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J Neurosci. Author manuscript; available in PMC 2008 Nov 7.
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PMCID: PMC2408742

The Dystrophin Dp186 Isoform Regulates Neurotransmitter Release at a Central Synapse in Drosophila


The Dystrophin protein is encoded by the gene whose mutation in humans underlies Duchennes muscular dystrophy, a disease characterized by progressive muscle wasting. A number of Duchenne patients also exhibit poorly understood mental retardation, likely associated with loss of a brain-specific isoform. Furthermore, although Dystrophin isoforms and the related Utrophin protein have long been known to localize at synapses, their functions remain largely unknown. In Drosophila, we find that the CNS-specific Dp186 isoform localizes to the embryonic and larval neuropiles, regions rich in synaptic contacts. In the absence of Dp186, evoked, but not spontaneous, presynaptic release is significantly enhanced. Increased presynaptic release can be fully rescued to wildtype levels by expression of a Dp186 transgene in the postsynaptic motoneuron, indicating that Dp186 likely regulates a retrograde signaling pathway. Potentiation of synaptic currents in the mutant also occurs when cholinergic transmission is inhibited or in the absence of Glass Bottom Boat (Gbb) or Wishful Thinking (Wit), a TGF-β ligand and receptor respectively, both previously implicated in synaptic retrograde signaling. These results are consistent with the possibility that Dp186 modulates other, non-Gbb/Wit dependent, retrograde signaling pathways required to maintain normal synaptic physiology.

Keywords: Dystrophin, Drosophila, retrograde signaling, CNS, gbb, interneuronal synapse


Mutations in the human dystrophin gene cause Duchenne muscular dystrophy (DMD), a common human genetic disease, characterized by progressive muscle wasting (Hoffman et al., 1987). Dystrophin is part of a large multi-protein complex, the Dystrophin glycoprotein complex (DGC, reviewed in Lapidos et al., 2004), which stabilizes the muscle during contraction by linking the actin cytoskeleton to the extracellular matrix. Dystrophin and associated proteins bind a variety of signaling molecules (reviewed in Rando, 2001), indicating that the DGC likely also acts as a scaffold for components of inter- and intracellular signaling pathways.

A third of DMD patients also present cognitive defects (reviewed in Anderson et al., 2002), raising the possibility that, in addition to their roles in the musculature, dystrophin and its partially functionally redundant ortholog, utrophin, also have critical functions in the nervous system. Consistent with this, several Dystrophin isoforms and Utrophin are localized to synaptic regions in the mammalian brain, retina and at the neuromuscular junction (NMJ) (reviewed in Blake et al., 2002). Further indications that dystrophin-like proteins play roles at a variety of synapses include the observations that a) Utrophin-deficient mice display NMJ structural abnormalities (Deconinck et al., 1997a; Grady et al., 1997a), b) NMJs lacking Dystrophin and Utrophin display disrupted AChR clustering (Deconinck et al., 1997b; Grady et al., 1997b), c) Dystrophin-deficient C. elegans animals are hyperactive due to the delocalization of an acetylcholine transporter and the subsequent failure to clear acetylcholine (Kim et al., 2004), d) absence of the post-synaptically localized Drosophila DLP2 Dystrophin isoform results in increased presynaptic neurotransmitter release at the larval NMJ (van der Plas et al., 2006) and e) mice lacking the large dystrophin isoform required for muscle integrity exhibit deficits in both long-term spatial and recognition memory (Vaillend et al., 2004). These results suggest that alterations in synaptic function, resulting from dystrophin deficiency, may underlie DMD-associated cognitive defects. The precise roles of Dystrophin at any synapse, however, remain to be elucidated.

We have employed Drosophila melanogaster, with its amenability to genetic analysis and highly orthologous single dystrophin gene, to examine the roles of Dystrophin at a central synapse. Here, we describe a role of the CNS-specific Dp186 isoform at an identified motoneuron-interneuron synapse. We found that decreased post-synaptic expression of Dp186 results in increased evoked, but not spontaneous, presynaptic neurotransmitter release, similarly to that observed in a previous study where the absence of the Dystrophin DLP2 isoform from the muscle resulted in increased neurotransmitter release by the motoneuron. However, retrograde modulation of presynaptic release by Dp186 at this central synapse did not require the TGF-β signaling pathway previously implicated in DLP2-mediated modulation at the neuromuscular junction. Thus, although the Dp186 and DLP2 Dystrophin isoforms are required to maintain wildtype neurotransmitter release levels at the interneuronal synapse and NMJ, respectively, they apparently do so via different signaling pathways.

Materials and Methods

Fly stocks

Flies were fed on apple juice agar supplemented with yeast. Wildtype was Canton-S. RN2-Gal4 was used to selectively express UAS driven transgenes in aCC and RP2 (Baines, 2003; Fujioka et al., 2003; Baines, 2004). Expression of RN2-Gal4 begins in early stage 16 embryos, preceding the onset of synaptogenesis (Baines and Bate, 1998). B19-Gal4 bears the Gal4 open reading frame downstream of the choline acetyltransferase promoter sequence and is selectively expressed in cholinergic neurons (Salvaterra and Kitamoto, 2001). The gbb1 allele, described previously (Wharton et al., 1999), was rebalanced over Cyo::GFP to allow unequivocal identification of mutant larvae. A transheterozygous combination of witA12 and witB11 was used as described (McCabe et al., 2003). Ok6-Gal4 (Aberle et al., 2002) drives expression in most, if not all, larval motoneurons. UAS-mCD8-GFP (Lee and Luo, 1999), when driven by Ok6-Gal4, localizes to the membranes of cell bodies, axons and dendrites of the motoneurons (Sanchez-Soriano et al., 2005). Elav-GAl4 drives expression throughout the embryonic and larval CNS (Luo et al., 1994). The chats2 allele was used to reduce cholinergic transmission. Recordings from chats2 were performed using early 2nd instar larvae raised at 18°C, a temperature at which cholinergic signaling is significantly reduced (Salvaterra and McCaman, 1985). Df{3R}Exel6184, which uncovers the entire dys locus and several additional proximal genes, was obtained from the Bloomington Stock Center.

Generation of Dp186 mutants, UAS-RNAi-Dp186 and UAS-Dp186 transgenic fly lines

A P-element excision screen (Tower et al., 1993) was used to generate Dp186 mutant fly lines. The GE20705 P-element line (obtained from GenExel, Daejon, South Korea) was used as a starting point for the excision screen; the transposon is inserted 250 bps 5′ of the Dp186 initiator ATG (Fig. 1A). Imprecise excisions, removing genomic sequences flanking the original P-element, were generated by crossing in a source of transposase. Two lines were used for further analyses, dysDp186 166.3, and dysDp186 30.3 (Fig. 1A). Sequence analyses indicated that dysDp186 166.3 bears a 1.2 kb deletion removing the Dp186 ATG codon and most of the unique first exon of Dp186 and dysDp186 30.3 bears a 0.9 kb deletion.

Figure 1
The structure of the Drosophila dystrophin gene and the location of the deletions in the dysDLP2 E6, dysDp186 166.3 and dysDp186 30.3 mutants

To reduce Dp186 expression levels in a tissue specific manner, we generated transgenic fly lines that express double stranded (ds)-RNA targeting Dp186 unique sequences under Gal4 control. The UAS-RNAi-Dp186 construct contains the unique Dp186 sequences from basepairs 74 to 714 (Genbank Accession Number NM_169863) cloned into a pUAST (Brand and Perrimon, 1993) derivative bearing the mub intron between the Dp186 specific sequences (van der Plas et al., 2006). Multiple independent transgenic lines were generated using standard P-element transformation techniques and two lines, 3 and 8, were used in these studies.

To express Dp186 in a tissue-specific manner, we generated fly lines expressing the full length Dp186 cDNA sequence under Gal4 control in the pUAST P-element vector. Multiple transgenic lines were obtained and two lines, 6.1 and 12.1, were used in these studies.


Anti-Dp186 rabbit antisera were raised against a histidine-tagged fusion protein containing unique Dp186 sequences (amino acids 15-170 of Genbank accession number AAK15257; van der Plas et al., 2006).

Immunohistochemistry was performed as described previously (Dekkers et al., 2004). Anti-Dp186 (1: 2500), anti-Synapsin (1:1000; Klagges et al., 1996), anti-Bruchpilot Nc82 antibody (1:50; Hofbauer, 1991; Developmental Studies Hybridoma Bank), anti-GFP (1:1000; Roche Diagnostics, Almere, The Netherlands), Alexa Fluor 488 Phalloidin (1:200; Invitrogen, Breda, The Netherlands) and Alexa Fluor-conjugated secondary antibodies (1: 300; Invitrogen, Breda, The Netherlands) were used as indicated. The samples were visualized and photographed using confocal and/or standard epi-fluorescence microscopy.

Dissection of Larvae

First (within 4 hrs of hatching) and second instar larvae were dissected and central neurons accessed as described (Baines and Bate, 1998). Larvae were viewed using a water immersion lens (total magnification 800X) combined with Nomarski optics (BX51 WI microscope, Olympus Optical, Tokyo, Japan).


Whole cell voltage clamp recordings were performed using thick-walled borosilicate glass electrodes (GC100TF-10, Harvard, Edenbridge, UK), fire-polished to resistances of between 15 and 20mΩ. Recordings were made using an Axopatch-1D amplifier controlled by pClamp 8.1 via a Digidata 1322A A/D converter (Molecular Devices, Sunnyvale, CA). Cells were identified based on their invariant size and dorsal position in the ventral nerve cord. After breakthrough, currents were measured for a period of time ranging from between at least 2 and for no longer than 4 minutes from a minimum of 8 cells for each genotype tested. Although currents can be recorded for longer than 5 minutes, cells often begin to show increased leak currents after this duration of whole cell recording. Traces were filtered at 2 KHz and sampled at 20 KHz. Spontaneous miniature currents were recorded in the presence of 0.1 μM Tetrodotoxin (TTX; Alomone labs, Jerusalem, Israel). Miniature currents were identified based on a fast rise and slower decay and on having amplitudes of at least twice background (2-3 pA). Amplitudes of both evoked and miniature currents were measured using Minianalysis 6.0.3 (Synaptosoft, Decatur, GA). Composite averaged current amplitude was calculated by grouping all individual currents recorded in each genotype tested. All recordings were performed at room temperature (22-24°C). External saline consisted of: (in mM) NaCl (135), KCl (5), MgCl2.6H2O (4), CaCl2.2H2O (2), N-Tris[hydroxymethyl]methyl-2-amonoethanesulfonic acid (TES, 5), sucrose (36), pH 7.15. Internal patch solution consisted of (in mM): KCH3SO3, (140), MgCl2.6H2O (2), EGTA (2), KCl (5), HEPES (20), pH 7.4.


Data were compared using a non-paired t-test. Results were deemed significant at P ≤ 0.05 (*) or P ≤ 0.01 (**). All values shown are mean ± SEM.


The Dystrophin Isoform Dp186 is expressed in the embryonic and larval synapse-rich neuropiles

The mammalian dystrophin gene is a large, highly conserved gene that encodes a number of isoforms expressed from distinct promoters (reviewed in Blake et al., 2002). The Drosophila melanogaster dystrophin (dys) gene encodes six known isoforms which, similarly to their mammalian orthologs, are expressed predominantly in the musculature and nervous system (Greener and Roberts, 2000; Neuman et al., 2001; Dekkers et al., 2004; Neuman et al., 2005; Fig. 1A). All Drosophila isoforms bear the highly conserved cysteine-rich Dystrophin carboxyterminal region (Fig. 1). The aminotermini of three large isoforms, DLP1, -2 and -3 include the actin-binding domain and spectrin repeats found in the large mammalian protein, while the aminotermini of the smaller isoforms, Dp186, Dp205 and Dp117, do not resemble those of the smaller mammalian isoforms. The Drosophila DLP2 isoform is expressed predominantly in the musculature and accumulates at the sarcomeres and at the postsynaptic side of the NMJ (van der Plas et al., 2006). Previous RNA in situ analyses revealed that Dp186 is highly expressed in the embryonic CNS (Neuman et al., 2001; Dekkers et al., 2004). We raised rabbit polyclonal antisera against sequences unique to Dp186 (Materials and Methods) and examined the localization of the Dp186 protein during the embryonic and larval stages. Dp186 protein was first detected in the embryonic CNS at stage 13 and is expressed throughout embryonic development, localizing to regions near the longitudinal connectives (Fig. 2B). These regions likely represent the synapse-rich dorsal neuropile, since the presynaptic Bruchpilot protein, recognized by mAb Nc82 (Kittel et al., 2006; Wagh et al., 2006), is also present there (Fig. 2A and C).

Figure 2
Dp186 protein is expressed in synapse-rich regions of the embryonic and larval neuropiles

In 3rd instar larval CNS, Dp186 protein is found in the neuropile and accumulates in distinct areas in the two brain lobes (Fig. 2D). In addition, the protein can be observed in three bilaterally symmetric clusters, likely within the thoracic neuromeres, which are located at the lateral sides of the neuropile. Staining is not evident in the dysDP186 166.3 mutant (Fig. 2E). We performed double labeling of anti-Dp186 with an antibody recognizing the presynaptically-localized Synapsin protein (Klagges et al., 1996). We found that Dp186 and Synapsin are present in close proximity in the synapse-rich neuropile (Fig. 2F-H), but Synapsin does not appear to as be highly expressed in the sets of lateral clusters within the presumptive thoracic neuromeres as Dp186. (Fig. 2F-H). We also examined a postsynaptic marker localized to motoneuron dendrites in double labelings with anti-Dp186. UAS-mCD8-GFP driven by a motoneuron-specific Gal4 driver (OK6-Gal4) results in the localization of GFP to the membranes of motoneuron cell bodies, dendrites and axons (Landgraf et al., 2003; Sanchez-Soriano et al., 2005). Double labeling with anti-Dp186 shows co-localization of GFP and Dp186 proteins (Fig. 2I-K), indicating that Dp186 is present at interneuron/motoneuron synapses. To further characterize Dp186 localization in the presumptive thoracic neuromeres, we used fluorescently-tagged phalloidin to stain F-actin in the larval neuropile, and found that Dp186 co-localized with F-actin most strongly in the lateral cluster extremities (Fig. 2L-N).

Lack of Dp186 results in an increase in synaptic currents in motoneurons

To study the role of the Dp186 dystrophin isoform during CNS development, we generated mutant fly lines that lack Dp186 protein. We used a P-element mobilization strategy starting with a P-element, GE20705, inserted 250 bps upstream of the Dp186 ATG initiator codon to generate Dp186-specific deletions (Materials and Methods; Fig. 1). Two lines were generated, dysDp186 166.3 and dysDp186 30.3, which both lack detectable levels of Dp186 protein (Fig. 2E) and mRNA (Supplemental Fig. 1), but have wildtype levels of the Dystrophin DLP2, Dp117 and Dp205 isoform mRNAs (Supplemental Fig. 1). The lack of the large muscle-specific DLP2 Dystrophin isoform results in a significant increase in evoked neurotransmitter release at the Drosophila NMJ (van der Plas et al., 2006). To determine whether synaptic transmission in the CNS is similarly regulated by the Dp186 CNS-specific Dystrophin isoform, voltage clamp recordings (Vh -60mV) were made from either the aCC or RP2 motoneurons (no differences were observed between these neurons). In wildtype backgrounds, such recordings show inward synaptic currents that are relatively long-lived (500-1000 msec) and have an average amplitude of 76 ± 3.3 pA (Fig. 3A-C). These excitatory synaptic currents, which are cholinergic in nature and action potential-dependent, result from the synchronous activity of interneurons that are presynaptic to the motoneurons (Baines and Bate, 1998; Baines, 2003). These currents arise from the combined synaptic output of more than one interneuron and are not the result of single action potentials (Baines, 2003). Identical recordings in dysDp186 166.3, an allele which bears a deletion encompassing most of the unique first Dp186 exon, revealed that synaptic currents were significantly increased in amplitude (127 ± 4.4 pA, P ≤ 0.01, Figs. 3B, C). Cumulative probability plots of individual synaptic currents, that better show the range of current amplitudes recorded, revealed that there is a significant increase in amplitudes of the majority of the individual currents measured, compared to the wildtype control (Fig. 3D). Synaptic current amplitude was also significantly increased in dysDp186 30.3, a second independent imprecise excision allele lacking Dp186 (117 ± 3.5 pA, P ≤ 0.01, Fig. 3C). Similarly high currents were observed both in dysDp186 166.3/+ heterozygotes (152 ± 3.4 pA, P ≤ 0.01) and in Df(3R)Exel6184/+ heterozygotes (139 ± 2.4 pA, P ≤ 0.01); this deficiency uncovers the entire Dystrophin locus. Together, these data indicate that Dp186 is haploinsufficient in maintaining the wildtype electrophysiology of this interneuronal synapse. In contrast, synaptic currents in dysDLP2 E6, which lacks the DLP2 muscle-specific isoform, but expresses Dp186 at wildtype levels (van der Plas et al., 2006), were no different in amplitude compared to wildtype (75 ± 2.5 pA, P > 0.05, Fig. 3C). Evoked synaptic currents at the NMJ were, however, significantly elevated in dysDLP2 E6 (van der Plas et al., 2006). The frequency of action potential-dependent synaptic currents recorded in aCC/RP2 was not significantly altered in either dysDp186 166.3 or dysDp186 30.3 compared to wildtype (21.1 ± 4.2, 19.3 ± 3.2 and 21 ± 3.8, currents per min, respectively, P > 0.05), suggesting that the degree of synaptic connectivity of these two motoneurons with presynaptic interneurons remains unaltered. There are a number of conceivable mechanisms that might underlie an increase in evoked synaptic transmission between presynaptic interneurons and aCC/RP2. Two possibilities are that there is an increase in evoked release of ACh from presynaptic terminals or an increased postsynaptic sensitivity to this neurotransmitter. To distinguish between these two possibilities, recordings were repeated in the presence of TTX (0.1μM). All evoked transmitter release was blocked under these conditions (Fig. 4A). Analysis of the TTX-insensitive miniature synaptic currents (mepsc) in aCC/RP2 showed no difference in amplitude between dysDp186 166.3, dysDp186 30.3 and wildtype controls (Fig. 4B, C). This result is consistent with a lack of change in postsynaptic sensitivity to neurotransmitter in the mutant. The frequency of mepsc’s was, however, significantly increased in both dysDp186 166.3 and dysDp186 30.3 compared to the control (Fig. 4D). An increase in mepsc frequency is consistent with, and indeed predictive of, a heightened probability of presynaptic vesicle release.

Figure 3
Synaptic currents in motoneurons are increased in the absence of Dp186
Figure 4
Loss of Dp186 results in increased mepsc frequency, but no change in amplitude

RNA interference and rescue experiments reveal a predominantly postsynaptic role for Dp186

Our data indicates that Dp186 is required for normal synaptic signaling between interneurons and motoneurons in the CNS. To better localize the site of action of Dp186, we adopted two approaches. First, we used targeted expression of an RNA interference (RNAi) transgene to reduce the endogenous levels of Dp186 protein specifically either pre- or postsynaptically Second, we attempted to rescue the Dp186 mutant electrophysiological phenotype by tissue-specific expression of a wildtype Dp186 transgene. Targeted expression of both RNAi and rescue constructs was achieved through use of two well-characterized Gal4 drivers. B19-Gal4 drives expression in all cholinergic neurons, including those interneurons that are presynaptic to aCC/RP2 (Baines, 2004). RN2-Gal4 is an even-skipped promoter-Gal4 transgene that drives expression in the aCC and RP2 motoneurons (Fujioka et al., 2003; Baines, 2004). The efficiency of the RNA-interference was determined by quantitative RT-PCR and Dp186 expression levels were found to be reduced to one-third to one-half of wildtype levels when the UAS-RNAi-Dp186 constructs were expressed throughout the larval neuropile by use of the pan-neuronal Elav-Gal4 driver (Supplemental Figure 2).

Expression of the Dp186-RNAi transgene was sufficient to phenocopy the Dp186 mutant phenotype: synaptic currents were significantly increased, when expressed in either presynaptic cholinergic neurons or in the postsynaptic motoneurons aCC/RP2 (Fig. 5A). The increase in synaptic currents observed was, however, significantly greater when the RNAi transgene was expressed in the postsynaptic aCC/RP2 than in the presynaptic interneuron (pre vs post P ≤ 0.01). It should be noted that synaptic currents are potentiated by the presence of the Dp186-RNAi transgene alone (i.e. in the absence of Gal4), suggesting that its ‘leaky’ pan-neuronal expression might, in part, contribute to the effect observed when using either the presynaptic B19-Gal4 or postsynaptic RN2-Gal4 driver. However, Gal4-dependent expression results in clearly significantly increased synaptic current amplitude relative to the UAS control level.

Figure 5
Dp186 is required in postsynaptic motoneurons

Significant rescue of the Dp186 mutant phenotype by expression of a UAS-Dp186 transgene was, by contrast, only observed following expression in the postsynaptic aCC/RP2 motoneurons and not when expression was limited to the presynaptic cholinergic interneurons compared to the UAS-transgene control (Fig. 5B; P ≤ 0.01). Thus, the reduction in current amplitude observed following presynaptic expression of Dp186 was not significantly different from that seen in the UAS control, which is presumably again the result of basal Gal4-independent expression of UAS-Dp186. Taken together, our data are consistent with a predominantly postsynaptic role for Dp186 in the regulation of the strength of central motoneuron synaptic excitation, however, given the effect of presynaptic Dp186-directed dsRNA expression observed, we cannot rule out Dp186 roles in the interneurons or their upstream partners (see Discussion).

Gbb/Wit signaling is not required for increased synaptic transmission in the Dp186 mutant

The observation that Dp186 is required in postsynaptic motoneurons in order to regulate the efficacy of presynaptic neurotransmitter release is consistent with the existence of a retrograde signal. Studies have shown that such a signaling mechanism, based on the secretion of the BMP ligand Gbb, is active at both the NMJ and at this motoneuron-interneuron synapse in the CNS (Aberle et al., 2002; Marques et al., 2002; McCabe et al., 2003; Baines, 2004). A more recent study has, however, refined the action of gbb signaling at the NMJ by showing that it is required to establish the competence of the motoneuron to respond to another non-gbb retrograde signal which itself is presumably responsive to synaptic change (Goold and Davis, 2007). To determine whether the increase in synaptic currents observed in aCC/RP2 in the Dp186 mutant is reliant on gbb retrograde signaling, we performed whole cell recordings in larvae lacking both Dp186 and gbb.

As previously reported (Baines, 2004), synaptic currents in aCC/RP2 were significantly reduced in homozygous gbb1 mutants compared to heterozygous controls (54 ± 1.5 pA vs. 79.5 ± 2.2, Fig. 6A). In the presence of only a single copy of the wildtype gbb gene, the absence of Dp186 (gbb1/Cyo; dysDp186 166.3) was sufficient to increase synaptic currents to a level (128 ± 2.6 pA, Fig 6A) comparable to that observed in the Dp186 mutant with wildtype levels of gbb expression (Fig. 3C). Recordings from aCC/RP2 in the doubly homozygous gbb1;dysDp186 166.3 mutant similarly display increased synaptic currents relative to the gbb1 control, albeit to lower absolute values (82 ± 3.2 pA, P ≤ 0.01, Fig. 6A). These results are consistent with the effect of Gbb and removal of Dp186 being additive and as such acting through separate non-interacting pathways. This is because the presence of Gbb increases synaptic current amplitude by ~ 28 pA (WT - gbb1 values) while removal of Dp186 results in an increase of ~ 79 pA (dysDp186 166.3 - WT). The combined total of 107 pA is comparable to the value we observed following removal of Dp186 in the presence of gbb (128 pA). We therefore conclude that, while the absolute levels of synaptic current increases due to the absence of Dp186 are limited in the absence of gbb1, potentiation of synaptic currents in the absence of Dp186 is unlikely to require gbb-dependent signaling. Gbb retrograde signaling at this interneuronal synapse may instead represent a parallel pathway required for other aspects of synaptic maturation or function.

Figure 6
aCC/RP2 synaptic current increase in Dp186 mutants is independent of Gbb/Wit signaling and ongoing cholinergic neurotransmission

To further investigate requirements for gbb in the dysDp186 166.3 phenotype, we examined the consequences of overexpressing Dp186 in the presence or absence of gbb. Surprisingly, postsynaptic expression of UAS-Dp186 in a wildtype background resulted in significant potentiation of synaptic currents (Fig. 6B). Thus, either loss or gain of Dp186 function results in increased synaptic currents indicating that specific absolute levels of Dp186 are required for normal synaptic function. That the RNAi knockdown of Dp186 (which is only 50-70% effective) also results in larger synaptic currents than the complete removal of Dp186 supports this hypothesis. Although this result was unexpected, it nevertheless allowed us to further test for gbb requirements. Consistent with the effects of dysDp186 166.3 in the absence of gbb (Fig. 6A), potentiation of synaptic currents due to overexpression of Dp186 was also independent of gbb (Fig. 6B). Moreover, overexpression of Dp186 in the gbb null background potentiated synaptic currents to levels similar to those seen with loss of Dp186 in an otherwise wildtype background (c.f. Fig. 6A and B), indicating that Dp186 levels must be tightly regulated for its wildtype function. Taken together, these data strongly suggest that Dp186 regulation of synaptic function does not act through the previously described Gbb-dependent retrograde signaling mechanism at this synapse (Baines, 2004).

Retrograde signaling at the NMJ requires the Type II TGF-β receptor, Wit (McCabe et al., 2003; Goold and Davis, 2007). To determine whether Wit is required in retrograde signaling at this interneuron-motoneuron synapse, we recorded synaptic currents in the absence of Wit (Fig. 6C). In contrast to the requirement for Wit at the NMJ, central synaptic transmission was unaffected by the absence of this receptor. Moreover, postsynaptic expression of dsRNA directed against Dp186, which increased synaptic currents in the wildtype background (Fig. 5A), also did so in the absence of wit (Fig. 6C).

Finally, we tested the possibility that Dp186-dependent regulation of presynaptic neurotransmitter release is activity-dependent, as was previously shown for Gbb at this synapse (Baines, 2004). Cholinergic neurotransmission was significantly reduced by the utilization of a temperature-sensitive allele of choline acetyltransferase (chats2). In comparison to controls containing the wildtype cha gene, synaptic currents recorded in chats2 2nd instar larvae raised at 18° C are significantly reduced (Fig. 6D) consistent with the demonstrated reduction in enzyme activity at this temperature (Salvaterra and McCaman, 1985). Overexpression of gbb in the chats2 background fails to potentiate synaptic currents; however postsynaptic expression of Dp186-directed dsRNA significantly increased synaptic currents in this same background (Fig. 6D). This strongly suggests that the increase in neurotransmitter release caused by the absence of Dp186 was not dependent on cholinergic neurotransmission and, as such, differs from that observed when gbb is overexpressed postsynaptically. These data further suggest that gbb and Dp186 act through independent, but possibly parallel, pathways.


The cognitive impairments displayed by many DMD patients (reviewed in Anderson et al., 2002; Culligan and Ohlendieck, 2002) and the localization of Dystrophin to synapse-rich regions (reviewed in Blake and Kroger, 2000) are consistent with Dystrophin roles in the CNS. Mutations primarily in the sequences encoding the highly conserved common carboxyterminal domain of Dystrophin have been correlated with human mental deficits (Moizard et al., 1998), suggesting that mutation of one of the shorter CNS-specific Dystrophin isoforms may underlie mental impairment. Mental defects have, however, also been observed in patients (Hinton et al., 2000) and in the mdx mouse model (Vaillend et al., 2004), lacking only the large Dystrophin isoform. Therefore, the relative roles of the various Dystrophin isoforms in normal CNS function remain unclear.

We show in this study that the Drosophila Dp186 isoform is expressed in the CNS and is required to maintain normal synaptic physiology. It is found in the synapse-rich neuropile and in its absence, evoked presynaptic neurotransmitter release is significantly increased at an identified interneuron-motoneuron central synapse, without apparent change in postsynaptic receptor field sensitivity. Transgenic RNA interference and rescue experiments indicate that Dp186 is required primarily in the postsynaptic motoneuron to maintain wildtype presynaptic release levels. The effects of altering postsynaptic levels of Dp186 on presynaptic neurotransmitter release are consistent with a role for Dp186 in retrograde signaling but, as we show, this signaling is not dependent on gbb, previously implicated in interneuronal retrograde signaling or wit, shown to act at the NMJ.

Dystrophin Dp186 accumulates at synaptic regions of the CNS

During embryogenesis, the Dp186 protein is found close to the longitudinal axon bundles of the ventral nerve cord. The presence of the presynaptic protein, Bruchpilot (Kittel et al., 2006; Wagh et al., 2006) in this domain indicates that it is rich in synaptic contacts which include the motoneuron dendrites and their interneuronal inputs. In the third instar larval neuropile, Dp186 is also in close proximity to the presynaptic Synapsin protein (Klagges et al., 1996). Double stainings performed with anti-Dp186 and membrane-associated GFP (mCD8-GFP, Lee and Luo, 1999) expressed in motoneurons, reveal colocalization, further suggesting that Dp186 is synaptically-localized. The density of synaptic contacts in these regions precluded us from evaluating the precise degree of colocalization between Dp186 and these presynaptic markers at the level of light microscopy, however these data support our hypothesis that Dp186 is synaptically-localized.

Dystrophin Dp186 is required for wildtype synaptic physiology

In a previous study, we observed that the absence of the large postsynaptically-expressed Dystrophin DLP2 isoform resulted in increased presynaptic neurotransmitter release at the Drosophila NMJ (van der Plas et al., 2006). To address whether Dp186 might play a similar role at interneuronal synapses, we generated mutant lines lacking Dp186 protein and evaluated the electrophysiology of a well-characterized synapse between the aCC/RP2 motoneurons and their presynaptic cholinergic interneurons (Baines et al., 2001; Baines, 2003). Unlike the NMJ motoneurons, the presynaptic cholinergic neurons cannot be directly stimulated to allow evaluation of evoked responses. However, in the preparation used, endogenous evoked responses, that form part of the motor pattern generator, occur at defined frequencies from late embryogenesis onwards (Baines et al., 2002), allowing the recording of endogenous evoked responses.

Recordings of such endogenous evoked currents in Dp186 mutants reveal that they are significantly increased in amplitude, but not frequency, relative to wildtype controls and a mutant lacking DLP2. Recordings performed in the presence of TTX, which allows measurement of spontaneous mepsc events in the absence of evoked responses, indicate that the postsynaptic AChR field is apparently unaffected by the absence of Dp186. Together with the increased frequency of mepsc observed in the Dp186 mutant, these findings support the hypothesis that evoked presynaptic neurotransmitter release is significantly elevated in the absence of Dp186.

Tissue-specific rescue experiments revealed that postsynaptic, but not presynaptic, expression of Dp186 in the dysDP186 mutant background rescued presynaptic release to wildtype levels. Therefore, Dp186 is apparently predominantly required postsynaptically. Our results from the transgenic RNA interference approach are less unambiguous, but do show that the largest increase in presynaptic release occurred when post-synaptic Dp186 expression levels were decreased. Presynaptic expression of Dp186-RNAi, however, also resulted in increased release, albeit to lower levels. A possible explanation is that decreased Dp186 expression in first order interneurons that drive motoneurons might, in turn, elevate their own excitation from second order interneurons (to which they are postsynaptic). The resultant increased activity in these first order interneurons might manifest itself by increasing the synaptic excitation of downstream motoneurons. While many details of the circuitry that form the motor pattern generator are lacking, first order interneurons that synapse directly with motoneurons are indeed reliant on second order interneurons for synaptic excitation (Carhan et al., 2004). At present, markers suitable for evaluating whether Dp186 is present at these upstream synapses are not available.

Dystrophin and the retrograde control of presynaptic release

The requirement for Dp186 in motoneurons for normal function of presynaptic cholinergic interneurons is consistent with the regulation of neurotransmitter release by a retrograde signal derived from the targets. Recent studies have shown that presynaptic release at these synapses is regulated, at least in part, by BMP signaling (Baines, 2004). Moreover, increasing expression of the BMP ortholog, Gbb, in postsynaptic motoneurons is sufficient to significantly increase synaptic current amplitudes, achieving levels similar to those observed in the Dp186 null mutants (Baines, 2004). We therefore examined the effects of eliminating Gbb signaling upon the Dp186 electrophysiological phenotype. In the absence of gbb, the relative increase in synaptic currents caused by the lack of expression of Dp186 was similar to that seen in the dysDp186 mutant alone, suggesting that Dp186 regulates presynaptic neurotransmitter release independently of gbb. The failure of the loss of gbb to suppress the increases in central synaptic currents observed following overexpression of Dp186 further indicates the apparent independence of these two pathways.

Previous studies have implicated wit in the proper morphological development of the NMJ synapse and retrograde signaling there (Aberle et al., 2002; Marques et al., 2002; McCabe et al., 2003) and in establishing the competence of motoneurons to respond to muscle-derived retrograde signals (Goold and Davis, 2007). Potentiation of synaptic transmission at the NMJ due to postsynaptic loss of the DLP2 Dystrophin isoform was previously shown to require wit (van der Plas et al., 2006). In the CNS, we find that there is seemingly no corresponding requirement for wit for increased neurotransmitter release at the Dp186-deficient synapse. Furthermore, contrary to what was observed at the NMJ, wit is apparently not required for wildtype electrophysiology. Clearly then, although the absence of a specific Dystrophin isoform at either the NMJ or a central synapse results in increased neurotransmitter release, the underlying mechanisms likely differ, perhaps reflecting the use of different retrograde signaling pathways.

We observe that dysDp186-dependent potentiation of central synaptic currents persists when cholinergic neurotransmission is significantly reduced. This, again, is in contrast to the effect of postsynaptic overexpression of gbb under the same conditions. There are two implications of this data; first, it further supports the hypothesis that Dp186 and gbb independently regulate central synaptic transmission. Second, this data predicts the existence of two types of retrograde signaling pathways regulating presynaptic function, one activity-dependent and the other, constitutive. Indeed, our data are consistent with the possibility that Dp186 controls the ongoing release of a negative regulator of synaptic function. Conceivably, the strength of synaptic transmission will be regulated by the interplay of these and other mechanisms. The identity of the signal regulated by Dp186 is not known but it may be one or more of the six related BMP signaling molecules present in Drosophila: Dpp, Screw, Activin, Activin-like protein, Myoglianin and Maverick (Lo and Frasch, 1999; Keshishian and Kim, 2004) or it is possibly unrelated to BMP/TGF-β signaling. It is not currently clear by what mechanisms reduced postsynaptic expression levels of Dp186 potentiate presynaptic neurotransmitter release. The increased mepsc frequency, but not amplitude, observed in the mutant is consistent with an increase in quantal content caused by heightened probability of release. Additional possibilities include increased synchronicity of release from multiple interneurons that drive these motoneurons. Alternatively, individual interneurons might have increased numbers of presynaptic motoneuron contacts. We consider the latter possibility less probable as we observe that evoked synaptic current amplitude, but not frequency, is affected in the Dp186 mutant. This indicates that the synaptic connectivity between interneurons and motoneurons is apparently normal in the absence of Dp186.

In summary, the Drosophila Dystrophin Dp186 isoform is required predominantly postsynaptically for wildtype neurotransmitter release levels at an identified cholinergic central synapse. Our previous study of the DLP2 isoform revealed that its absence from the muscle increased the probability of presynaptic release at the glutamatergic NMJ (van der Plas et al., 2006). Therefore, these two postsynaptically-localized Dystrophin isoforms are required for the appropriate regulation of presynaptic release at two different types of synapses, each utilizing a different neurotransmitter. Furthering our understanding of the role of the Dystrophin isoforms in synaptic transmission in Drosophila should yield insights into evolutionarily-conserved roles of dystrophin in the nervous system and perhaps shed light on the poorly understood mental retardation presented by a significant subset of DMD patients.

Supplementary Material

Supplementary Figures


We thank E. Buchner, M. Fujioka, J. B. Jaynes, K. A. Wharton and the Bloomington Stock Center for fly stocks and antibodies and B. van Veen, M. Bansraj, A. de Jong and L. Dekkers for help with the experiments. We also acknowledge J. Plomp, S. Potikanond and G. Pilgram and the reviewers for providing insightful suggestions on the manuscript and B. Gerber and R. Stocker for their kind advice. This work was supported by The Wellcome Trust (R.A.B.) and a ‘Pionier’ grant # 900-02-003 of the ‘Nederlandse Organisatie voor Wetenschappelijk Onderzoek, “N.W.O.” (J.N.N.).


Section: Molecular and Cellular

Section Editor: Dr. Chris McBain


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