Figure 1.
SV2A-EGFP is trafficked to presynaptic terminals.
Neurons expressing either EGFP or an SV2A-EGFP fusion protein were fixed and processed for immunolabeling. In neurons expressing EGFP green fluorescence was present throughout the length of neurites, whereas in neurons expressing SV2A-EGFP green fluorescence was punctate. Immunolabeling with anti-SV2 and anti-synaptotagmin revealed that the two proteins co-localized. Immunolabeling with anti-synaptotagmin and anti-synaptophysin revealed that synaptotagmin co-localized with synaptophysin. The co-localization of these three presynaptic proteins with SV2A-EGFP indicates that the exogenous fusion protein is trafficked to presynaptic terminals.
Figure 2.
Overexpression of SV2A-EGFP in wild-type neurons results in reduced EPSC amplitude and decreased synaptic depression.
A) Representative traces, average EPSC amplitude, and average EPSC charge transfer for cultured wild-type (WT) neurons expressing EGFP or SV2A-EGFP. B) (top) Representative traces of EPSCs induced by a 10 Hz, 2.5 sec stimulus in WT cells expressing EGFP or SV2A-EGFP. (bottom) Plot of averaged, normalized EPSC amplitudes in response to a 10 Hz stimulus train. Data are means ± SEMs. The numbers of cells recorded from is indicated in parentheses and is representative of at least 10 independent cultures. Asterisks indicate a significant difference in measurement when compared to control (student's t-test; *p<0.05).
Figure 3.
Addition of levetiracetam reverses the effect of SV2A overexpression in wild-type neurons.
Responses to a 2.5 sec, 10 Hz stimulus train recorded from WT neurons expressing EGFP or SV2A-EGFP that were incubated for 6–10 hrs in (A) 32 µM leviteracetam, (B) 100 µM leviteracetam, (C) 32 µM piracetam, or (D) 100 µM piracetam. Neurons incubated in 32 µM or 100 µM leviteracetam displayed normal WT-like synaptic depression. For each neuron, responses were normalized to the first response in the stimulus train. Graphs show mean ± SEM. The numbers of cells recorded from are indicated in parentheses and are representative of 10(A), 6(B), 5(C), and 4(D) independent cultures.
Figure 4.
Levetiracetam has no effect on wild type neurons and does not reverse the SV2A/B knockout neurotransmission phenotype.
Normalized averaged EPSC amplitude, normalized averaged EPSC charge transfer, and plot of normalized responses to a 2.5 sec, 10 Hz train recorded from neurons cultured (A) WT or (B) SV2A/B double knock-out mice (DKO). Neurons were incubated for 6–10 hrs with vehicle (water) or 100 µM levetiracetam. Shown are mean values ± SEMs. The numbers of cells recorded from are indicated in parentheses and are representative of 4(A), and 7(B) sets of cultures. Drug treatment had no significant effect on any measure.
Figure 5.
Effects of SV2-EGFP expression and levetiracetam treatment on synaptic levels of SV2 and synaptotagmin.
A) Neuronal processes labeled with an antibody against SV2 (all isoforms) revealed punctuate immunofluorescence along the lengths of the processes. Labeling was more intense in cells expressing SV2A-EGFP (SV2A) than in wild-type cultures infected with EGFP (EGFP-control). B) Expression of SV2A-EGFP increases the amount of SV2 at synapses and treatment with levetiracetam restores normal intensity levels. Shown is a graph of the average intensity of SV2 immunolabeling in individual puncta presumed to be synapses. Incubation with 100 µM levetiracetam (Lev) for 6–8 hours restored control levels of SV2. C) Expression of SV2A-EGFP increases the amount of total SV2 in neurites, and treatment with levetiracetam does not reverse the increase. Shown is a graph of the fluorescent immunolabeling of SV2 measured as the total intensity of fluorescence over the total length of neuronal processes in each image. Incubation with 100 µM levetiracetam (Lev) for 6–8 hours did not appear to affect on the amount of total SV2 expressed in the neuronal processes. D) Overexpression of SV2A increases the amount of synaptotagmin at synapses and treatment with levetiracetam restores normal intensity levels. Shown is a graph of the average intensity of synaptotagmin immunolabeling in individual puncta. Incubation with 100 µM levetiracetam (Lev) for 6–8 hours restored control levels of synaptotagmin. E) Overexpression of SV2A increases the total amount of synaptotagmin in neurites and treatment with levetiracetam restores normal levels. Shown is a graph of the fluorescent immunolabeling of synaptotagmin measured as in C. Incubation with 100 µM levetiracetam (Lev) for 6–8 hours restored normal synaptotagmin levels. F) Graph of the number of fluorescent puncta measured per length of neurite in neurons labeled with anti-synaptotagmin. G) Levetiracetam does not affect the co-localization of SV2 and synaptotagmin. Shown are graphs of the fluorescent intensity ratios of SV2 to synaptotagmin in synapses of the indicated cultures. Protein levels were measured using fluorescent immunolabeling. Bars represent the mean ± SEM. Numbers inside the bars indicate the number of images analyzed and are the same for B and C, and D and E. Data for B–E are from 4 separate cultures/replicates and for G from two cultures/replicates. To control for differences in antibody labeling between repetitions of the study, the intensity of each synaptic punctum was normalized to the average of all control (EGFP-infected) puncta within the same replicate. Asterisks indicate a significant difference in measurement when compared to EGFP-expressing neurons incubated with vehicle (one-way ANOVA followed by Fisher's LSD test; **p<0.01).