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First published online October 12, 2006
doi: 10.1242/10.1242/jcs.03194

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The synapsin domain E accelerates the exoendocytotic cycle of synaptic vesicles in cerebellar Purkinje cells

Anna Fassio¹, Daniela Merlo^1,*, Jonathan Mapelli², Andrea Menegon³, Anna Corradi¹, Maurizio Mete¹, Simona Zappettini¹, Giambattista Bonanno^1,4, Flavia Valtorta³, Egidio D'Angelo² and Fabio Benfenati^1,5,

¹ Center of Neuroscience and Neuroengineering, Department of Experimental Medicine, University of Genoa, Italy
² Department of Cellular and Molecular Physiology and Pharmacology, University of Pavia, Italy
³ San Raffaele Scientific Institute, `Vita Salute' University and I.I.T. Unit of Molecular Neuroscience, Milan, Italy
⁴ Center of Excellence for Biomedical Research, University of Genoa, Italy
⁵ Unit of Neuroscience, The Italian Institute of Technology, Morego Central Laboratories, Genoa, Italy

View larger version (79K): [in a new window]   Fig. 1. Construction and PC-specific expression of the L7-pepE transgene. (A) The L7-pepE transgene was made by insertion of a synthetic mini-gene coding for the highly conserved 25 C-terminal amino acids of synapsin Ia (pepE; see box) into the L7AUG vector. (B) PCR analysis of DNA prepared from tail biopsies revealed insertion of the transgene in four distinct transgenic (Tg-1, Tg-2, Tg-3, Tg-4) mouse lines. WT, wild-type. (C) The expression of the L7-pepE transgene was assessed by RT-PCR experiments performed on total RNA prepared from the cerebella of wild-type (wt) and two L7-pepE (Tg-1 and Tg-2) mouse lines. Samples that were not incubated with reverse transcriptase (RT) are shown as negative controls. HPRT was used as an internal control. (D) In situ hybridization was performed on sagittal (a,b) or coronal (c-f) sections from the brains of wild-type (a,c,e) and Tg-1 (b) or Tg-2 (d,f) transgenic mice using a 35S-labelled L7-pepE antisense oligonucleotide probe. The hybridization signal, undetectable in wild-type sections, is intense in transgenic sections and exclusively restricted to the cerebellar cortex. At higher magnification (e,f), grains counterstained with cresyl violet are concentrated in PC somata. Bars, 2.8 mm (a,b); 1.2 mm (c,d); 100 µm (e,f).

View larger version (81K): [in a new window]   Fig. 2. Cerebellar anatomy and innervation of DCN neurons by Purkinje cells are preserved in L7-pepE mice. (A) Sections of wild-type (a,c,e) and Tg-2 (b,d,f) mice were stained with Cresyl Violet (a,b) or with an anti-calbindin D-28K antibody (c-f). The cerebella of transgenic and control mice are indistinguishable when considering size, foliation, layering of cerebellar cortex, morphology, density and arrangement of PC. Bar, 600 µm (a-d); 100 µm (e,f). (B) Double immunofluorescence with anti-calbindin D-28K (a,b,e,f) and anti-synapsin pepE (G304; c,d,g,h) antibodies in cerebellar sections from wild-type (a,c,e,g) and Tg-2 (b,d,f,h) mice. Synapsin pepE immunoreactivity in the cerebellar cortex (a-d) and DCN (e-h) of both groups has a typical distribution with intense staining of the molecular layer, negative PC somata and intense staining at the granule cell layer and in the DCN. Bar, 100 µm (a-d); 200 µm (e-h). (C) High magnification of wild-type (a) and Tg-2 (b) sections double-immunostained with anti-calbindin D-28K (green) and anti-synaptophysin (red) antibodies. PC terminals appear yellow because of the positive staining for both antibodies. Bar, 10 µm. (D) Number (mean ± s.e.m.) of synaptophysin-positive terminals and calbindin- and synaptophysin-positive terminals (PC terminals) in the DCN was calculated as described in Materials and Methods (n=4 per genoptype). PC nerve terminals represented 49±3.7% (means ± s.e.m.; n=4) and 51±2.7 (means ± s.e.m.; n=4) of total synaptophysin-positive DCN terminals in wild-type and Tg-2 mice, respectively.

View larger version (51K): [in a new window]   Fig. 3. The recombinant pepE is expressed in Purkinje cells and is targeted to nerve terminals. (A) Tg-2 PC double immunostained for calbindin (a) and pepE (b). PepE immunoreactivity is absent from the cell body (arrow) and concentrated in presynaptic boutons. Bar, 100 µm. (B) Primary cerebellar (CB) and neocortical (CTX) cultures from Tg-2 mice were solubilized at 8-10 DIV and subjected to Tris-tricine SDS-PAGE. Expression of synapsin I (10.22 antibody; Syn I), actin, calbindin, VAMP2 and recombinant pepE (G304 antibody) is shown. The first lane on the left contained synthetic pepE (50 ng). (C) Primary wild-type hippocampal neurons were transfected to overexpress either GFP-synapsin Ia (a), GFP-domain E (b) or GFP alone (c). Bar, 200 µm. (D) Expression and targeting of GFP-synapsin Ia (a) or GFP-domain E (b) were followed by counterstaining neurons with VAMP2 antibodies (c,d). Both synapsin Ia and domain E GFP chimeras are transported along the axon and localized in en-passant varicosities and VAMP2-positive axon terminals (see arrows in e,f). Bar, 100 µm.

View larger version (21K): [in a new window]   Fig. 4. Motor coordination is altered in L7-pepE mice. (A) Upper panel, representative footprint patterns of a wild-type and a L7-pepE mouse. Lower panel, comparison of step widths, step lengths and maximum differences in stride length (mdsl) between wild-type (black bars; n=20) and Tg-2 (grey bars; n=20) animals. Locomotion is similar in both groups and no ataxic phenotype is observed. (B) Vertical pole scores (means ± s.e.m.), recorded as described in Materials and Methods, are reported for wild-type (black bars; n=23), Tg-2 (light grey bars; n=23) and Tg-1 (dark grey bars; n=10) mice. Each animal was tested in three consecutive trials. *P<0.05, Newman-Kleus multiple comparison test. (C) Rotarod performance of wild-type (, n=9), Tg-1 (, n=9) and Tg-2 (, n=8) mice in the constant speed mode. The time (means ± s.e.m.) mice remained on the rotating rod at the various speeds are reported. *P<0.05, Newman-Kleus multiple comparison test. Inset, rota-rod motor learning of wild-type (, n=10) and Tg-2 (, n=10) mice in the constant acceleration mode. Individual learning curves were fitted using linear regression [Y-intercept, 162±19 for WT mice and 151±19 for Tg-2 mice (P=0.7); slope, 11.2±3.3 for wild-type mice and 1.5±2.9 for Tg-2 mice (P<0.05)]. The performances in the initial and final trials of the learning curve were significantly different only in wild-type mice (P<0.005), but not in Tg-2 mice (P=0.26, Student's t test).

View larger version (17K): [in a new window]   Fig. 5. [3H]-GABA release from L7-pepE DCN synaptosomes is impaired in response to ionomycin, but it is more efficient after repetitive depolarization. (A) DCN synaptosomes from wild-type (black bars), Tg-1 (light grey bars) and Tg-2 (dark grey bars) were depolarized with 12, 15 or 35 mM KCl. Results are expressed as stimulus-evoked overflow, means ± s.e.m. of 5-10 independent experiments. (B) DCN synaptosomes from wild-type, Tg-1 and Tg-2 mice were treated as in A, except that [3H]-GABA release was induced by 0.5 µM ionomycin. Bars are means ± s.e.m. of 6-8 independent experiments. *P<0.05, Dunnett's multiple comparison test vs wild-type. (C) DCN synaptosomes from wild-type (), Tg-1 () and Tg-2 () mice were treated as in A and subjected to three sequential depolarizing pulses with either 15 mM (left panel) or 35 mM (right panel) KCl. [3H]-GABA release evoked by each pulse is expressed in percent of the release evoked by the first stimulus (means ± s.e.m. of four to five independent experiments). *P<0.05 and **P<0.001, one way ANOVA followed by Duncan's multiple comparison test.

View larger version (26K): [in a new window]   Fig. 6. Depression induced by short trains at 100 Hz is less intense in L7-pepE mice. (A) Whole-cell patch-clamp recordings of P8-P10 DCN neurons from wild-type (left panel) and Tg-2 (right panel) mice. IPSC trains were obtained by stimulating PC axons at 100 Hz for 200 milliseconds. Facilitation is apparent and depends on temporal summation of IPSCs. Each train is the average of ten tracings to reduce synaptic variability. (B) Average of independent recordings performed as described above in wild-type (, n=4) and Tg-2 (, n=4) mice. Amplitudes were normalized to the first IPSC and fittings were performed with an exponential function of the form y(t)=Axe–t/+y0. Note the slower depression and the higher steady-state level observed in mutant animals. (C) Time constant () and steady-state amplitude (y0) of depression are shown as means ± s.e.m. for wild-type (white bars; n=4) and Tg-2 (black bars; n=4) mice. *P<0.05, Student's unpaired t test.

View larger version (32K): [in a new window]   Fig. 7. L7-pepE PC terminals exhibit milder depression and faster recovery after prolonged high-frequency stimulation. (A) Effect of prolonged stimulation trains (50 Hz for 2 minutes) on IPSC amplitude. A representative experiment shows the faster and more intense synaptic depression in wild-type mice (left) with respect to Tg-2 mice (right). (B) Left panel, IPSC amplitudes during the prolonged stimulation are expressed in percent of the first IPSC. The IPSC decrease was smaller in L7-pepE (, n=4) than in wild-type (, n=5) mice. Statistical analysis was performed on the steady-state levels (50-120 seconds) using the Student's unpaired t test (P<10–8). Right panel, the number of failures during prolonged stimulation trains was lower in L7-pepE (, n=4) than in wild-type (, n=5) mice. Data are reported as means ± s.e.m. Statistical analysis was performed on the steady-state levels (50-120 seconds) using the Student's unpaired t test (P<10–13).

View larger version (14K): [in a new window]   Fig. 8. The kinetics of release is faster in L7-pepE mice. (A) Representative IPSCs recorded from DCN neurons from wild-type and Tg-2 mice. IPSC traces were normalized to identical peak amplitudes. (B) Representative IPSCs recorded from PC-DCN synapses showing the shortening of the synaptic delay in IPSC in Tg-2 mice with respect to wild-type mice. (C) The histogram shows the mean values (± s.e.m.) for IPSC rise, decay and delay times obtained from wild-type (white bars; n=9) and Tg-2 (black bars; n=7) mice. IPSC rise time was measured as the time needed to rise from 10% to 90% of peak amplitude. IPSC decay time was evaluated by exponential fitting of the decay with the exponential function y(t)=Axe–t/. Statistical analysis was performed using the Student's unpaired t test; *P<0.05.

View larger version (86K): [in a new window]   Fig. 9. SV density and distribution is altered in L7-pepE mice. (A) Representative electron micrographs of PC terminals on DCN somata from wild-type (WT, left panel) and Tg-2 (right panel) mice. Bar, 1 µm. (B) Distribution of SVs in PC terminals from wild-type (black bars; n=60) and Tg-2 (grey bars; n=54) mice. The absolute number of SVs (means ± s.e.m.) located within successive 50 nm shells from the active zone (AZ) is shown. The frequency distribution of SVs as a function of the distance from the AZ was analyzed using the Kolgomorov-Smirnov and Mann-Whitney tests; P<0.05 for the 50-100 nm shell; P<0.01 for the intervals between 400 and 1400 nm. Inset, to analyze the distribution pattern of SVs, the number of SVs in the various shells was expressed in percent of the total SV number for each PC terminal of wild-type (WT, upper panel) and Tg-2 (lower panel) mice. The solid traces and dotted lines represent the mean and s.e.m. values, respectively.