To quantitatively test whether inputs of multiple transmitter classes can direct postsynaptic dendrite arbor growth, we needed an identified central neuron that displays a stereotyped dendritic morphology between animals, contains different segregated synaptic input domains from presynaptic partners of different transmitter classes, and allows measuring dendritic length and branch numbers in control animals as well as following selective manipulation of synaptic input to different parts of its developing arbor. The identified adult flight motoneuron 5 (MN5) () of Drosophila melanogaster fulfills these requirements. First, MN5 displays a stereotyped dendritic morphology ( Figure 1 A) with metric and topological features that are sufficiently constant across animals to allow statistical comparison of measurements between control and genetic manipulation. MN5 contains a fixed number of 23 dendritic subtrees that all arise from the primary neurite, tile the input space, and, on average, comprise 4,000 ± 577 dendritic branches with a total length of 6,716 ± 731 μm (means ± SD) (). Second, MN5 receives synaptic input of different transmitter classes. The main excitatory input is cholinergic through the Dα7 nicotinic acetylcholine receptor (Dα7nAChR) (), whereas the main inhibitory input is GABAergic to the Rdl GABAreceptor. We have previously shown () that more than 75% of all cholinergic inputs to the Dα7nAChR are targeted to the proximal dendritic domain of MN5 ( Figure 1 B, dotted red oval), whereas more than 75% of all GABAergic inputs to the Rdl GABAR are targeted to dendrites originating from the distal part of the primary neurite (distal domain; Figure 1 B, dotted blue ovals). Third, genetic tools allow selective manipulation of synaptic input to the GABAergic or to the cholinergic domain of MN5.

We used 30 geometric MN5 dendrite reconstructions () to quantify dendrites in the distal GABAergic and in the proximal cholinergic input domains. The average distance between the most proximal (in the cholinergic input domain) and the most distal (in the GABAergic input domain) dendritic sub-tree branching off the primary neurite is 52 ± 4 μm (mean ± SD, n = 30), thus highly stereotyped between animals. We used the midpoint of this distance as landmark to divide each geometric dendrite reconstruction into a proximal ( Figure 1 C, red, mainly cholinergic inputs) and a distal half ( Figure 1 C, blue, mainly GABAergic inputs). Quantification of MN5 dendrite reconstructions from nine different control animals demonstrated that, on average, the total dendritic length of 6,416 ± 423 μm ( Figure 1 D, white bar) was distributed to equal amounts to proximal ( Figure 1 D, red bar) and distal dendrites ( Figure 1 D, blue bar). Similarly, in controls half of the 3,998 ± 512 branches were proximal, the other half were distal dendrites ( Figure 1 E). Therefore, during normal development dendritic building material is assigned in equal proportions to the excitatory cholinergic ( Figure 1 C, red) and to the inhibitory GABAergic domain ( Figure 1 C, blue). However, although mean branch numbers and dendrite length were statistically identical in both input domains, in individual animals the relative sizes of both domains to each other showed some variation ( Figures 1 D and 1E, individual data points are depicted as circles, the lines connect proximal and distal length values of the respective neurons). This may indicate developmental plasticity in assigning dendrites to both input domains, but there were no hints for systematic size increases of one over the other input domain in controls. We next tested whether synaptic input to either dendritic domain could locally shape dendrite growth.

GABAergic and Cholinergic Synapses Compete for Dendritic Building Material

Ryglewski et al., 2014a Ryglewski S.

Kilo L.

Duch C. Sequential acquisition of cacophony calcium currents, sodium channels and voltage-dependent potassium currents affects spike shape and dendrite growth during postembryonic maturation of an identified Drosophila motoneuron. Kuehn and Duch, 2013 Kuehn C.

Duch C. Putative excitatory and putative inhibitory inputs are localised in different dendritic domains in a Drosophila flight motoneuron. Kuehn and Duch, 2013 Kuehn C.

Duch C. Putative excitatory and putative inhibitory inputs are localised in different dendritic domains in a Drosophila flight motoneuron. All MN5 dendrites () as well as cholinergic and GABAergic inputs form during pupal life (). We employed targeted genetic manipulation to selectively alter cholinergic or GABAergic synaptic activity in different parts of the developing MN5 arbor. In order to increase cholinergic synaptic drive to the proximal dendritic domain, we overexpressed Dα7nAChRs as UAS transgene postsynaptically in MN5 under the control of P103.3-GAL4. Correct localization of UAS-Dα7nAChR has previously been confirmed by comparison with immunolabeling of native Dα7nAChRs (). Therefore, Dα7nAChR overexpression increases receptor number but does not change localization predominantly to the proximal dendritic domain of MN5. Although we did not precisely quantify the amount of Dα7nAChR overexpression by immuno EM following expression under the control of P103.3-GAL4, we estimated an increase of 23% ± 11% of Dα7nAChR immuno-positive puncta in flight motoneuron dendrites on the light microscopy level ( Figures S1 A, S1B, S1D, and S1E). Co-labeling of native and GFP-tagged receptors revealed that expression of Dα7nAChR-GFP under the control of P103.3-GAL4 resulted in ∼40% GFP-positive and 60% native receptors in flight motoneuron dendrites ( Figures S1 F and S1G).

Figure 2 Competition for Dendrites by Cholinergic and GABAergic Synaptic Drive Show full caption A Rs (blue). The separation point for proximal and distal dendrites is defined as the midpoint between the origins of the most proximal cholinergic and the most distal GABAergic sub-tree (see A Rs by targeted overexpression of UAS-Rdl (B) increased the quantity of distal (blue) in relation to proximal (red) dendrites. By contrast increasing the availability of both receptors (C; UAS-Rdl; UAS-Dα7) had no effect. Scale is 10 μm. (A–C) Representative reconstructions color coded for proximally originating dendritic sub-trees with high densities of Dα7nAChRs (red) and distally originating dendrites with high densities of Rdl GABARs (blue). The separation point for proximal and distal dendrites is defined as the midpoint between the origins of the most proximal cholinergic and the most distal GABAergic sub-tree (see Figure 1 C). Increasing the availability of nAChRs by targeted overexpression of UAS-Dα7 (A) increased the quantity of proximal (red) in relation to (blue) distal dendrites. Vice versa, increasing the availability of GABARs by targeted overexpression of UAS-Rdl (B) increased the quantity of distal (blue) in relation to proximal (red) dendrites. By contrast increasing the availability of both receptors (C; UAS-Rdl; UAS-Dα7) had no effect. Scale is 10 μm. (D and E) Quantification of dendritic length (D) and the number of branches (E) revealed significant redistribution of dendrites from the distal GABAergic domain to the proximal cholinergic domain following increased availability of Dα7nAChRs, but total arbor length and branch numbers remained unchanged. Vice versa, significant redistribution of dendrites from the proximal cholinergic to the distal GABAergic domain were observed following increased availability of GABA A Rs, again total arbor size remained unchanged. No significant dendrite redistribution occurred upon increasing GABA A Rs and Dα7nAChRs. (F) The ratio of dendrite length in the proximal cholinergic relative to the distal GABAergic domain was 1.02 ± 0.11 in controls (bar 1, white). Intra-neuronal dendrite redistribution from the distal GABAergic to the proximal cholinergic domain are depicted by a ratio significantly larger than 1.0 and were caused by overexpression of Dα7nAChRs (bar 2, red), increasing presynaptic cholinergic neuron activity by activation of TrpA1 channels (ChAT-TrpA1 at 29°C, bar 3, orange), and in heterozygous Rdl mutants (Rdl− × Rdl+, bar 7, purple). Redistribution from the proximal cholinergic to the distal GABAergic domain resulted in a ratio smaller than 1.0 and was caused by overexpression of Rdl receptors (UAS-Rdl, bar 6, blue), and in heterozygous Dα7nAChR mutants (Dα7− × Dα7+, bar 5, pink). No significant dendrite redistribution occurred in controls with TrpA1 channel activation (ChAT-TrpA1 at 22°C, bar 4, gray), following co-expression of Rdl and Dα7nAChRs (UAS-Rdl; UAS- Dα7, bar 8, blue/red), and with activation of presynaptic cholinergic neurons in heterozygous Dα7nAChR mutants (ChAT-TrpA1 × Dα7−, bar 9, orange/pink). (D)–(F) show means and SDs. Circles depict individual data points, lines connect data points for cholinergic and GABAergic domains from the same animals. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; n.s. p > 0.1, ANOVA with Newman-Keuls post-hoc test. See also Figure S1 for an estimate of the amount of Dα7nAChR overexpression, Figure S2 for quantitative branch order analysis in controls and following Dα7 overexpression, and Figure S3 for increasing the magnitude of intra-neuronal dendrite shift by further increasing Dα7 receptor overexpression. The addition of ∼20% of postsynaptic Dα7nAChRs caused a significant increase in the length of proximal dendrites ( Figures 2 A and 2D ), i.e., the cholinergic dendritic domain. Importantly, total dendritic length was not affected by this manipulation. The reason for constant total dendrite length was that a 20% increase of proximal dendrites was accompanied by a 20% reduction of distal dendrites with mainly GABAergic inputs ( Figure 2 D). Consequently, the proximal cholinergic domain was on average 40% larger than the distal GABAergic domain ( Figures 2 D and 2F). The same was measured for the number of dendritic branches ( Figure 2 E). Importantly, all animals with increased Dα7nAChR expression showed increased branch numbers and dendrite length in the proximal cholinergic domain ( Figures 2 D and 2E). By contrast, controls showed no preferences for size increases of one over the other domain, despite some variation in the relative sizes of both dendritic domains (see Figures 1 D and 1E). Therefore, increasing receptor availability for cholinergic input caused a significant redistribution of dendrites from the GABAergic to the cholinergic input domain. Branch order analysis revealed that this intra-neuronal dendrite shift affected branch orders higher than ten (see Figure S2 ).

A Rs in MN5 caused the exact opposite effect: an increase in dendritic length and branch numbers in the distal GABAergic domain that came at the cost of a reduced arbor in the proximal cholinergic domain. All individuals tested showed increased dendritic length ( A or Dα7nAChR density by ∼20% caused an intra-neuronal shift of ∼20% of dendrites away from the input domain with only native receptors. Vice versa, overexpression of Rdl GABARs in MN5 caused the exact opposite effect: an increase in dendritic length and branch numbers in the distal GABAergic domain that came at the cost of a reduced arbor in the proximal cholinergic domain. All individuals tested showed increased dendritic length ( Figures 2 B and 2D) and branch numbers ( Figure 2 E) in the distal GABAergic domain. Again, neither total dendritic length nor the total number of branches were changed ( Figures 2 D and 2E). With respect to the amount of Rdl overexpression, we yielded similar estimates for increased receptor density as described above for overexpression of Dα7nAChR (data not shown). Therefore, an increase of either Rdl GABAor Dα7nAChR density by ∼20% caused an intra-neuronal shift of ∼20% of dendrites away from the input domain with only native receptors.

Vonhoff et al., 2013 Vonhoff F.

Kuehn C.

Blumenstock S.

Sanyal S.

Duch C. Temporal coherency between receptor expression, neural activity and AP-1-dependent transcription regulates Drosophila motoneuron dendrite development. A receptors in heterozygous Rdl mutants caused a shift of dendrites to the cholinergic domain ( A Rs had no effect ( Additional genetic manipulations further supported these findings. First, reducing the amount of available Dα7nAChRs in heterozygous Dα7 mutants caused a redistribution of dendrites to the GABAergic domain ( Figure 2 F). We did not use Dα7 homozygous null mutants, because MN5 total dendritic length is significantly reduced in these animals (). Second, a reduction of available GABAreceptors in heterozygous Rdl mutants caused a shift of dendrites to the cholinergic domain ( Figure 2 F). Third, redistribution of dendrites to the cholinergic input domain was induced not only by overexpression of receptors but also by activating presynaptic cholinergic neurons during pupal life by temperature shifts to 29°C following expression of TrpA1 channels under the control of ChAT-GAL4 ( Figure 2 F). And finally, no dendrite redistribution was observed following TrpA1-activation of presynaptic cholinergic neurons in a Dα7 heterozygous mutant background ( Figure 2 F). Therefore, intra-neuronal dendrite redistribution could either be induced by manipulation of presynaptic activity or by changing postsynaptic receptor availability. These manipulations altered neither total dendritic length nor the total number of branches through the dendritic tree. Furthermore, our data indicate that GABAergic and cholinergic inputs locally direct dendrite growth in a competitive manner, because overexpression of both Dα7nAChRs and Rdl GABARs had no effect ( Figures 2 C–2F).

Boerner and Duch, 2010 Boerner J.

Duch C. Average shape standard atlas for the adult Drosophila ventral nerve cord. Upon supplying an estimated 20% of extra postsynaptic neurotransmitter receptors, we observed an average 20% intra-neuronal dendrite shift to the domain with increased receptor density. At least for Dα7nAChRs, we have indications that this effect can be enhanced by further increasing receptor density. Expression of UAS-Dα7Rs under the control of the stronger motoneuron driver, D42-GAL4, yielded an estimated 40% increase over control of receptor density in flight motoneuron dendrites ( Figures S1 C–S1E). Expression was restricted mainly to flight motoneurons by inclusion of ChAT-GAL80 to inhibit GAL4 in all cholinergic interneurons (). Increasing Dα7nAChR density in the cholinergic input domain by ∼40% caused a significantly stronger dendrite shift to that domain as compared to an estimated 20% of Dα7nAChR density ( Figure S3 ). In controls, the proximal to distal size ratio is on average 1.02 ± 0.11. Increasing Dα7nAChR density by ∼20% yields an average proximal to distal domain size ratio of 1.39 ± 0.10, and increasing Dα7nAChR density by ∼40% yields a ratio of 1.81 ± 0.19 ( Figure S3 D). This indicated that cholinergic synaptic input might locally direct dendrite growth in a dose-dependent manner. Although both drivers express from pupal stage P5 to adulthood, expression in cholinergic interneurons was inhibited, and expression was restricted mainly to motoneurons, we cannot exclude the possibility that some indirect effects may have been caused by spatial or temporal differences in Dα7nAChR expression in both sets of experiments. Although dendrites may contain output synapses, we judge indirect effects via feedback into the flight circuit upon receptor expression in motoneurons unlikely. In general, central output synapses are rare in insect motoneurons, and we have not found presynaptic specializations in Drosophila wing motoneuron dendrites by expression of fluorescently tagged proteins, which reliably label presynaptic specializations in the axon terminals (not shown). However, without a rigorous electron microscopic study we cannot completely exclude the possibility of some output synapses from these dendrites.

We next addressed part of the mechanism by which cholinergic and GABAergic synaptic inputs compete for postsynaptic dendrites, to then test functional consequences for motoneuron firing patterns during behavior.