Impaired Pre-Motor Circuit Activity and Movement in a Drosophila Model of KCNMA1-Linked Dyskinesia

Abstract

Background: Paroxysmal dyskinesias (PxDs) are characterized by involuntary movements and altered pre-motor circuit activity. Causative mutations provide a means to understand the molecular basis of PxDs. Yet in many cases, animal models harboring corresponding mutations are lacking. Here we utilize the fruit fly, Drosophila, to study a PxD linked to a gain-of-function (GOF) mutation in the KCNMA1/hSlo1 BK potassium channel.

Objectives: We aimed to recreate the equivalent BK (big potassium) channel mutation in Drosophila. We sought to determine how this mutation altered action potentials (APs) and synaptic release in vivo; to test whether this mutation disrupted pre-motor circuit function and locomotion; and to define neural circuits involved in locomotor disruption.

Methods: We generated a knock-in Drosophila model using homologous recombination. We used electrophysiological recordings and calcium-imaging to assess AP shape, neurotransmission, and the activity of the larval pre-motor central pattern generator (CPG). We used video-tracking and automated systems to measure movement, and developed a genetic method to limit BK channel expression to defined circuits.

Results: Neuronal APs exhibited reduced width and an enhanced afterhyperpolarization in the PxD model. We identified calcium-dependent reductions in neurotransmitter release, dysfunction of the CPG, and corresponding alterations in movement, in model larvae. Finally, we observed aberrant locomotion and dyskinesia-like movements in adult model flies, and partially mapped the impact of GOF BK channels on movement to cholinergic neurons.

Conclusion: Our model supports a link between BK channel GOF and hyperkinetic movements, and provides a platform to dissect the mechanistic basis of PxDs. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Keywords: BK channel; Drosophila; central pattern generator; locomotion; paroxysmal dyskinesia; pre-motor circuit; slowpoke.

FIG. 1

(A) Alignment of residues surrounding hSlo1 D434 (arrow) with orthologous BK α‐subunits from bilateral species spanning >540 million years of evolutionary divergence. (B) Schematic illustrating the procedure to generate the slo ^E366G and slo ^loxP alleles via ends‐out homologous recombination. The region surrounding exon 10 of the slo locus, which encodes the E366 residue, and corresponding targeting arms to induce homologous recombination, are shown. (C) Sanger sequence verification of the presence or absence of the A > G mutation in slo ^E366G/+ and slo ^loxP/+ flies via allele‐specific polymerase chain reaction (PCR). (D) RNAseq‐based quantification of slo ⁺ (GAG) and slo ^E366G (GGG) mRNAs from slo ^E366G/+ heterozygous head tissue. (E) Illustration of location of SLO‐positive axonal tracts (arrowheads in F) in the adult nervous system. CNS, central nervous system. (F) SLO channel expression in the slo ^loxP/+, slo ^E366G/+, and slo null (slo ⁴) backgrounds, imaged in region noted in (E). Scale, 20 μm. (G) Illustration showing morphology of large ventral lateral neurons (l‐LN_vs) labeled with PDF promoter‐driven RFP (PDF::RFP) and location of patch‐clamp recording sites. (H) Average action potential (AP) waveforms in l‐LN_vs. Darker and lighter shades show mean and standard error of the mean (SEM). (I–L) l‐LN_v AP and afterhyperpolarization (AHP) parameters. Values of n are noted. Error bars: 95% confidence interval (CI). *P< 0.05, **P < 0.005, ***P < 0.0005, ns – P > 0.05, unpaired t‐test with Welch's correction (D, I, J, L), Mann–Whitney U test (K). [Color figure can be viewed at wileyonlinelibrary.com]

FIG. 2

(A) Illustration of the electrophysiological protocol used in the larval preparation. A sharp intramuscular recording electrode records from abdominal segment A3 of the longitudinal body wall muscle 6. Motoneurons innervating the body wall muscles are severed just below the ventral nerve cord (VNC) and excitatory junction potentials (EJPs) are evoked by stimulating the severed end of the motoneurons innervating muscle 6, A3. Abdominal segments A2−A8 are shown. NMJ, neuromuscular junction. (B–E) Representative EJPs from slo ^loxP/+ and slo ^E366G/+ larvae at high and low [Ca²⁺]_e. (F) Mean EJP amplitudes at various [Ca²⁺]_e in slo ^loxP/+ and slo ^E366G/+ larvae (x‐axis shown as log₁₀). (G–H) Mean mEJP amplitude (G) and inter‐event interval (H) across a range of [Ca²⁺]_e. (I) Representative mEJPs from slo ^loxP/+ and slo ^E366G/+ larvae at different [Ca²⁺]_e. (J–M) Representative paired‐pulse waveforms across a range of [Ca²⁺]_e. PPF, paired‐pulse facilitation. (N) Paired‐pulse ratio shown as EJP2/EJP1 at various [Ca²⁺]_e in slo ^loxP/+ and slo ^E366G/+ larvae (x‐axis shown as log₁₀). Values of n are noted. Error bars: mean ± SEM. *P < 0.05, **P < 0.005, ns – P > 0.05, two‐way ANOVA with Sidak's multiple comparisons test (F, N). ns – P > 0.05, Mann–Whitney U test (G, H). [Color figure can be viewed at wileyonlinelibrary.com]

FIG. 3

(A) Illustration of GCaMP6m‐labeled motoneuron cell bodies and dendritic regions in the ventral nerve cord (VNC) and location of recording area around motoneurons in the abdominal A4 segment of the VNC. (B) Representative images showing GCaMP6m‐labeled motoneuron cell bodies and dendrites in the VNCs of slo ^loxP/+ and slo ^E366G/+ larvae, and location of motoneuron dendrites on the left‐ (LHS) and right‐hand side (RHS) of abdominal segments 4–7. (C, D) Representative traces of GCaMP6m fluorescence over 300 seconds in slo ^loxP/+ (C) and slo ^E366G/+ (D) motoneuron dendrites within abdominal segment 4. Arrows in C indicate fictive turns, where the LHS and RHS motoneuron dendrites exhibit opposing patterns of excitation. (E, F) Line‐based kymographs showing rhythmic alterations in GCaMP6m fluorescence within dendritic domains of slo ^loxP/+ (E) and slo ^E366G/+ (F) motoneurons in abdominal segments 4–7. (G–J) Parameters of fictive locomotor patterns. Values of n are shown. Data are derived from n = 11 slo ^loxP/+ and slo ^E366G/+ larvae. (K) Illustration of the electrophysiological protocol used. Motoneuron axons innervating the body wall are left intact. Postsynaptic excitatory junction potentials (EJPs) are thus elicited via activation of motoneurons by the upstream larval central pattern generator (CPG). (L–M) Representative traces of spontaneous firing from slo ^loxP/+ (L) and slo ^E366G/+ (M) larvae. (N) Percentage of slo ^loxP/+ and slo ^E366G/+ larvae showing burst firing, irregular firing, or no firing. (O) Number of bursts in 5 minutes of recording. (P) Duration of bursts, only including recordings in which at least one burst occurred (derived from n = 11 larvae). Error bars: mean and 95% confidence interval (CI). *P < 0.05, ***P < 0.0005, Mann–Whitney U test (G–J, O) or unpaired t‐test with Welch's correction (P). [Color figure can be viewed at wileyonlinelibrary.com]

FIG. 4

(A‐C) Schematics of phone‐based video‐tracking of Drosophila larvae foraging across an agar plate (A), the DART (Drosophila Arousal Tracking) video‐tracking system (B; DAC: digital to analog converter), and the DAM system (C), which counts breaks of an infrared beam bisecting a DAM monitor. (D) Overlaid traces of foraging paths traveled by individual slo ^loxP/+ (n = 26) and slo ^E366G/+ (n = 28) L3 larvae during 1 minute of free movement. (E, F) Mean distance traveled (E) and number of turns (F) over 1 minute between slo ^E366G/+ and slo ^loxP/+ L3 larvae. (G, H) Overall movement in adult male slo ^loxP/+ and slo ^E366G/+ flies measured by the DART (G) and DAM (Drosophila Activity Monitor) (H) systems. (I, J) Number (I) and duration (J) of movement bouts during 0–1 hours following lights‐on in slo ^loxP/+ and slo ^E366G/+ adult male flies. (K) Cumulative distribution of speeds in slo ^loxP/+ and slo ^E366G/+ males during 0–1 hours following lights‐on. Data are derived from 100 slo ^loxP/+ and 94 slo ^E366G/+ males. (L) Plot of locomotor speed in slo ^loxP/+ and slo ^E366G/+ males before and after an applied mechanical stimulus (red dotted line). (M) Peak speed in slo ^loxP/+ and slo ^E366G/+ males in the 1‐minute period following a mechanical stimulus. Values of n are shown. Error bars: mean and 95% confidence interval (CI). **P < 0.005, ***P < 0.0005, unpaired t‐test with Welch's correction (E, G), Mann–Whitney U test (F, H–J, M), or Kolmogorov–Smirnov test (K). [Color figure can be viewed at wileyonlinelibrary.com]

FIG. 5

(A) Model of the dysc‐based system for indirect, cell‐specific control of SLO E366G expression. (B) Locomotor activity in slo ^E366G/+ and slo ^loxP/+ adult males with wild‐type or loss‐of‐function (dysc ^s168) dysc alleles. (C, D) Cell type‐specific restoration of DYSC in slo, dysc double mutants. Cell types associated with each promoter‐Gal4 driver are shown. Columns of blue circles denote the experimental genotypes in each dataset. Smaller dots in each graph represent measurements derived from individual adults. Values of n are noted. Error bars: mean and 95% confidence interval (CI). **P < 0.005, ***P < 0.0005, Kruskal−Wallis test with Dunn's post hoc test. [Color figure can be viewed at wileyonlinelibrary.com]