Article Figures & Data
Figures
- Figure 2.
Temperature dependence of larval locomotor activity. Larvae were reared at the indicated temperatures and video taped as mid third instar larvae (feeding stage) either from the wall of culture vials (a) or from the surface of isothermal agar plates (b-e). Shown are the following locomotor parameters during stretches of fast-forward locomotion (see Materials and Methods) of size-matched animals: moved distance per contraction wave (stride-length), frequency of contraction waves (stride-frequency), and the average speed of locomotion. To estimate the overall locomotor activity of larvae, we monitored the crawling distances (see Materials and Methods) of size-matched animals over 45 min (a, b) or 10 min (c-e). a, b, At a given temperature, wild-type animals showed similar locomotor parameters during stretches of fast-forward locomotion on both culture vials and agar plates. However, because of the strong differences of larval behavior within the food slurry of culture vials and on agar plates, the overall larval crawling distance was significantly larger on 25°C agar plates than in 25°C food vials. At 29°C the measured parameters of fast-forward locomotion were significantly higher than at 18°C, resulting in a significantly larger larval crawling distance per 10 min interval on 29°C agar plates versus 18°C agar plates (c). d, In dglurIIA-ko mutants the stride length remained unaltered at 18 and 29°C, whereas stride frequency and speed of locomotion showed significant temperature-dependent changes. However, these temperature-dependent differences did not significantly alter the crawling distance. e, pabpP970/+ mutants showed enhanced locomotor parameters during fast-forward locomotion at 18°C; however, the crawling distance over 10 min remained similar to wild type. Rearing at 29°C further increased the locomotor parameters and resulted in similarly larger crawling distances as in wild-type larvae. The number of animals was as follows: locomotor parameters: a, 21, 40, 42; b, 11, 13, 25; d, 5, 6; e, 11, 18; crawling distance: a, 4; b, 20, c,: 9, 8; d, 9, 19; e, 16, 11. Data represent means ± SEM.
- Figure 1.
Increased outgrowth of NMJs at 29°C rearing temperature. a,b, Confocal images of anti-HRP-labeled NMJs (muscle 6/7, abdominal segment 2) of wild-type larvae reared at 18°C (a) or 29°C (b). Scale bar, 20 μm. c-e, Quantification of NMJ size on muscle 6/7 of abdominal segment 2 (see Materials and Methods) in wild-type larvae (c), dglur-IIA-ko animals (dglurIIAAD9/df(2L)clh4) (d), and pabpP970/+ larvae (e) that have been reared at the indicated temperatures. Larvae reared at 29°C developed significantly larger NMJs than animals raised at 25 or 18°C (*p < 0.001). This effect was particularly prominent in pabpP970/+ larvae but significantly suppressed in dglur-IIA-ko animals (p << 0.001). Note that pabpP970/+ larvae showed a strong increase in bouton outgrowth at 25°C, whereas NMJs of wild-type and dglur-IIA-ko animals developed simple NMJs at this rearing temperature. Data represent means ± SEM.
- Figure 6.
Ultrastructural effects of elevated locomotor activity. a, Representative transmission electron microscopy image of a type Ib bouton of a wild-type larva (muscle 6/7, abdominal segment 2). Marked are synapses (dense areas between arrowheads), the subsynaptic reticulum (ssr), and a presynaptic dense body (T-bar, arrow). Large sequential series of such images were used to reconstruct junctional branches and analyze ultrastructural changes in larvae reared at 18 or 29°C (Table 1; see Materials and Methods). b, Rearing at 18 and 29°C resulted in a similar density of synapses with T-bars (i.e., active zones; gray bars) and without T-bars (white bars). Because 29°C larvae develop more synapse-harboring boutons, this lead to a significant increase in the total number of active zones per NMJ compared with 18°C reared larvae (black bars; p < 0.001). Data are taken from Table 1 and given as means ± SEM.
- Figure 3.
Experience-dependent strengthening of Drosophila NMJs. Two-electrode voltage-clamp recording of eEJCs and mEJCs from muscle 6 of abdominal segment 2. a, Shown are representative traces of mEJC recordings (top panels) and average traces of 10 consecutively recorded eEJCs of animals raised at either 18 or 29°C. b, All locomotor-stimulated animals showed a significantly larger junctional quantal content and thus enhanced junctional signal transmission compared with controls. This effect was already maximal after 2 hr of enhanced locomotor activity. Larval locomotor activity was acutely enhanced by transferring 18°C reared mid third instar larvae (feeding stage) onto agar plates (29°C) for 2 and 4 hr; chronic locomotor enhancement was achieved by continuously rearing larvae at 29°C (Fig. 2). From these animals we measured the muscle 6 input resistances Rin as an estimate of the relative muscle sizes (bottom panel, gray bars) and the amplitudes of spontaneous mEJCs and stimulation evoked eEJCs (top panel, gray and black bars). The derived junctional quantal content (mean eEJC amplitude/mean mEJC amplitude) (bottom panel, black bars) gives an estimate of the number of presynaptic vesicles released per action potential and thus represents a measure of the efficacy of evoked junctional signal transmission. Note that the slight but significant reduction of mEJCs in 4 hr stimulated animals is likely caused by the somewhat smaller muscle cells (larger Rin) of this animal population. Because the eEJC amplitudes are reduced proportionally in these cells, the quantal content is restored. The number of analyzed cells per experimental condition is given in b, below the top panel. c, Intracellular recordings of mEJPs and eEJPs in wild-type larvae and the temperature-sensitive paralytic mutant parats1 revealed that 2 hr of enhanced larval locomotor activity at permissive temperature (22°C agar plates) results in a consistent and significant strengthening of junctional signal transmission in both genotypes (wild type, n = 7; parats1, n = 6) compared with their 18°C reared siblings (wild type, n = 11; parats1, n = 9). These larvae were then treated with a temperature-shift protocol (20 min at 34°C followed by maintained 29°C on agar plates) that results in immediate paralysis of parats1 mutants, whereas wild-type larvae continue vigorous locomotion. Within 2 hr of paralysis the eEJP amplitudes of parats1 mutants dropped to the control value (n = 5), whereas signal transmission at wild-type NMJs was enhanced further (n = 6). The mEJP amplitudes of all three experimental conditions were similar (wild type 18°C:1.17 ± 0.07 mV; 2 hr 22°C plate:1.06 ± 0.08 mV; +2 hr paralysis protocol: 1.06 ± 0.05 mV; parats1 18°C: 1.02 ± 0.12 mV; 22°C plate: 0.91 ± 0.06 mV; +2 hr paralysis protocol: 1.14 ± 0.11 mV). d, The quantal content (expressed as percentage of control genotypes) is plotted as a function of the normalized NMJ size. Wild-type larvae that have been chronically reared at either 18 or 29°C develop NMJs with a typical relationship between NMJ size and transmission strength (black and white pentagons). A similar relationship has been described previously in several model genotypes of junctional plasticity, such as the transgenic overexpression of the glutamate receptor subunit DGluR-IIA (black and white circles) and the pabpP970/+ mutant (black square and white triangle) (Sigrist et al., 2002). However, wild-type animals that experienced acute locomotor stimulation showed enhanced junctional signal transmission in the absence of additional growth (white squares). Data represent means ± SEM. t test results: *p < 0.001; #p < 0.005.
- Figure 4.
Experience-dependent regulation of subsynaptic protein synthesis. a, Large eIF4e aggregates (Sigrist et al., 2000) appear locally within and close to the subsynaptic reticulum of junctional boutons (arrows). Scale bar, 5 μm. b-e, Quantification of large subsynaptic eIF4e aggregates (Sigrist et al., 2002). b, Larvae that showed vigorous locomotor activity on 18°C agar plates for 7 hr developed significantly more eIF4e-positive boutons than controls in standardized culture vials at 18°C. c, After a temperature-shift protocol, which can be used to paralyze the temperature-sensitive mutants parats1 for ∼3 hr (20 min at 34°C, then 29°C), wild-type animals showed vigorous locomotor activity and a significant increase in the number of eIF4e-positive boutons compared with 18°C reared animals (black bars). In contrast, the number of large subsynaptic eIF4e aggregates remained unaltered in paralyzed parats1 mutants compared with those at permissive 18°C (gray bars). d, Time course of subsynaptic eIF4e accumulation after the experimental induction of high locomotor activity by transferring larvae from standardized 18°C cultures vials onto agar plates (29°C). We observed a significant increase in the number of eIF4e-labeled boutons within 1 hr of high locomotor activity. e,pabpP970/+ mutants show a slight but not significant increase in the number of subsynaptic eIF4e aggregates in 18°C reared animals; rearing at 29°C results in a very large and highly significant increase of eIF4e aggregates. Data are plotted as means ± SEM; the numbers of analyzed segments are within bars or below symbols.
- Figure 5.
Experience-dependent upregulation of postsynaptic DGluR-IIA expression precedes the downregulation of Fasciclin II (Fas II). Shown are confocal images of boutons double labeled with antibodies recognizing the cell adhesion molecule Fasciclin II (left panels and green channel) and the postsynaptic glutamate receptor subunit DGluR-IIA (middle panels with enlarged insets and red channel). a, Control animals, which have been reared at constant 18°C in standardized larval cultures, show strong perisynaptic Fas II expression (arrowhead) and few DGluR-IIA positive synapses. b, After 4 hr of vigorous locomotor activity on 29°C agar plates, the perisynaptic Fas II expression was essentially unaltered (compare green channels), whereas DGluR-IIA showed an increased, ring-shaped immunoreactivity (arrows) at preexisting synapses (holes in Fas II expression, arrows). c, Larval rearing at 29°C, which is associated with chronically enhanced locomotor activity, leads to an enhanced overall DGluR-IIA immunoreactivity at an increased number of postsynaptic patches. The perisynaptic Fas II immunoreactivity is significantly downregulated (arrowhead, compare green channels). Scale bar, 5 μm. d, Quantification of immunofluorescence signals at NMJs (see Materials and Methods) revealed a significantly enhanced immunoreactivity of postsynaptic DGluR-IIA and a reduced immunoreactivity of Fas II in animals reared at 29°C compared with their 18°C reared sibling. Note that the enhanced DGluR-IIA immunoreactivity is caused by an increased number of DGluR-IIA patches and obviously stronger fluorescence signals per DGluR-IIA patch.
Tables
Rearing condition Characteristic 18°C 29°C Number of branches 6 5 Total length of branches (μm) 40.1 48.7 Total junction surface (μm2) 420.9 506.9 Number of dense areas 222 191 Number of dense areas with one T-bar (% of all) 116 (52.3%) 95 (49.7%) Number of dense areas with more than one T-bar (% of all) 19 (8.6%) 30 (15.7%) Number of dense areas without T-bar (% of all) 87 (39.2) 66 (34.6%) Mean size of dense areas (μm2) 0.33 ± 0.04 0.35 ± 0.02 Number of dense areas per surface area (μm2) 0.56 ± 0.03 0.42 ± 0.06 Number of dense areas with T-bars per surface area (μm2) 0.32 ± 0.04 0.27 ± 0.02 Number of dense areas with more than one T-bar per surface area (μm2) 0.05 ± 0.01 0.06 ± 0.01 Number of dense areas without T-bar per surface area (μm2) 0.23 ± 0.03 0.16 ± 0.03 Size of entire NMJ (arbitrary units) (number of boutons per muscle surface area) 1.50 ± 0.06 2.49 ± 0.07* Total number of active zones per entire NMJ 100% ± 4% 129% ± 4%* Summary of scored and derived data obtained from serial reconstructions of junctional branches (Sigrist et al., 2002). NMJs of wild-type larvae, which have been raised at either 18 or 29°C, distribute their active zones (dense areas with presynaptic T-bars) at similar densities (dense areas with T-bars per surface area). Note the unproportionally high number of complex synapses (number of synapses with more than 1 T-bar) in 29°C reared larvae. Larvae reared at 29°C develop larger NMJs and therefore harbor significantly more active zones than NMJs of 18°C reared animals (*p<0.01). The total number of active zones per NMJ was extrapolated by multiplying the measured NMJ sizes of each rearing condition (Fig. 1c) with the respective mean density of active zones. All data were obtained from individual branches and then combined to mean values (±SEM) per rearing condition.
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