Intra-neuronal Competition for Synaptic Partners Conserves the Amount of Dendritic Building Material

Abstract

Brain development requires correct targeting of multiple thousand synaptic terminals onto staggeringly complex dendritic arbors. The mechanisms by which input synapse numbers are matched to dendrite size, and by which synaptic inputs from different transmitter systems are correctly partitioned onto a postsynaptic arbor, are incompletely understood. By combining quantitative neuroanatomy with targeted genetic manipulation of synaptic input to an identified Drosophila neuron, we show that synaptic inputs of two different transmitter classes locally direct dendrite growth in a competitive manner. During development, the relative amounts of GABAergic and cholinergic synaptic drive shift dendrites between different input domains of one postsynaptic neuron without affecting total arbor size. Therefore, synaptic input locally directs dendrite growth, but intra-neuronal dendrite redistributions limit morphological variability, a phenomenon also described for cortical neurons. Mechanistically, this requires local dendritic Ca²⁺ influx through Dα7nAChRs or through LVA channels following GABA_AR-mediated depolarizations. VIDEO ABSTRACT.

Keywords: Drosophila; GABA; acetylcholine; competition; dendrite; development; excitation inhibition balance; flight; motoneuron; synapse.

Figure 1. Equal Dendrite Distribution to the Proximal Cholinergic and Distal GABAergic Domain of the Identified Motoneuron 5

(A) MN5 dendrite structure as maximum intensity projection view from a representative confocal image stack. MN5 is a unipolar neuron with a primary neurite arising from the soma. All dendritic sub-trees and the axon emerge from the primary neurite. (B) Geometric dendrite reconstruction from the image stack shown in (A). Soma is omitted. Predominantly cholinergic (>75% of all inputs to the Dα7nAChR) and GABAergic (>75% of all inputs to the Rdl GABA_AR) input domains are color coded and encircled by dotted lines. All branches with high densities of Dα7nAChRs (cholinergic input domain, red) arise from the proximal part of the primary neurite. All dendrites with high densities of GABAergic input synapses to the Rdl receptor (GABAergic input domain, blue) arise from the distal part of the primary neurite (Kuehn and Duch, 2013). Therefore, dendrites that originate from the proximal part of the primary neurite receive mostly cholinergic input whereas dendrites originating from the distal part of the primary neurite receive mostly GABAergic input. (C) The distance between the most proximal cholinergic and the most distal GABAergic dendritic sub-tree is 52 ± 4 μm (mean and SD from 30 MN5 reconstructions). We defined the exact midpoint between these two dendritic sub-trees as the midline between the proximal cholinergic (red) and the distal GABAergic input domain (blue). This reproducibly divided all reconstructions into distal and proximal parts by the identical method without introducing bias. In all experiments, the midpoints were determined blindly without any knowledge of the experimental group. (D and E) Quantification revealed equal dendritic length (D) and branch numbers (E) in the cholinergic (red) and in the GABAergic (blue) input domains of control MN5 (means and SD; p > 0.8; paired two-sided t test; circles show individual data points, lines connect data points from both input domains for each individual animal).

Figure 2. Competition for Dendrites by Cholinergic and GABAergic Synaptic Drive

(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 GABA_ARs (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 1C). 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 GABA_ARs 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_ARs, again total arbor size remained unchanged. No significant dendrite redistribution occurred upon increasing GABA_ARs 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.

Figure 3. Global versus Local Dendritic Ca²⁺ Signals

(A) In situ Ca²⁺ imaging of MN5 with targeted expression of GCaMP6s reveals global Ca²⁺ signals during a train of action potentials as evoked by somatic ramp current injection. Upper image is at rest and lower image during a train of action potentials as induced by somatic ramp current injection (original images overlaid as false color code for gray values from 70 to 240). Eight regions of interest (ROIs) for ΔF/F measurement are depicted by numbered ovals. (B) Simultaneous somatic current clamp (upper trace) and Ca²⁺ imaging recordings from all eight ROIs before, during, and after a train of action potentials reveals global increases in ΔF/F. Note different scale for ROIs 4 and 5 (soma and primary neurite), which displayed the largest amplitude signals upon firing. (C) In situ Ca²⁺ imaging of the same MN5 with identical ROIs as in (A) at rest (upper image) and during a brief (5 ms) puff of nicotine (10⁻⁵ M) onto a distal dendrite (lower image). (D) Simultaneous somatic current clamp (upper trace) and Ca²⁺ imaging recordings from all eight ROIs before, during, and after three consecutive nicotine puffs to ROI1. Nicotinic AChR activation in ROI1 caused locally restricted dendritic Ca²⁺ signals. See also original data movies for global (Movie S1) and local (Movie S2) dendritic signals as well as Figure S4 and Movies S3 and S4 for local Ca²⁺ signals at different dendritic sites.

Figure 4. GABA Induces Depolarization, LVA Ca²⁺ Channel Activation, and Dendritic Ca²⁺ Signals during Immature Stages

(A) During pupal life somatic current clamp recordings revealed depolarizations upon brief (5 ms) puffs of GABA (10⁻⁴ M) onto dendrites. (B) For some high order branches within the GABAergic domain GABA puffs caused large amplitude depolarizations with spikelets. GABA-induced spikelets displayed distinctly different amplitudes and shapes as compared to spontaneously occurring action potentials as recorded from the soma (see inset). (C and D) Sharp electrode recordings were used to determine the chloride reversal potential in adult (C) and pupal (D) MN5. (C) Representative responses to GABA puffs at differentmembranepotentials revealeda chloride reversal potential of −74mV for the adult flight MN5, more than 10m V more hyperpolarized than the average resting membrane potential (−63 mV). (D) By contrast, at P12 the chloride reversal was at −54 mV, about 10 mV more depolarized than resting membrane potential. (E) Quantification revealed that resting membrane potential remained unchanged through development (−63 ± 2 mV), but chloride reversal was significantly shifted from values more positive than resting during pupal life (−54 ± 3 mV) to values more negative than resting in mature stages (−74 ± 4 mV). (F) GABA-induced depolarizations are amplified by Ca²⁺ influx through LVA channels. Sharp electrode recording from a representative pupal stage P12 MN5 held at −70 mV displayed a large depolarizing response to dendritic GABA puffs (lower traces, black). Pharmacological blockade of LVA DmαG Ca²⁺ channels by amiloride (10⁻³ M) significantly reduced amplitude and duration of this GABA response (lower traces, red). No response to GABA puff was observed when holding the cell at the chloride reversal potential (middle traces). A biphasic response, hyperpolarization followed by depolarization, was observed when holding membrane potential at −45 mV (upper traces, black). Blockade of LVA channels isolated the GABA-induced hyperpolarizing response (upper traces, red). (G–I) During pupal life GABA puffs (10⁻⁴ M) cause somatic depolarizations but local dendritic Ca²⁺ signals at the site of GABA_AR activation. Ca²⁺ imaging of parts of MN5 dendrites before (G) and after (H) GABA puff to ROI 1. (I) Simultaneous somatic current clamp recording (upper trace) and Ca²⁺ imaging from ROIs 1–8 as defined in (G) and (H) in response to three GABA puffs to ROI 1 (for signal spread, see Movie S5). See also Figures S5D–S5F for pharmacological evidence that GABA-mediated depolarizations required chloride channels. Local dendritic Ca²⁺ responses to GABA puffs at different dendritic sites are shown in Figure S6 and Movie S6.

Figure 5. GABA-Induced LVA Channel Activation Is Required for Dendrite Redistribution

(A and B) Representative MN5 dendrite reconstructions with color-coded proximal cholinergic (red) and distal GABAergic dendritic domain (blue) following overexpression of Rdl GABA_ARs (UAS-Rdl) in a control background (A) and in a DmαG LVA Ca²⁺ channel null mutant background (B, DmαG^null;UAS-Rdl). (C) Quantification revealed a statistically significant dendrite shift from the proximal to the distal domain upon overexpression of Rdl GABA_ARs in a control background. No dendrite shift to the distal domain was observed upon overexpression of Rdl GABA_ARs in a DmαG null mutant background. By contrast, lack of LVA Ca²⁺ channels caused a slight but significant shift to the proximal cholinergic domain.

Figure 6. Intra-neuronal Dendrite Shifts Impair Adaptive Adjustments of Firing Rates

(A) Wing beat detection by laser beam during tethered flight. (B) Simultaneous recording of MN5 from its target flight muscle fiber (lower trace) and wing beat (WB) recording during tethered flight. Threshold-based event detection and instantaneous frequencies are depicted above the original MN5 and WB recordings. (C and D) No statistical differences were detected in mean MN5 firing frequencies (C) or mean wing beat frequencies (D) during tethered flight in controls (white bars), following overexpression of Dα7nAChRs (red bars), Rdl GABA_ARs (blue bars), or both (gray bars). (legend continued on next page) (E) Tethered flight durations were not statistically different between receptor overexpression with intra-neuronal dendrite shift and controls. Boxplots in (C)–(E) depict medians, quartiles, and 10 and 90 percentiles (n.s., p > 0.1, Kruskal-Wallis ANOVA). (F) In controls (left) and following overexpression of both Dα7nAChRs and Rdl GABA_ARs (right) wing beat frequencies correlated significantly with motoneuron firing rates (R², Spearman’s rank correlation coefficient). No correlation was detected following overexpression of either Rdl GABA_ARs or Dα7nAChRs alone (middle). (G) Optomotor modulation of MN5 firing rates were elicited by using a visual grating pattern that moved either up or down. (H) Upward and downward movement simulated decreasing or increasing flight altitude and resulted in compensatory adjustments of MN5 firing frequencies in controls (upper trace) and following overexpression of both Rdl GABA_ARs and Dα7nAChRs (lower trace). Following overexpression of Rdl GABA_ARs or Dα7nAChRs and intra-neuronal dendrite shift to the respective dendritic domain, adjustments of frequencies in response to visual stimulation were significantly altered (second and third trace from top). (I) Quantification showed that in controls (white bar), following overexpression of both receptors (gray bar), and with conditional expression of Dα7nAChRs in the adult stage only (red and white bar), upward movement caused firing rate increases by ~9 Hz (upward bars, means ± SD) whereas downward movement caused firing rate decreases by ~2 Hz (downward bars, means ± SD). Following increased Rdl GABA_ARs and dendrite shifts to the GABAergic domain, upregulation of firing rate was slightly but significantly smaller (blue bar), and downregulation of firing rate was significantly increased (blue bar). Following increased Dα7nAChRs and dendrite shifts to the cholinergic domain upregulation of firing rates was significantly reduced by ~65% and downregulation was significantly impaired (red bar, ANOVA, Newman-Keuls post hoc test, p ≤ 0.05).