns, either spontaneously, or due to clockwise rotating optomotor stimuli, the fly increases its wing beat amplitude on the left side and decreases it on the right side (blue lines). The phase relationship of b1 motor neurons spikes to the wing beat cycle is inverted, compared to left turns (black recordings, lb1, rb1). After punishment on left turning torque, right turning optomotor response amplitude is unchanged.

Main question: how can the b1 motor neuron by modified in a way that only left-turning torque is affected, when the b1 motor neuron is involved in both turns? Easy answer: floor/ceiling effects.

The primary antibody used for HSP70 detection was ordered as a rat anti-HSP70 antibody. However, the information sheet stated that the antibody was produced in rats, while the tube label claimed it was produced in mice. To clarify the origin of the primary antibody and determine which secondary antibody could be used in future experiments, four Western blots were performed using the delivered anti-HSP70 antibody as the primary antibody and a different secondary antibody for each blot.

The following secondary antibodies were tested: goat anti-mouse with fluorescence detection at 800 nm, goat anti-rat at 800 nm, donkey anti-rat at 700 nm and goat anti-mouse at 680 nm. The results indicated that both anti-mouse and anti-rat secondary antibodies bound to the delivered primary antibody, despite their different target species.

A message from the manufacturer later confirmed that the primary antibody was indeed a rat anti-HSP70 antibody. Therefore, the observed binding of the anti-mouse secondary antibodies were unexpected. One possible explanation is sequence similarity between rat and mouse immunoglobins, which may allow some cross-reactivity.

In addition to the cross-reactivity, an unexpected effect on the protein marker was observed. The green sizing bands disapperared when the goat anti-rat secondary antibody was used. The reason for this effect remains unclear.