Naked dense bodies provoke depression

Abstract

At presynaptic active zones (AZs), the frequently observed tethering of synaptic vesicles to an electron-dense cytomatrix represents a process of largely unknown functional significance. Here, we identified a hypomorphic allele, brpnude, lacking merely the last 1% of the C-terminal amino acids (17 of 1740) of the active zone protein Bruchpilot. In brpnude, electron-dense bodies were properly shaped, though entirely bare of synaptic vesicles. While basal glutamate release was unchanged, paired-pulse and sustained stimulation provoked depression. Furthermore, rapid recovery following sustained release was slowed. Our results causally link, with intramolecular precision, the tethering of vesicles at the AZ cytomatrix to synaptic depression.

Figure 1.

Impaired vesicle tethering at AZs of a bruchpilot mutant (brp^nude) lacking the last 17 C-terminal amino acids. A, Chemical induction of premature stop codons resulted in truncated versions of the BRP protein (BRP; dark blue; boxes indicate coiled-coil domains) of ∼99% (brp^nude; gray), of ∼70% (brp^1.3; green), and of ∼50% length (brp^5.45; blue). Below, Illustration of the BRP protein (blue) within the AZ (red: Ca²⁺ channels; gray: synaptic vesicle) and the corresponding positions of the truncation (arrows) (Fouquet et al., 2009). B, All three BRP mutants (brp^nude, brp^1.3; brp^5.45) showed severely impaired survival rates and walking skills. C, In the brp^nude allele, a single base mutation at the C-terminal position 1724 leads to a premature stop codon and a BRP protein (BRP^nude) lacking the last 17 of 1740 aa. D, STED images of neuromuscular junctions stained with a C-terminal BRP antibody (green; BRP^Nc82) and simultaneous confocal images of an N-terminal BRP antibody (magenta; BRP^N-term). The distribution of both antibody signals appears unaltered at brp^nude synapses (right) compared to controls (left). Arrows and arrowheads indicate planar and vertical AZs, respectively. E, Examples of conventionally embedded AZs of control and brp^nude. Note fewer tethered vesicles in brp^nude. F, Height and length of the platform of the electron-dense cytomatrix (T-bar) for control and brp^nude. G, The number of docked vesicles per AZ section for control and brp^nude (n = 22 and 25 AZs, respectively) and the number of vesicles found within three shells (see inset) each of 50 nm thickness surrounding the AZ (n = 20 and 31 AZs, respectively). Scale bars: D, 1 μm; E, 200 nm.

Figure 2.

Normal Ca²⁺ channel clustering and basal release at brp^nude synapses. A, GluRIID labels (magenta, confocal) were used to quantify the Ca²⁺ channel (Cac^GFP, green, STED) clusters in control animals (top row) and brp^nude mutants (below). The Cac signals at synapses that appeared ideally planar (n = 16 each) were averaged after alignment with the postsynaptic GluRIID signal (right panels) or themselves (center panels). While the example images were scaled up individually, all four averages were scaled up with the same factor. B, Intensity profiles of the average Cac images in black for controls and in gray for brp^nude mutants with SE bars and the corresponding Gaussian fits (standard deviations given by σ) in gray. C, Examples of EPSCs elicited at 0.2 Hz in control (black), brp^nude (gray), brp^1.3 (green), and brp^5.45 mutants (blue; average of 10 each) in 1.0 mm Ca²⁺. The average peak EPSC amplitudes and rise and decay kinetics were normal in brp^nude mutants. In brp^1.3 and brp^5.45 mutants EPSC amplitudes were reduced and rise times increased (n = 22, 18, 4, and 12 for control, brp^nude, brp^1.3, and brp^5.45, respectively). D, Example traces of mEPSCs of control (black), brp^nude (gray), brp^1.3 (green), and brp^5.45 mutants (blue). The average mEPSC amplitudes were normal in brp^nude, brp^1.3, and brp^5.45 mutants (n = 6, 6, 4, and 6 for control, brp^nude, brp^1.3, and brp^5.45, respectively). Scale bar in A, 250 nm.

Figure 3.

Tethering vesicles to the AZ dense body prevents synaptic depression. A, Examples of EPSCs elicited by a paired-pulse protocol with 30 ms interpulse interval in control (black) and brp^nude (gray). B, Average paired-pulse ratios plotted versus interpulse interval in 1.0 mm Ca²⁺ for control (black, n = 12) and brp^nude (gray, n = 9; asterisk indicates significant differences) superimposed with exponential fits (time constants are indicated). Inset, 0.6 mm Ca²⁺. C, Average peak EPSC amplitudes during the 60 Hz train for control (black, n = 21) and brp^nude (gray, n = 20). Inset, Average amplitudes of the first 5 EPSCs of the train. The steady-state amplitude (average of EPSC amplitudes marked by the bracket) was significantly reduced in brp^nude mutants compared to controls. D, Steady-state amplitudes normalized to the steady-state amplitude of the respective control, for brp^nude (gray), brp^1.3 (green), brp^5.45 (blue), and Cac RNAi synapses (red) in extracellular Ca²⁺ concentrations that lead to normal (“rescued”) basal EPSC amplitudes ([Ca²⁺]_rescue) (see supplemental Fig. S2, available at www.jneurosci.org as supplemental material). E, Average peak EPSC amplitudes during the first rapid component of recovery after the train (τ₁) for control (black, n = 21) and brp^nude (gray, n = 20) fitted exponentially (lines). Time constants with SEs are indicated. Bottom, The time constants of the first component of recovery normalized to the corresponding time constant of the control (color code as in D). F, Corresponding average peak EPSC amplitudes during the second slower component of recovery after the train (τ₂) with corresponding average data.