Postnatal Increases in Axonal Conduction Velocity of an Identified Drosophila Interneuron Require Fast Sodium, L-Type Calcium and Shaker Potassium Channels

GF conduction velocity increases over the first day of adult life

On each side of the Drosophila CNS the GFS consists of a GF interneuron that receives sensory information to its dendrites located in the brain and relays this information through a descending axon to two thoracic motor sub-circuits: the GF-TTM system, controlling the extension of mesothoracic legs during escape jumping, and the GF-DLMs system, controlling wing downstroke during flight initiation (Fig. 1A). In the GF-TTM and GF-DLM pathways, the GF makes mixed electrical and chemical (cholinergic) synapses with the TTM jumping motoneuron (TTMn) and the peripherally synapsing interneuron (PSI; Phelan et al., 1996; Sun and Wyman, 1996; Allen et al., 1999; Blagburn et al., 1999; Allen and Murphey, 2007). The PSI synapses directly onto the axons of the five motoneurons (MN1–5) that innervate the DLMs. MN1–4 innervate the ventral-most four DLM fibers, whereas MN5 innervates the two dorsalmost DLM fibers (Fig. 1A; King and Wyman, 1980; Koto et al., 1981; Ikeda and Koenig, 1988; Sun and Wyman, 1997; Koenig and Ikeda, 2005; Allen et al., 2006). The GF to DLM flight muscle pathway comprises one synapse (PSI to MN1–5) more than the GF to TTM pathway (Fig. 1A), thus adding the time for one chemical synaptic transmission onto the signal delay between GF and muscle. Therefore, the typical escape response is a jump followed by flight.

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Figure 1.

The GF axonal conduction velocity increases 80% during postnatal maturation. A, GFS schematic depiction at 1hPE (Ai) and 24hPE (Aii) control flies. Average latency ± SEM for GF-DLM5–6, GF-TTM, MN5-DLM5–6, and TTMn-TTM branches are indicated between dotted lines. Lower boxes on GF indicate the latency in the GF axon. Upper boxes on GF indicates the GF axonal speed. The percentage of the GF speeding (80%) during the first day of the fly life is presented in the gray-black arrow. B, Ten overlapping sweeps of APs recorded from DLM5–6 muscles, after GF (Bi) or MN5 (Bii) stimulation, and from TTM muscle, after GF (Biii) or TTMn (Biv) stimulation, at 1hPE (gray) and 24hPE (black) flies. Times above double arrows indicate the latency, which is measured as the interval of time between the stimulus artifact (black arrow) and the onset of the initial phase of muscle potential (white arrow). Underlined time values show the difference in latency measurements between the two stages. C, Latency measurements in the GF-DLM5–6 and GF-TTM pathways (Ci), in the MN5-DLM5–6 and TTMn-TTM sub-pathways (Cii), in the GF axon (Ciii), and measurements of the GF axonal speed (Civ), at 1hPE (50% black), 12hPE (70% black), and 24hPE or 48hPE (black) flies. Underlined time values between dotted arrows indicate the latency difference between1hPE and 24hPE. Data are shown as means ± SEM (Ci–Ciii). Dots on box plots showcase the measurements from individual flies (Civ). Asterisks indicate p values from one-way ANOVA with post hoc Dunnett’s tests (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., p > 0.19). St, stimulation site; Re, recording site; S, stimulus; R, record.

To investigate whether the GF interneuron and/or the downstream motor circuits are subject to functional changes during early postnatal life, circuit performance was compared by electrophysiological recordings in newly eclosed (1hPE) and 1-d-old mature flies (24hPE). We bypassed all sensory neurons and synaptic input computation times in the GF dendrites by direct electrical stimulation of the GF interneuron. Electrical stimulation of the GF elicits a muscle potential in the TTM and in the DLM5–6 muscles (Fig. 1Ai,Aii). The response latency (the time between GF stimulation and muscle potential; Fig. 1B) is the sum of the durations for AP conduction through the GF, PSI, and respective motoneuron axons, plus the time for synaptic transmission. As stated above, for the DLM branch, the PSI to MN5 synaptic delay adds to this latency (Tanouye and Wyman, 1980; Engel and Wu, 1992; Kadas et al., 2012). Therefore the latency of the GF-DLM5–6 (1.45 ± 0.03 ms) pathway is significantly (statistical test and p values are provided in figure legends) longer than that of the GF-TTM (1.13 ± 0.02 ms) pathway (Fig. 1Aii,Bi,Biii,Ci), although the net effect size is only ∼0.3 ms, indicating that fast chemical synaptic transmission between PSI and MN5 takes only about one third of a millisecond. Please note that synaptic transmission between the GF and the TTMn is dominated by the electrical component of the mixed synapse. If the electrical component is blocked, the chemical component of the GF/TTMn synapse increases the latency in the GF-TTM pathway almost to the level of the GF-DLM5–6 pathway (Allen and Murphey, 2007; Pézier et al., 2016).

Comparing the response latency of the GF-DLM5–6 branch between 1-h-old and 1-d-old flies reveals a significant shortening during the first day of postnatal life from 1.66 ± 0.02 ms at 1hPE to 1.45 ± 0.03 ms at 24hPE (Fig. 1Ai,Aii,Bi,Ci). Similarly, 1-d-old flies exhibit a shorter GF-TTM latency response (1.13 ± 0.02 ms) in comparison to 1-h-old flies (1.32 ± 0.02 ms; Fig.1Ai,Aii,Biii,Ci). Therefore, during the first day of adult life (1–24hPE), response latency of both branches of the GFS is decreased by ∼0.2 ms, which equals to an improvement of 14% for the GF-DLM branch (0.21 ms) and 17% for the GF-TTM branch (0.19 ms; Fig. 1Bi,Biii,Ci). Additional measurements at 48 h post-eclosion indicated that postnatal maturation was completed by 24 h because response latency did not further decrease at 48 h (Fig. 1Ciii,Civ). Measurements at 12 h revealed intermediated values as compared to 1 and 24 h post-eclosion (Fig. 1Ciii,Civ)., thus indicating that conduction velocity is likely steadily increased during the first day of adult life. Although net effect size is only ∼0.2 ms, this increase in conduction velocity may be of biological relevance considering that it is close to the normal difference of information processing speed through the GF-TTM versus the GF-DLM path, which ensures that jumping precedes flight initiation (The GF to DLM flight muscle pathway comprises one synapse (PSI to MN1–5) more than the GF to TTM pathway (Fig. 1A), thus adding the time for one chemical synaptic transmission onto the signal delay between GF and muscle. Therefore, the typical escape response is a jump followed by flight.). A similar net decrease in latency in both GFS branches seems important to maintain the time difference in jump and succeeding flight initiation, and it indicates functional maturation of common circuit elements (axons/synapses). We next aimed at pinpointing the cellular site that underlies the postnatal increases in information transfer.

To test for potential maturation of the motoneurons and/or the neuromuscular synapses, we stimulated the motoneurons directly by inserting tungsten electrodes into the thoracic nerve cord and recorded the responses from the DLM and the TTM muscle, respectively. The latency between motoneuron activation and muscles responses of 1-h-old flies was not significantly different from that of 1-d-old flies, for both, the DLM branch (1hPE, 0.95 ± 0.03 ms and 24hPE, 0.97 ± 0.03 ms; Fig. 1A,Bii,Cii) and the TTM branch (1hPE, 0.80 ± 0.04 ms and 24hPE, 0.84 ± 0.02 ms; Fig. 1A,Biv,Cii). Hence, motoneuron axonal conduction speed and neuromuscular transmission delay do not undergo postnatal changes. This leaves GF axonal conduction speed as cause for postnatal speeding of GF.

The AP conduction time in the GF axon can be estimated by subtracting the TTMn-to-TTM latency from the GF-to-TTM latency. Note that the GF to TTMn synapse is dominated by electrical transmission and, thus, does not add notable time to the latency. According to this calculation the GF axonal conduction duration decreased by 80% during the first day of postnatal period, from 0.52 ± 0.04 ms in 1hPE flies to 0.29 ± 0.03 ms in 24hPE flies (∼0.23 ms; Fig. 1A,Ciii; Table 1). Considering that the axon of the GF is ∼0.6 mm long, this equals to an increase in axonal conduction velocity from 1.15 ± 0.09 m/s at 1hPE to 2.07 ± 0.21 m/s in 24PE flies (Fig. 1A,Civ; Table 2). We next aimed at addressing the ionic basis of this postnatal increase in axonal conduction velocity of the GF interneuron.

Voltage-gated sodium and L-type calcium channels contribute to the GF conduction velocity increase postnatally

Potential mechanisms underlying postnatal increases in axonal conduction velocity are (1) an enlargement in axon diameter (this would result in decreased axial resistance, thus increasing the length constant λ; Hodgkin, 1954); (2) improving glial wrapping (this would result in increased membrane resistance, thus increasing λ; Moore et al., 1978); and (3) hypothetically, changes in the complement, properties, and/or densities of ion channels located in the axonal membrane. First, axon diameter changes can be ruled unlikely because it has been suggested that the axon of the GF reaches its final diameter before eclosion (Allen et al., 1998). Indeed, confocal images demonstrate that the GF outer axon diameter (∼7 μm) is not different between 1hPE and 24hPE flies (Fig. 2). But note that CLSM just uncovers the outer axon diameter. EM would be required to rule out small changes in the effective inner diameter that determines axoplasmatic resistance. However, we feel that subtle differences in effective axon diameter that might be hidden to CLSM diameter measurements are difficult to reconcile with the observed 80% increase in conduction velocity. Second, the GF is a non-myelinated invertebrate axon, thus making major effects of glial insulation on AP conduction speed unlikely. Please note that GF axonal conduction velocity is roughly similar to that of unmyelinated C-fibers in vertebrates (see Discussion). Therefore, we hypothesized that postnatal changes in the expression of axonally localized voltage-gated ion channels may underlie postnatal AP conduction velocity increases in the GF.

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Figure 2.

The GF axon diameter does not increase postnatally, and GFP-RNAi decreases GFP fluorescence similarly at 1 h and 24 h post-eclosion. A, Representative confocal image stacks of the GF interneuron axons between the neck connectives and the terminals in the VNC at 1hPE (A) and at 24hPE (B). To avoid potential histology artifacts images were taken live in saline with a 60× water dipping lens from freshly dissected animals with UAS-cd4-tomato expression in the GF. White boxes indicate areas shown as selective enlargements in Ai, Bi. C, Quantification from 14 axons at each stage shows that GF axon diameter is similar at 1hPE and 24hPE (p > 0.6, Student’s t test). VNC, ventral nerve cord. D–G, In comparison to control (D), targeted GFP RNAi knock-down reduces GFP fluorescence at both stages tested, 24hPE (E) and 1hPE (F). G, Quantification of mean gray levels in eight bit tiff images of the GF axon (0–254 gray levels) reveals a significant reduction by ∼70% but no differences between both stages tested (ANOVA with Newman–Keuls post hoc testing); ***p < 0.001; n.s., p > 0.3.

To test this, we manipulated the expression levels of selected ion channels specifically in the GF by targeting UAS-transgenes with a split-GAL4 driver that expresses solely in the GF interneurons (von Reyn et al., 2014), and compared axonal conduction velocity at 1 h (1hPE) and at 24 h (24hPE) post adult eclosion. This approach ideally requires identical levels of RNAi knock-down efficacy in the GF interneuron at both stages. To exclude largely different expression levels of Gal4 at both staged, we estimated knock-down efficacy in the GF by expressing UAS-RNAi for GFP under the control of the GF selective split-GAL4;UAS-mcd8-GFP driver and compared fluorescence intensity at both stages (Fig. 2D–G). At 1hPE, targeted expression of UAS-GFP-RNAi decreased mean GFP fluorescence intensity in the GF interneuron highly significantly (Fig. 2D,F) by ∼70% (Fig. 2G), thus demonstrating significant effects of RNAi at 1hPE. GFP fluorescence was not further decreased at 24hPE (Fig. 2D,F,G). Although this approach provides only a rough estimate of RNAi knock-down efficacy, and generalization for different RNAi constructs is not readily possible, these data indicate that UAS-RNAi expression in the GF may provide roughly similar knock-down efficacy at 1hPE and at 24hPE. However, this does not exclude different knock-down efficacies of UAS-RNAi constructs for different ion channels. Four of the five UAS-RNAi constructs for different ion channels used in this study (see below) have previously been confirmed to cause 50–70% knock-down efficacy in larval Drosophila motoneurons by means of electrophysiology and/or immunocytochemistry (see Materials and Methods). Taken together, it seems plausible to assume that each RNAi constructs used caused knock-down of the respective channels in the GF neuron, and that for a given ion channel knock-down efficacy was similar at both stages tested.

We first tested the contribution of voltage-gated sodium channels for axonal conduction velocity. Fast sodium channels are required for AP generation and propagation in the GF (Tanouye et al., 1981; Tanouye and Ferrus, 1985; von Reyn et al., 2014). The only gene encoding fast voltage-gated sodium channels in Drosophila is paralytic (para) or DmNa_v (Feng et al., 1995; Mee et al., 2004). We lowered the amount of para expression in the GF interneuron by targeted expression of para-RNAi. For both stages tested (1hPE and 24hPE), this resulted in a significant decrease in axonal conduction velocity in the GF interneuron (Fig. 3A,B), which was calculated from measurements of the response latency increases in the GF-TTM pathway with and without para RNAi (Fig. 3Bi,Biii,Ci). The latency between TTMn and TTM was not affected because the RNAi knock-down was targeted to the GF only (Fig. 3Bii,Biv,Cii). Keeping in mind that the GF axonal conduction time equals the time difference between the GF-TTM and the TTMn-TTM latencies, para RNAi knock-down in the GF increases axonal conduction time from 0.52 ± 0.04 to 0.69 ± 0.04 ms at 1hPE and from 0.29 ± 0.03 to 0.47 ± 0.03 ms at 24hPE (Fig. 3Ciii; Table 1). This corresponds to a 32% decrease in GF axonal conduction velocity at 1hPE (1.15 ± 0.09 m/s in control to 0.87 ± 0.05 m/s in para RNAi knock-down) but in a 62% decrease in conduction velocity at 24hPE (from 2.07 ± 0.21 m/s in control to 1.28 ± 0.08 m/s in para RNAi knock-down; Fig. 3A,Civ; Table 2). Therefore, para RNAi had a stronger effect in more mature GF interneurons. However, even in the presence of para RNAi we found a postnatal decrease in GF conduction time of 0.21 ms (from 0.69 ± 0.04 to 0.47 ± 0.03 ms; Fig. 3Ciii; Table 1), corresponding to a postnatal speeding of only 47% (Fig. 3Aii; Table 2), roughly half the postnatal speeding found in control (80%; Figs. 3Ai; Table 2).

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Figure 3.

Para-RNAi decreases postnatal conduction velocity speeding in the GF. A, GF schematic depiction at 1hPE (gray) and 24hPE (black) in controls (Ai) as compared to 1hPE (orange) and 24hPE (red) in flies with para RNAi (Aii). GF axonal conduction velocity (dotted arrow) is given for each experimental group. The percentage of the GF postnatal speeding in control (gray-black arrow, 80%) is strongly reduced by para RNAi knock-down (orange-red arrow, 47%). B, Representative TTM muscle APs recorded after GF (Bi) or TTMn (Bii) stimulation at 1hPE (gray) and at 24hPE (black) in control flies as compared to para RNAi knock-down (Biii, GF stimulation; Biv, TTMn stimulation) at 1hPE (orange) and at 24hPE (red). Times above double arrows and between dotted lines indicate the latency between GF (or TTMn) and TTM. Latency differences between stages are underlined for control and for para RNAi knock-down. C, Latency measurements in the GF-TTM pathway (Ci), in the TTMn-TTM sub-pathway (Cii), in the GF axon (Ciii) and measurements of the GF axonal speed (Civ), at 1hPE (gray) and 24hPE (black) in controls and at 1hPE (orange) and 24hPE (red) in flies expressing para RNAi transgene. Underlined times between dotted arrows indicate the latency difference between the two stages in control and para RNAi knock-down. Data are shown as means ± SEM (Ci–Ciii). Dots on box plots showcase the measurements from individual flies (Civ). Asterisks indicate p values from one-way ANOVA with post hoc Dunnett’s tests (*p < 0.05, ***p < 0.001, ****p < 0.0001, n.s., p > 0.29).

Since GF AP conduction velocity was decreased on lowering the amount of Para channels, we hypothesized that conversely, an upregulation of voltage-gated sodium channel expression will increase GF axonal conduction velocity. Although Drosophila Para channels have been functionally characterized in Xenopus oocytes (Lin et al., 2009), UAS-para transgenes have so far not been successfully expressed in flies (Lin and Baines, 2015). Therefore, we drove expression of a transgene encoding the bacterial sodium channels, NaChBac, specifically in the GF (GF split-Gal4). Functional sodium current through NaChBac expressed in Drosophila neurons and muscles has previously been demonstrated (Luan et al., 2006; Nitabach et al., 2006). We predicted that an increase in the total number of (bacterial + native) sodium channels will increase axonal conduction velocity. Indeed, for both stages tested (1hPE and 24hPE), the latency in the GF-TTM pathway was significantly lower in flies expressing the NaChBac transgene than in control ones (Fig. 4Bi,Biii,Ci). Again, given that expression was restricted to the GF interneuron, the latency between TTMn and TTM remained unchanged following NaChBac transgene expression (Fig. 4Bii,Biv,Cii). Thus, in contrast to the increased axonal conduction time on para RNAi knock-down, the expression of extra sodium channels significantly decreased conduction time in the GF axon of 1hPE flies (0.52 ± 0.04 vs 0.35 ± 0.02 ms) and of 24hPE flies (0.29 ± 0.03 vs 0.16 ± 0.03 ms; Fig. 4Ciii; Table 1), and therefore, increased GF conduction speed (1.15 ± 0.09 vs 1.71 ± 0.10 m/s) at 1hPE and (2.07 ± 0.21 vs 3.75 ± 0.70 m/s) at 24hPE (Fig. 4A,Civ). Therefore, with extra sodium (NaChBac) channel expression, the postnatal increase in AP conduction velocity is further enhanced to 119% (Fig. 4Aii; Table 2), as compared to the 80% enhancement in control (Figs. 4Ai, 8; Table 2). To sum up, sodium channels control axonal conduction speed in the GF and may contribute to postnatal maturation.

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Figure 4.

Expression of extra sodium channels (NaChBac) increases postnatal conduction velocity speeding in the GF. A, GF schematic depiction at 1hPE (gray) and 24hPE (black) in controls (Ai) as compared to 1hPE (light orange) and 24hPE (neon red) in flies expressing NaChBac channels (Aii). GF axonal conduction velocity (dotted arrow) is given for each experimental group. The percentage of the GF postnatal speeding in control (gray-black arrow, 80%) is strongly increased by NaChBac channels expression (light orange-neon red, 119%). B, Representative TTM muscle APs recorded after GF (Bi) or TTMn (Bii) stimulation at 1hPE (gray) and at 24hPE (black) in control flies as compared to flies expressing NaChBac channels (Biii, GF stimulation; Biv, TTMn stimulation) at 1hPE (light orange) and at 24hPE (neon red). Times above double arrows and between dotted lines indicate the latency between GF (or TTMn) and TTM. Latency differences between stages are underlined for control and for NaChBac channels expression. C, Latency measurements in the GF-TTM pathway (Ci), in the TTMn-TTM sub-pathway (Cii), in the GF axon (Ciii) and measurements of the GF axonal speed (Civ), at 1hPE (gray) and 24hPE (black) in controls and at 1hPE (light orange) and 24hPE (neon red) in flies expressing NaChBac transgene. Underlined times between dotted arrows indicate the latency difference between the two stages in control and flies expressing NaChBac sodium channels. Data are shown as means ± SEM (Ci–Ciii). Dots on box plots showcase the measurements from individual flies (Civ). Asterisks indicate p values from one-way ANOVA with post hoc Dunnett’s tests (**p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., p > 0.15).

We next tested possible contributions of other voltage-gated ion channels localized in the axon. It has recently been demonstrated that L-type calcium channels encoded by Dmca1D localize to motoneuron axons, where they augment high frequency firing (Kadas et al., 2017). Targeted DmCa1D RNAi knock-down, specifically in the GF, dramatically increased response latency in the GF-TTM pathway at both stages examined (Fig. 5Bi,Biii,Ci). Given that the TTMn and its neuromuscular junction with TTM (TTMn/TTM) do not contribute to that change in response latency (Fig. 5Bii,Biv,Cii), the data demonstrate that DmCa1D RNAi knock-down significantly increases GF axonal conduction time, both at 1hPE (0.52 ± 0.04 vs 0.73 ± 0.05 ms) and at 24hPE (0.29 ± 0.03 vs 0.64 ± 0.04 ms; Fig. 5Ciii; Table 1). Noticeably, this corresponds to a decrease in GF axonal conduction speed of 40% (1.15 ± 0.09 vs 0.82 ± 0.06 m/s) at 1hPE and a much higher decrease of 120% (2.07 ± 0.21 vs 0.94 ± 0.06 m/s) at 24hPE (Fig. 5A,Civ; Table 2). Consequently, in DmCa1D RNAi knock-down flies postnatal speeding of GF axonal conduction velocity was reduced to just 15% (Fig. 5Aii; Table 2), as compared to 80% in control (Fig. 4Ai, 8; Table 2). Hence, in addition to reducing conduction speed at both stages tested, DmCa1D plays a major role in postnatal conduction velocity speeding.

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Figure 5.

DmCa1D-RNAi almost eliminates postnatal conduction velocity speeding in the GF. A, GF schematic depiction at 1hPE (gray) and 24hPE (black) in controls (Ai) as compared to 1hPE (cyan) and 24hPE (blue) in flies with DmCa1D RNAi (Aii). GF axonal conduction velocity (dotted arrow) is given for each experimental group. The percentage of the GF postnatal speeding in control (gray-black arrow, 80%) is extremely reduced by DmCa1D RNAi knock-down (cyan-blue arrow, 15%). B, Representative TTM muscle APs recorded after GF (Bi) or TTMn (Bii) stimulation at 1hPE (gray) and at 24hPE (black) in control flies as compared to DmCa1D RNAi knock-down (Biii, GF stimulation; Biv, TTMn stimulation) at 1hPE (cyan) and at 24hPE (blue). Times above double arrows and between dotted lines indicate the latency between GF (or TTMn) and TTM. Latency differences between stages are underlined for control and for DmCa1D RNAi knock-down. C, Latency measurements in the GF-TTM pathway (Ci), in the TTMn-TTM sub-pathway (Cii), in the GF axon (Ciii) and measurements of the GF axonal speed (Civ), at 1hPE (gray) and 24hPE (black) in controls and at 1hPE (cyan) and 24hPE (blue) in flies expressing DmCa1D RNAi transgene. Underlined time values between dotted arrows indicate the latency difference between the two stages in control and DmCa1D RNAi knock-down. Data are shown as means ± SEM (Ci–Ciii). Dots on box plots showcase the measurements from individual flies (Civ). Asterisks indicate p values from one-way ANOVA with post hoc Dunnett’s tests (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., p > 0.05).

Shaker and Slowpoke potassium channels control the GF conduction velocity but only Shaker contributes to its postnatal increment

In addition to having identified two voltage-gated channels mediating inward current that increase AP conduction velocity, we also tested for potential roles of potassium outward currents. We tested the Drosophila homologs of the mammalian K_V1 and K_V4 voltage-gated potassium channels, such as Shaker and Shal, and the BK channel homolog Slowpoke. In Drosophila motoneurons, Shaker and Shal mediate fast activating, fast inactivating voltage-gated potassium currents (Ryglewski and Duch, 2009), while Slowpoke underlies transient calcium-activated potassium currents (Kadas et al., 2015). Expression of each of these potassium channels was reduced by selective expression of the respective UAS-RNAi transgenes specifically in the GF interneuron (von Reyn et al., 2014).

Axonal localization of Shaker potassium channel in Drosophila DLM flight MNs has previously been demonstrated by immunocytochemistry (Ryglewski and Duch, 2009). Although light microscopy lacks sufficient spatial resolution to unambiguously assign labeled ion channel proteins to the membrane of an identified neuron in situ, high resolution confocal laser microscopy of UAS-tomato expressing GF axons in animals with endogenously GFP-tagged Shaker channels suggested that Shaker channels likely localize to the GF axonal membrane (Fig. 6A–C). Representative projection views show the GF axons between the cervical connectives and their axon terminals in the thoracic neuromere, as well as many additional Shaker positive descending axons (Fig. 6A). Single optical sections (0.3-µm section thickness) reveal patches of GFP tagged Shaker channels that co-localize with segments of UAS-tomato positive GF axonal membrane (Fig. 6B,C), whereas the lumen of the large diameter GF axons is mostly devoid of Shaker label. This indicated functional Shaker channel localization to the GF axonal membrane.

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Figure 6.

Shaker channels are localized in the GF axon and Sh-RNAi decreases postnatal conduction velocity speeding. A–Aii, Projection views of a representative confocal image stack from the GF with UAS-cd4-tomato expression (red) and Shaker channel expression as visualized by an endogenous GFP tag (green). Dotted white boxes in Ai indicate the areas which are selectively enlarged in B, C and shown as z-projections of 1 µm (three optical sections). White arrowheads demark areas with overlap of patches of GF axonal membrane (red) and Shaker-GFP label. The GF axonal lumen is mostly devoid of Shaker-GFP label. D, GF schematic depiction at 1hPE (gray) and 24hPE (black) in controls (Di) as compared to 1hPE (violet) and 24hPE (purple) in flies with Sh RNAi (Dii). GF axonal conduction velocity (dotted arrow) is given for each experimental group. The percentage of the GF postnatal speeding in control (gray-black arrow, 80%) is strongly reduced by Sh RNAi knock-down (violet-purple arrow, 36%). E, Representative TTM muscle APs recorded after GF (Ei) or TTMn (Eii) stimulation at 1hPE (gray) and at 24hPE (black) in control flies as compared to Sh RNAi knock-down (Eiii, GF stimulation; Eiv, TTMn stimulation) at 1hPE (violet) and at 24hPE (purple). Times above double arrows and between dotted lines indicate the latency between GF (or TTMn) and TTM. Latency differences between stages are underlined for control and for Sh RNAi knock-down. F, Latency measurements in the GF-TTM pathway (Fi), in the TTMn-TTM sub-pathway (Fii), in the GF axon (Fiii), and measurements of the GF axonal speed (Fiv), at 1hPE (gray) and 24hPE (black) in controls and at 1hPE (violet) and 24hPE (purple) in flies expressing Sh RNAi. Underlined times between dotted arrows indicate the latency difference between the two stages in control and Sh RNAi knock-down. Data are shown as means ± SEM (Fi–Fiii). Dots on box plots showcase the measurements from individual flies (Fiv). Asterisks indicate p values from one-way ANOVA with post hoc Dunnett’s tests (*p < 0.05, ***p < 0.001, ****p < 0.0001, n.s., p > 0.15).

Targeted RNAi knock-down of shaker (sh) in the GF significantly increased the latency in the GF-TTM pathway at both stages, although the effect is smaller at 1hPE (Fig. 6Ei,Eiii,Fi). Again, motoneuron axonal conduction and neuromuscular transmission were not significantly affected by the expression of sh RNAi in the GF interneuron (Fig. 6Eii,Eiv,Fii). Sh RNAi knock-down in the GF of 1hPE flies did not cause a statistically significant change in the GF conduction time (0.56 ± 0.05 vs 0.52 ± 0.04 ms of control; Fig. 6Fiii; Table 1). By contrast, sh RNAi knock-down in the GF of 24hPE flies significantly enhanced the GF conduction time (0.29 ± 0.03 vs 0.41 ± 0.04 ms of controls; Fig. 6Fiii; Table 1). Consequently, sh RNAi knock-down decreased GF axonal conduction speed significantly at 24hPE (2.07 ± 0.21 vs 1.46 ± 0.14 m/s), but not at 1hPE (1.15 ± 0.09 vs 1.07 ± 0.10 m/s; Fig. 6A,Fiv). Given that sh RNAi knock-down reduced GF conduction at 24hPE by 42%, but only by 7% at 1hPE (Table 2), postnatal conduction speeding was only 36% (Fig. 6Dii; Table 2), as compared to 80% in control (Fig. 6Di; Table 2).

Targeted RNAi knock-down of shal in the GF caused a small but statistically significant increase of the latency recorded in the GF-TTM pathway at 1hPE (Fig. 7Bi,Biii,Ci), without affecting the latency between TTMn and TTM (Fig. 7Bii,Biv,Cii). By contrast, it did not significantly affect response latency of the GF-TTM pathway at 24hPE (Fig. 7Bi,Biii,Ci). Similar to sh RNAi knock-down, the almost negligible increase in response latency in the GF-TTM pathway at 1hPE did not account for a statistically significant increase in the GF conduction time (Fig. 7Ciii). Indeed, shal RNAi knock-down had only small effects on GF conduction velocity, a 6% at decreases at 1hPE (1.15 ± 0.09 vs 1.09 ± 0.06 m/s) and a 14% decrease at 24hPE (2.07 ± 0.21 vs 1.82 ± 0.17 m/s; Fig. 7A,Civ; Table 2). Therefore, Shal RNAi knock-down lowered postnatal speeding in GF conduction velocity only minimally, from 80% in controls (Figs. 7Ai; Table 2) to 67% (Fig. 7Aii; Table 2).

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Figure 7.

Shal-RNAi does not affect postnatal conduction velocity speeding in the GF. A, GF schematic depiction at 1hPE (gray) and 24hPE (black) in controls (Ai) as compared to 1hPE (violet) and 24hPE (purple) in flies with Shal RNAi (Aii). GF axonal conduction velocity (dotted arrow) is given for each experimental group. The percentage of the GF postnatal speeding in control (gray-black arrow, 80%) is not significantly reduced by Shal RNAi knock-down (violet-purple arrow, 67%). B, Representative TTM muscle APs recorded after GF (Bi) or TTMn (Bii) stimulation at 1hPE (gray) and at 24hPE (black) in control flies as compared to Shal RNAi knock-down (Biii, GF stimulation; Biv, TTMn stimulation) at 1hPE (violet) and at 24hPE (purple). Times above double arrows and between dotted lines indicate the latency between GF (or TTMn) and TTM. Latency differences between stages are underlined for control and for Shal RNAi knock-down. C, Latency measurements in the GF-TTM pathway (Ci), in the TTMn-TTM sub-pathway (Cii), in the GF axon (Ciii) and measurements of the GF axonal speed (Civ), at 1hPE (gray) and 24hPE (black) in controls and at 1hPE (violet) and 24hPE (purple) in flies expressing Shal RNAi transgene. Underlined time values between dotted arrows indicate the latency difference between the two stages in control and Shal RNAi knock-down. Data are shown as means ± SEM (Ci–Ciii). Dots on box plots showcase the measurements from individual flies (Civ). Asterisks indicate p values from one-way ANOVA with post hoc Dunnett’s tests (*p < 0.05, ***p < 0.001, ****p < 0.0001, n.s., p > 0.09).

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Figure 8.

Slo-RNAi affects slightly postnatal conduction velocity speeding in the GF. A, GF schematic depiction at 1hPE (gray) and 24hPE (black) in controls (Ai) as compared to 1hPE (mint green) and 24hPE (green) in flies with Slo RNAi (Aii). GF axonal conduction velocity (dotted arrow) is given for each experimental group. The percentage of the GF postnatal speeding in control (gray-black arrow, 80%) is moderately reduced by Slo RNAi knock-down (mint green-green arrow, 56%). B, Representative TTM muscle APs recorded after GF (Bi) or TTMn (Bii) stimulation at 1hPE (gray) and at 24hPE (black) in control flies as compared to Slo RNAi knock-down (Biii, GF stimulation; Biv, TTMn stimulation) at 1hPE (mint green) and at 24hPE (green). Times above double arrows and between dotted lines indicate the latency between GF (or TTMn) and TTM. Latency differences between stages are underlined for control and for Slo RNAi knock-down. C, Latency measurements in the GF-TTM pathway (Ci), in the TTMn-TTM sub-pathway (Cii), in the GF axon (Ciii) and measurements of the GF axonal speed (Civ), at 1hPE (gray) and 24hPE (black) in controls and at 1hPE (mint green) and 24hPE (green) in flies expressing Slo RNAi transgene. Underlined time values between dotted arrows indicate the latency difference between the two stages in control and Slo RNAi knock-down. Data are shown as means ± SEM (Ci–Ciii). Dots on box plots showcase the measurements from individual flies (Civ). Asterisks indicate p values from one-way ANOVA with post hoc Dunnett’s tests (**p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., p > 0.1).

Finally, RNAi knock-down of slowpoke (or slo) significantly increased GF-TTM pathway latency (Fig. 8Bi,Biii,Ci) and axonal conduction time in the GF (Fig. 8Ciii) at both stages tested (1hPE and 24hPE). Again, TTMn to TTM was not affected (Fig. 8Bii,Biv,Cii) by slo RNAi knock-down in the GF. Given that slo RNAi exerted a slightly stronger effect at 24hPE than at 1hPE (56% decrease in the GF conduction speed at 24hPE vs 35% at 1hPE; Table 2), the postnatal speeding of the GF conduction velocity in the presence of slo RNAi knock-down was reduced from 80% in control to 56% with slo RNAi expression (Fig. 8A,Civ; Table 2).

In summary, Shaker voltage-gated and Slowpoke calcium-activated potassium channels significantly increase axonal conduction velocity in the GF. However, in the presence of constant RNAi expression, postnatal speeding of the GF axonal conduction is affected much stronger with sh RNAi than with slo RNAi (see Fig. 6A vs Fig. 8A). Knock-down of para reduces postnatal speeding to a similar amount as sh RNAi (see Fig. 3A vs Fig. 6A). By contrast, targeted expression of Dmca1D RNAi eliminates postnatal speeding of axonal conduction velocity almost completely (Fig. 5A). In sum, postnatal upregulation of both Shaker outward and sodium inward current likely contributes to the postnatal increase of the GF axonal conduction speed, but up-regulation of L-type calcium channels seems most important.