PPM2 flies showed light-avoidance in T-Maze experiments when tested in yellow light for 1 minute. In the JoyStick setting, where flies are tested for 10 one-minute periods flies displayed a shift from negative (light-avoidance) values in the beginning to no preference in the last training period. For this reason I started a set of T-maze experiments where flies could decide between light and dark tubes for 10 minutes. Since control flies were not available for this experiment but were testet in the 1-minute T-maze setting in parallel, they are not included yet.

FLP; GFP ♂1

FLP;GFP♂2

FLP;GFP♀1

W|TH-Gal4-UAS-GFP|♂1

W|TH-Gal4-UAS-GFP|♂2

W|TH-Gal4-UAS-GFP|♀

13.0273 (Red)

For all Figures the left side displays all 10 testing and training periods for the JoyStick experiment. The right side compares the PIs of the last training period between the groups. Graphics indicate whether red or yellow light was used.

13.0273 (Yellow)

SIFa (Red)

SIFa (Yellow)

The left-hand side of the figure displays the choice indices (CIs) for the different groups tested for 1 minute in the T-Maze setting. On the right-hand side, the preference indices (PIs) for the final training period in the JoyStick setting are shown. Since flies of the TH_Flp_p10;64H06 line were not blind, they could not be tested in the T-Maze setting. The upper part of the figure refers to experiments conducted with yellow light, while the bottom part to experiments with red light, as indicated by the graphics.

In previous posts I referred to the different dopaminergic neurons (DA neurons) with the names of the driver lines used for the crossings. The table below the figure connects the fly strains to the targeted neurons and gives the reference. Gr28bd+TrpA1 target heat sensing neurons and acted as a control, since flies expressing the chrisom channel in these heat sensing neurons would avoid light activation. Flies were fed with all-trans retinal (ATR) for 2 days before the experiments, to enable light activation of the targeted neurons. For the negative control ethanol was used.

In the T-Maze experiments, flies were tested for 1 minute without prior exposure to light, whereas the JoyStick results reflect preferences after nine 1-minute training periods. Therefore, the T-Maze experiments should be repeated using longer testing periods. Additionally, PIs from the initial training periods in the JoyStick experiments will be included to allow for better comparison.

Hulse et al. eLife 2021: https://doi.org/10.7554/eLife.66039

Xie et al. Cell Reports 2018: https://doi.org/10.1016/j.celrep.2018.03.068

1. Summary protein-protein aPKC interactions flybase results

aPKC: atypical protein kinase C/ Serine/threonine protein kinase

• function: encodes a member of the conserved Par complex, is required for apico- basal cell polarity in the germ line as well as in epithelial and neural precursor cells, for epithelial planar cell polarity and for cell proliferation.

• expression pattern:

• indirect flight muscle ((thoracic dorso-longitudinal muscles (DLM))
• high in MNs (70% in leg muscle motor neurons)

1.1 Potential aPKC protein interaction partners in MNs

1.2 Summary RNA-protein interactions aPKC-flybase

2. Promotor sequence analysis FoxP target genes

dFoxP

• function:  transcription factor expressed in the nervous system; involved in locomotion, operant self-learning and courtship behavior

• consensus seq FoxP: AAACAaATTTC (Santos et al., 2015; JASPAR data base)

• human ortholog:  Hsap\FOXP4, Hsap\FOXP1, Hsap\FOXP2, Hsap\FOXP3

• expression pattern:

• indirect flight muscle (thoracic dorso-longitudinal muscles (DLM))
• leg muscle motor neuron (high)

2.1 Potential FoxP target genes

AAACAAATTTC

insc – Inscuteable (insc)

• function: encodes an adaptor protein required for asymmetric cell division; interacts with the microtubule binding protein encoded by mud and the adaptor encoded by pins; also binds to the apical complex proteins encoded by baz, par-6 and aPKC and may recruit microtubule binding proteins to the apical cell cortex to induce apical-basal spindle orientation

•  human ortholog:  –

• expression in MNs: low

AAACACATTTC

CG15233 

• function: uncharacterized protein; putative target of stat and escargo, two master regulators of intestinal stem cells (Khanbabaei et al., 2023)

• human ortholog:  –

• expression pattern:
• indirect flight muscle
• low in MNs (leg muscle motor neuron)

bowl 

• function: putative transcription factor; leg joint formation, acting downstream of notch to pattern the leg tarsal segments;

• acts downstream of drm and lin during foregut and hindgut patterning and morphogenesis; involved in cell rearrangement during elongation of the embryonic hindgut; regulates expression of hindgut patterning genes to establish the small intestine region of the embryonic hindgut

• human orthologs: Hsap\EGR2, Hsap\KLF11, Hsap\EGR4, Hsap\ZBTB4, Hsap\KLF15, Hsap\EGR1, Hsap\OSR2, Hsap\OSR1, Hsap\ZBTB38, Hsap\ZBTB2, Hsap\EGR3, Hsap\SP2, EGR: early growth response genes, supress exessive immune responses dysfunctions associated with inflammatory autoimmunse diseases like multiple sclerosis (MS), type 1 diabetes and inflammatory bowel disease (Powrie and Coffman 1993; Liblau, Singer et al. 1995, Morita et al., 2016)

• expression pattern:
• indirect flight muscle
• low in MNs (leg muscle motor neuron)

AAACATATTTC

CG4477 

• function: peptidase S1 domain-containing protein; Serine-type endopeptidase   activity, involved in wing disc dorsal/ventral pattern formation; proteolysis,

• human ortholog: responsible for vital processes in man such as digestion, blood coagulation, fibrinolysis, development, fertilization, apoptosis and immunity

• expression pattern:
• low in MNs (leg muscle motor neuron)
• moderate expression in muscle cells  (indirect flight muscle )

CG30354- UQCR-11L

• function: cytochrome b-c1 complex subunit 6; component of the ubiquinol-cytochrome c reductase complex (complex III or cytochrome b-c1 complex) which is part of the mitochondrial respiratory chain; formation of the complex between cytochromes c and c1. UQCRH/QCR6 family. [a.k.a. FBgn0050354, UQCR-11L-PB, UQCR-11L-PA, CG30354],

• human ortholog: Hsap\UQCRH (Ubiquinol-Cytochrome C Reductase Hinge) protein

• expression pattern:
• indirect flight muscle
• low in MNs (leg muscle motor neuron)

AAACAGATTTC

CG10864 

• function: potassium ion leak channel activity; potassium channel activity; involved in potassium ion transmembrane transport; stabilization of membrane potential; two pore domain potassium channel (TC 1.A.1.8) family

• human orthologs: Hsap\KCNK4, Hsap\KCNK18, Hsap\KCNK5, Hsap\KCNK7, Hsap\KCNK2, Hsap\KCNK10, Hsap\KCNK12, Hsap\KCNK16, Hsap\KCNK17, Hsap\KCNK6, Hsap\KCNK1

• expression pattern:
• indirect flight muscle
• low in MNs (leg muscle motor neuron)

AAACAATTTC

Lhr – Lethal hybrid rescue (Lhr)

• function: encodes a protein required to repress transposable element and satellite DNA expression. It also has a gain-of-function phenotype of causing lethality in F1 male hybrids between D. melanogaster and D. simulans

• human orthologs: –

• expression pattern:
• indirect flight muscle
• low in MNs (leg muscle motor neuron)

Source: EPD The Eukaryotic Promoter Database

Because the effect of yaw torque training on optomotor responses (OMRs) is still very small for now (we work on improving that), I pooled the two groups in which aPKC was knocked out in either motor neuron (MN) b1 or MN b3, as both these two groups and their WTB x aPKC/Cas9 controls seem to learn just fine (torque preference text after 8 minutes of training):

Obviously, we still need to check the Gal4 driver lines are really targeting the right neurons, but assuming they are ok, it seems like neither an aPKC knock-out in b1 alone nor in b3 alone is sufficient to affect operant self-learning. Maybe this is due to b1 and b3 acting as an agonist/antagonist pair and if one of them fails to show plasticity, the other is sufficient on its own? Another explanation could be that the torque preference depicted above is mediated by other neurons than b1 or b3 and that the OMR modulation is gone in these flies. Because the OMR effect is so small, I pooled the two groups, threw out all flies that didn’t have at least an acceptable OMR and halfway accurate OMR parameter estimation and plotted the OMR traces of the remaining 35 flies after training:

So despite these flies learning well, the OMR does not seem modulated as one can see in WT flies. However, there my be a slight effect for the fly punished on right turning torque, perhaps? However, this group also has much larger errors, which I would need to check the reason for. The quantification of the OM symmetry does not show any hint of an effect, though:

Below the total evaluation before and after training. What is weird is that despite there being no effect after training, the correlation between torque preference and OMR asymmetry seems to be there – or is it just the three outliers?

Either way, when I pooled the control flies from this experiment with the same genotype from the last experiment to get to 42 flies, only the group that was punished on left-turning torque showed the modulation:

Accordingly, the quantification shows no difference ion the control group either:

And no significant correlation between the indices either:

All in all rather puzzling results that reinforce my view that the OMR effect is much too small to practically work with. That means one of the next goals must be to get this effect size increased by, perhaps, decreasing the strength of the optomotor stimulus?

As it seems the flies without aPKC in b1 or in b3 steering motor neurons seem to learn fine, I’ve decided to leave this dataset where it is:

But I will try and analyze their optomotor response in more detail, maybe these flies can dissociate between the spontaneous preference and the OMR plasticity?

The positive control was tested once without food and once with food to observe its effect on general behavior, particularly on learning behavior. NF = No Food / WF = With Food

It seems that panneuronal downregulation of the expression of the foraging gene impairs yow torque learning when flies were trained for 8 minutes.