The behavioral consequences of form3 mutations on peripheral sensory

and INF2 rescue studies

Given the defects observed with form3 disruption in CIV nociceptive neuron dendrites, we hypothesized that form3 mutant larvae may also exhibit reduced sensitivity to noxious thermal stimuli leading to aberrant peripheral sensitivity. CIV neurons function as polymodal nociceptors and are required to mediate a characteristic aversive body rolling (360˚C) response in larvae upon exposure to noxious heat or mechanical stimuli, which typically occurs with 1-3 sec after exposure (Im and Galko 2012). Therefore, to investigate the potential role of Form3 in the nocifensive behavioral response, we specifically knocked down form3 in CIV nociceptive neurons and

observed the rolling behavior as an output when challenged with a noxious heat stimulus (45˚C). Behavioral response was video recorded and subjected to quantitative analyses of latency to respond (time in sec) and overall percent responders. The maximal latency period for this behavioral response was set at 20 sec after stimulus and larvae which failed to exhibit a behavioral response in this time period were considered non-responders. The result was quite striking, displaying a nearly complete impairment in noxious heat evoked behavioral response as measured by changes in latency to response and overall number of responders (Fig. 4-3B,E,F), thereby revealing a loss of peripheral sensitivity. Moreover, this behavioral defect was not due to any general defect in locomotion as both control and form3 mutant larvae exhibit normal locomotor behavior (data not shown). Control larvae exhibit an average behavioral latency of ~2.5sec (larval rolling shown by curved body angle) in response to noxious heat (Fig. 4-3A,E,F). In contrast, CIV-specific inhibition of form3 function leads to a dramatic increase in the latency to respond (among those very few larvae which ever respond) (Fig. 4-3B,E,F). The majority of form3 mutant larvae were classified as non-responders as they fail to exhibit rolling behavior within the 20 sec assay period (Fig. 4-3B,F).

Next, we sought to determine whether overexpression of Form3 or INF2-FH1-FH2 may lead to changes in behavioral latency or number of responders when challenged with noxious heat (Fig. 4-3). We found that neither Form3 (Fig. 4-3C) nor INF2-FH1-FH2 (data not shown) overexpression in CIV neurons resulted in a significant change from controls with respect to latency (Fig. 4-3E), however we did observe a reduction in the total percentage of responders in both these conditions relative to controls (Fig. 4-3F). Among those larvae that failed to execute nocifensive rolling behavior, they instead displayed head thrashing behavior. This may suggest that the alterations in dendritic morphology observed with Form3 or INF2-FH1-FH2

overexpression in CIV neurons may impair normal processing of thermal stimuli resulting in an aberrant behavioral response.

Given the causative role of INF2 mutations in CMT disease and the observance of impaired distal sensation to thermal stimuli in CMT patients, we next tested the hypothesis that introduction of the INF2-FH1-FH2 transgene into the form3-IR mutant background will rescue the impaired behavioral responses to thermal nociceptive stimuli. Consistent with our hypothesis, we found that CIV expression of INF2-FH1-FH2 significantly rescued that behavioral latency defects observed in form3 knockdown larvae leading to an increase in the percentage of behavioral responders (Fig. 4-3D-F). While the introduction of INF2-FH1-FH2 only partially rescues the behavioral defects, it nonetheless supports a conserved role for Form3 and INF2 in regulating peripheral sensitivity to nociceptive stimuli.

Figure 4-4 form3 disruption in CIV neurons severely impairs heat-evoked nociceptive rolling behavior which can be rescued by introduction of INF2- FH1-FH2.

(A-D) Representative stills of noxious heat-evoked rolling behavior for the designated genotypes. (E) Latency to roll in seconds for the designated genotypes at 45˚C. (F) Percent responders for the designated genotypes at 45˚C. The number of larvae examined for quantitative analyses is indicated on the bar plot (F).

4.2.4 Future Directions

To assess the functional role(s) of Form3 in this behavior, we will examine whether Form3 is required for the sensory transduction of noxious thermal stimuli or action potential (AP) propagation. To investigate this question, we will perform optogenetic activation studies in combination with CIV-specific form3 knockdown using the ultrafast Channelrhodopsin variant ChETA. We predict that if Form3 is required at the sensory transduction stage, then optogenetic activation of CIV neurons will bypass the form3 mutant defect and evoke the stereotypical rolling response, whereas if Form3 functions in AP propagation, then optogenetic activation will be insufficient to elicit the rolling behavior. To investigate how form3 mutation may affect Ca2+ dynamics in CIV neurons in response to noxious heat, we will specifically express UAS-GCaMP6

(a genetically encoded calcium indicator) in combination with form3 knock down in CIV neurons. We predict that Form3-mediated defects in dendritic cytoskeletal architecture will significantly diminish noxious heat-evoked Ca2+ responses as measured by changes in GCaMP6 fluorescence. Should we observe defects in peripheral motor neurons in form3 mutants, similar analyses can be conducted assessing alterations in Ca2+ dynamics in both motor neurons and their target muscles via intersectional expression strategies involving the GAL4-UAS and LexA-LexAop binary expression systems.

4.3 Materials and Methods

4.3.1 Drosophila strains

Drosophila stocks were maintained at 25°C and raised on standard cornmeal-molasses-

agar diet. Fly strains used in this study include: (1) GAL4477,UAS-mCD8::GFP/CyO,tubP-

GAL80;GAL4ppk.1.9,UAS-mCD8::GFP; (2) ,UAS-form3-IR; (3) UAS-form3; (4) UAS-INF2-FH1-

FH2; and (5) UAS-INF2-Full length.Oregon R was used as a wild-type strain and crosses were

performed at 29°C.