Fig 1.
GCaMP imaging reports neuronal activity.
A) Expression pattern of GCamP5 using the RRa-Gal4 driver line. Paired aCC neurons (solid arrow) show clear segmental expression whilst RP2 neurons (dashed arrow) show weaker expression and only in some segments. B) Sample recording of simultaneous loose-patch (lower trace) and calcium-imaging (upper trace) of the same aCC motoneuron. C) Spike count from loose-patch recordings of aCC neurons plotted against the area under the curve (AUC) of calcium-imaging from the same motoneuron (accumulated data from n = 5). The line of fit shown has an R2 = 0.572.
Fig 2.
Isolated VNCs show patterned GCaMP activity.
A) Expression pattern of GCaMP in the OK371-Gal4 (vGlut) driver line. Regions where motoneuron dendrites overlap were chosen as regions of interest (ROI) to monitor segmental calcium levels over time. A ROI was also drawn outside of the VNC for background normalization (upper left corner). The Drosophila VNC is symmetrical across the midline (dotted white line). L/R HS = left and right hand side, respectively. B, C) Activity patterns observed from the left and right hand side of the same VNC. Waves starting at the posterior end of the VNC (FW, forward locomotion) and waves starting at the anterior end of the VNC (BW, backward locomotion) are evident in both sides whilst unilateral activity (turning, only visible in panel B) is only seen one side (labelled LHS).
Fig 3.
Proconvulsants increase synchronicity of neuron activity.
A) GCaMP5 fluorescence from single aCC neurons from consecutive segments show hyperactive and synchronised pattern changes following 4 min application of 4-AP (3 mM). Dotted line shows period when 4-AP was perfused and downward arrow indicates time 4-AP entered the bath. The time difference results from the length of tubing between gravity-driven perfusion system and the recording chamber. B) PTX (5μM) perfusion similarly increases neuron activity. Dotted line shows period when PTX was perfused and downward arrow indicates time PTX entered the bath. C, D) Analysis of number of synchronised aCC cells before and after 4-AP (C) and PTX (D) application (values are mean ± sem for n = 6 and 5, respectively). Synchrony was compared between a fixed ROI (labelled brown in the inset) and compared to all ROIs (red box on inset) and with those on the ipsilateral (green box) or contralateral (blue box) sides.
Fig 4.
bss1 mutants show increased synchronicity of neuronal activity.
A) Representative calcium-imaging traces from wildtype aCC neurons in two adjacent segments (i.e. ipsilateral, see inset). B) Results of cross-correlation for the traces from the recording shown in A). The peak of the curve (at 800 ms) shows the lag time which presents the best alignment of both traces. C) Peaks plotted for recording from multiple preparations show the majority (black) peak outside of the region considered to show synchronicity. Those that are considered synchronous (i.e. occur within ≤ 199 ms) are shown in red. D) A calcium-imaging trace from two adjacent aCC neurons in the bss1 background. Coincident peaks are marked with dashed lines. E) Cross-correlation for the traces from the recording shown in D). The peak of the curve (at 0 ms) is indicative of synchronised activity. F) Peaks plotted from multiple bss1 preparations. Synchronised peaks are shown in red and asynchronous in black.
Table 1.
Summary of results showing degree of synchrony on aCC activity imaged using GCaMP.
Fig 5.
anticonvulsive compounds reduce INaP.
A) INaP is increased in bss1 relative to wildtype (WT). INaP was evoked using a voltage protocol that maintained the aCC neuronal membrane at 0mV to inactivate INaT before dropping to -30mV to measure maximal INaP. Treatment with rapamycin reduces INaP back to WT level. The three recordings shown were chosen because they have the same amplitude of INaT. The dotted lines are drawn as indictors only to show amplitudes of INaT and INaP in WT. B) Ratio for INaP/NaT recorded in aCC for bss1 raised in food containing either rapamycin, dipyramidole, isethionate, antipain or etopiside (concentrations as per Table 1). Values for untreated bss1 and WT are shown for reference. C) Ratio for INaP/NaT for bss1 following expression of RNAi transgenes targeting raptor, phosphodiesterase 11 (PDE11), CDK4, serine-type peptidase (SP) or topoisomerase II (TopII). Values are means ± sem.