Differential regulation of active zone density during long-term strengthening of Drosophila neuromuscular junctions - PubMed (original) (raw)

Differential regulation of active zone density during long-term strengthening of Drosophila neuromuscular junctions

Dierk F Reiff et al. J Neurosci. 2002.

Abstract

In this study we established a transgenic Ca2+ imaging technique in Drosophila that enabled us to target the Ca2+ sensor protein yellow Cameleon-2 specifically to larval neurons. This noninvasive method allowed us to measure evoked Ca2+ signals in presynaptic terminals of larval neuromuscular junctions (NMJs). We combined transgenic Ca2+ imaging with electrophysiological recordings and morphological examinations of larval NMJs to analyze the mechanisms underlying persistently enhanced evoked vesicle release in two independent mutants. We show that persistent strengthening of junctional vesicle release relies on the recruitment of additional active zones, the spacing of which correlated with the evoked presynaptic Ca2+ dynamics of individual presynaptic terminals. Knock-out mutants of the postsynaptic glutamate receptor (GluR) subunit DGluR-IIA, which showed a reduced quantal size, developed NMJs with a smaller number of presynaptic boutons but a strong compensatory increase in the density of active zones. This resulted in an increased evoked vesicle release on single action potentials and larger evoked Ca2+ signals within individual boutons; however, the transmission of higher frequency stimuli was strongly depressed. A second mutant (pabp(P970)/+), which showed enhanced evoked vesicle release triggered by elevated subsynaptic protein synthesis, developed NMJs with an increased number of presynaptic boutons and active zones; however, the density of active zones was maintained at a value typical for wild-type animals. This resulted in wild-type evoked Ca2+ signals but persistently strengthened junctional signal transmission. These data suggest that the consolidation of strengthened signal transmission relies not only on the recruitment of active zones but also on their equal distribution in newly grown boutons.

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Figures

Fig. 1.

Fig. 1.

Transgenic expression of the Ca2+ sensor molecule yCam2 in_Drosophila_ neurons. A, yCam2 fluorescence in elav-Cam transgenes (see Materials and Methods) highlights all neurons in the larval brain, including motoneurons, their axonal projections to the hemisegments of the periphery, and the presynaptic terminals of NMJs at the larval body wall musculature (B). Note the lack of fluorescence in non-neuronal tissues. Scale bars: A, 100 μm;B, 10 μm. C, Simultaneous dual emission recording of yCam2 was performed by exciting yCam2 in presynaptic terminals of larval NMJs at 435 nm and isolating the emission fluorescence of ECFP and EYFP using a beam splitter and appropriate filters (see Materials and Methods). ECFP and EYFP fluorescence signals were recorded with two synchronously operated CCD cameras.

Fig. 2.

Fig. 2.

Presynaptic Ca2+ imaging at_Drosophila_ NMJs using transgenically expressed yCam2.A, yCam2 fluorescence in elav-Cam transgenes highlights all presynaptic terminals of larval NMJs. Boutons within the focal plane of the image (blue-circled areas) were selected for quantitative analysis. Scale bar, 15 μm. B, False color images encode positive and negative fluorescence changes of EYFP (530 nm) and ECFP (485 nm) during nerve stimulation, which are indicative of FRET between excited ECFP and EYFP.C_–_E, Quantitative analysis of yCam2 fluorescence changes during spike-train stimulation (red bars). C1, Consecutive recordings of the EYFP (▵) and ECFP (▴) emission intensities show opposing signal changes during stimulation (40 Hz, 1.3 sec; 5 m

m

Ca2+) resulting in an EYFP/ECFP ratio change of 28% (line). C2, All individual boutons marked in A show almost identical evoked fluorescence changes (thin lines), which on average (thick lines) reach Δ_F_/F peak values of +14.4 ± 0.5% (EYFP) and −11.0 ± 0.4% (ECFP). EYFP emission changes depend on extracellular Ca2+concentration (stimulus: 40 Hz, 1.3 sec; 119 boutons; 6 animals) (D) and on the frequency of presynaptic spike trains (stimulus: 1.3 sec; 1 m

m

Ca2+; 58 boutons; 4 animals) (E).

Fig. 3.

Fig. 3.

Simultaneous dual emission imaging allows a reliable analysis of yCam2 fluorescence changes on a single bouton level. A, Top panel shows a raw image of a yCam2-expressing NMJ (C155-Cam_dglurIIA_). Black arrows mark an underlying nerve. The false color images encode EYFP/ECFP ratio changes (Δ_R_/R; see Materials and Methods) before or after and during nerve stimulation (40 Hz; 3.5 sec; 1 m

m

Ca2+). Note that Δ_R_/R changes were detected only in presynaptic boutons and not in yCam2-expressing nerves and axons or areas outside of NMJs. B, The time course of Δ_R_/R changes is represented for a single bouton (A, white arrow) in a series of false color images (time in seconds is indexed) or plotted in C. We detected a rapid onset of Δ_R_/R changes that reached up to 40% on a single bouton level. Scale bars: A, 6 μm;B, 10 μm.

Fig. 4.

Fig. 4.

yCam2 expression in larval neurons does not affect the junctional physiology of wild-type and mutant animals. Representative traces of mEJCs (A1) and eEJCs (A2) of the symbolized genotypes (C). B, C, yCam2 expression did not alter the amplitudes of mEJCs or eEJCs in wild-type controls [elav-Cam (⋄); wild type (▪)]. The derived junctional quantal contents (mean eEJC divided by mean mEJC) therefore remained unchanged (p > 0.5). Likewise, yCam2-expressing mutant animals showed similar phenotypes as reported previously for the mutants themselves. C155-Cam_dglurIIA_NMJs (●) have significantly reduced mEJC amplitudes (#p ≪ 0.0001), unaltered eEJC amplitudes, and an increased junctional quantal content compared with C155-Cam controls (*p < 0.001). elav-Cam_pabp_NMJs (▴) show unaltered mEJCs, significantly larger eEJCs, and thus an increased junctional quantal content compared with elav-Cam controls (*p ≪ 0.001). Note that in both mutants the junctional quantal content and thus the rate of evoked vesicle release are similarly increased relative to wild type. The muscle input resistance (R_in) in C155-Cam_dglurIIA and elav-Cam_pabp_ is similar to that of wild-type animals (p = 0.35). The decreased _R_in of C155-Cam (■) muscles compared with wild type and the associated slight increase in mEJC (**p < 0.002) and eEJC amplitudes is likely attributable to the somewhat larger muscle sizes of C155 animals (our unpublished observations). The number of analyzed cells is shown in B.

Fig. 5.

Fig. 5.

Typical and atypical relationship between the strength and size of NMJs. A, B, A morphological analysis of Fasciclin II-labeled NMJs (see Materials and Methods) revealed that elav-Cam_pabp_ NMJs (▴;n = 27) develop more boutons and C155-Cam_dglurIIA_ animals (●; n = 17) develop fewer boutons than control larvae [elav-Cam (⋄;n = 43); C155-Cam (■**;**n = 21), wild type (▪; n = 33); *p ≪ 0.001], all of which have been reared under normalized culture conditions. Raising of wild-type larvae at 29°C (×; n = 39) resulted in a small but significant increase of NMJ size compared with animals reared at 28°C (▪; #p < 0.05). C, Consistent with previous findings without yCam2 expression, the strength of junctional signal transmission (junctional quantal content) correlated significantly with the size of NMJs (r = 0.98). This tight structure–function relationship was disrupted in C155-Cam_dglurIIA_ larvae (●), which showed elevated vesicle release from fewer presynaptic boutons. All data are plotted as means ± SEM.

Fig. 6.

Fig. 6.

Comparison of spike train-evoked presynaptic Ca2+ signals from NMJs of various genotypes.A, Simultaneous EYFP–ECFP imaging revealed that stimulation-evoked presynaptic Ca2+ dynamics (stimulus: 40 Hz, 3.5 sec, 1 mm Ca2+) are indistinguishable at elav-Campabp NMJs (195 boutons, 7 animals) and elav-Cam NMJs (130 boutons, 7 animals). Note that these genotypes differ in their junctional transmission strength, but they conform to the described correlation of junctional size and strength.B, The same stimulation protocol elicited a significantly larger EYFP/ECFP ratio change at C155-CamdglurIIA NMJs (318 boutons, 14 animals) compared with C155-Cam controls (283 boutons, 13 animals; *p ≪ 0.001). Data are given as means ± SEM (error bars are hidden within the_symbols_).

Fig. 7.

Fig. 7.

Comparison of the bouton ultrastructure of the indicated genotypes. A, Representative electron micrograph of an ultrathin section through a type Ib bouton (muscle 6, segment A2) of a dglurIIA-ko animal (dglurIIA AD9 /df(2L)clh4). Note that three of the four cut synaptic profiles (electron-dense areas between arrowheads) harbor a presynaptic T-bar profile (arrows). v, Presynaptic vesicles;m, mitochondrion; SSR, subsynaptic reticulum. B, C, Type Ib boutons of wild-type larvae and animals with genetically increased subsynaptic protein synthesis (pabpEP0310/df(2R)Pcl7) have similar dimensions, numbers of synaptic profiles, and presynaptic T-bar profiles per scored ultrathin section (B) and therefore show a similar number and density of cut active zone profiles (C, white and black bars, respectively; see Table 1). In contrast,dglurIIA-ko mutants showed a significant reduction in the measured presynaptic terminal area and a corresponding decrease in the number of cut synaptic profiles compared with wild-type and_pab_ mutants. Strikingly, the number of cut presynaptic T-bar profiles was significantly larger in DGluR-IIA-ko_mutants than in wild-type animals (B), resulting in a strong increase in the relative number and density of active zones per sampled section (C, white and_black bars, respectively; see Materials and Methods). Data (Table 1) are represented as branch means ± SEM.

Fig. 8.

Fig. 8.

Comparison of junctional signal transmission of the indicated genotypes during high-frequency stimulation.A, Representative average traces of eEJP recordings during repetitive stimulation at 7 Hz (9 stimuli). B, NMJs of elav-Cam_pabp_ animals (▴) faithfully transmitted presynaptic stimuli at frequencies of up to 20 Hz. At this frequency, wild-type NMJs (▪) showed considerable depression of postsynaptic potentials. C155-Cam_dglurIIA_ animals (●) showed strong depression of postsynaptic potentials at all examined stimulation frequencies. Data are given as means ± SEM.

Fig. 9.

Fig. 9.

Potential similarities of the cellular mechanisms underlying the persistent strengthening of signal transmission during development and plasticity of Drosophila NMJs.A, Under resting conditions, junctional boutons harbor T-bar-containing active zones at a certain density (Meinertzhagen et al., 1998) (Fig. 7). Nerve activity therefore results in a typical evoked Ca2+ influx (green) into presynaptic terminals (Fig. 6) and consequently in characteristic postsynaptic responses to released neurotransmitter (Figs. 4, 8). B, Reduced muscle depolarization during muscle growth (Petersen et al., 1997; Paradis et al., 2001) and elevated subsynaptic protein synthesis during activity-dependent long-term strengthening (Sigrist et al., 2000) generate retrograde signals that result in the recruitment of active zones to enhance evoked vesicle release. This leads to a transient increase in the density of active zones per bouton (Fig. 7), which is associated with an enhanced Ca2+ influx into presynaptic terminals (red) (Fig.6_B_). High active zone density and enhanced Ca2+ influx seem to compromise junctional signal transmission (as seen in dglurIIA-ko mutants) (Fig. 8). Active zone density and the associated Ca2+ dynamics are typically reset to normal by equally distributing active zones in newly grown boutons (C). Such a morphological consolidation of altered junctional signal transmission ensures that each active synapse can operate normally, although the total junctional signal transmission is strengthened (as seen in elav-Cam_pabp_ animals). In addition, these NMJs can now cycle through further rounds of long-term strengthening. Morphological consolidation of functional alterations therefore appears well suited for developing synaptic systems.

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