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.2006 Jan 11;26(2):372-80.
doi: 10.1523/JNEUROSCI.3997-05.2006.

Synaptotagmin IV does not alter excitatory fast synaptic transmission or fusion pore kinetics in mammalian CNS neurons

Affiliations

Synaptotagmin IV does not alter excitatory fast synaptic transmission or fusion pore kinetics in mammalian CNS neurons

Jonathan T Ting et al. J Neurosci..

Abstract

Synaptotagmin IV (Syt IV) is a brain-specific isoform of the synaptotagmin family, the levels of which are strongly elevated after seizure activity. The dominant hypothesis of Syt IV function states that Syt IV upregulation is a neuroprotective mechanism for reducing neurotransmitter release. To test this hypothesis in mammalian CNS synapses, Syt IV was overexpressed in cultured mouse hippocampal neurons, and acute effects on fast excitatory neurotransmission were assessed. We found neurotransmission unaltered with respect to basal release probability, Ca2+ dependence of release, short-term plasticity, and fusion pore kinetics. In contrast, expression of a mutant Syt I with diminished Ca2+ affinity (R233Q) reduced release probability and altered the Ca2+ dependence of release, thus demonstrating the sensitivity of the system to changes in neurotransmission resulting from changes to the Ca2+ sensor. Together, these data refute the dominant model that Syt IV functions as an inhibitor of neurotransmitter release in mammalian neurons.

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Figures

Figure 1.
Figure 1.
Upregulated Syt IV protein coimmunoprecipitates with synaptic vesicles in cultured neurons. Cultured neurons were infected with either the Syt IV or GFP viral constructs 15 h before harvesting and homogenization. The cleared cell lysate (250–500 μg of protein) was incubated with anti-synaptophysin or anti-synaptotagmin protein A Sepharose beads in the absence of detergent to immunoprecipitate (IP) intact synaptic vesicles (physin IP or Syt I IP, respectively). The control IP was performed with no antibody. Bound proteins were eluted and resolved on an SDS gel and then subjected to immunoblotting with the following antibodies (top to bottom): anti-SV2A, anti-Syt I, anti-Syt IV, anti-synaptophysin, and anti-porin. Cell lysate (1:50 of the IP input) served as a positive control.
Figure 2.
Figure 2.
Basal release probability is not altered by acute upregulation of Syt IV.A, Top, Representative EPSCs recorded from cultured autaptic mouse neurons expressing GFP, Syt IV, or R233Q. Delivery of a 1 ms step depolarization to +40 mV initiates an unclamped presynaptic action current (blanked for clarity) followed by an EPSC. The peak amplitude of each EPSC is indicated by a dotted line. Bottom, Summary of average EPSC size for uninfected wild-type neurons and neurons expressing the indicated proteins. The number of neurons in each group is indicated, and plotted values represent the mean ± SEM. *p = 0.04 (unpaired Student'st test).B, Top, Representative EPSCs recorded from cultured autaptic mouse neurons expressing GFP, Syt IV, or R233Q. The shaded region is the EPSC charge. Bottom, Summary of average EPSC charge for uninfected wild-type neurons and neurons expressing the indicated proteins. The number of neurons in each group is indicated, and plotted values represent the mean ± SEM. *p = 0.01 (unpaired Student'st test). n.s., Not significant.
Figure 3.
Figure 3.
Ca2+ dependence of release is not altered by acute upregulation of Syt IV, whereas R233Q-expressing neurons exhibit altered Ca2+ dependence of release.A, Top, Representative EPSCs recorded from a wild-type autaptic neuronin 1,2.5, and 10 mm external calcium. External magnesium concentration was held constant at 1.5 mm. Bottom, Relative EPSC size plotted versus external calcium concentration. (Note: in some cases, the plotted values are nearly indistinguishable.) The solid line is a best fit to the Dodge–Rahamimoff equation that enabled us to obtain an estimate of theKCa for uninfected wild-type neurons (KCa = 0.35 mm; see Materials and Methods). Neurons expressing GFP, Syt IV, or Syt I hadKCa values very similar to those for wild-type neurons (KCa = 0.36, 0.36, and 0.37 mm, respectively) and were well described by the wild-type calcium dependence curve.B, Relative EPSC size plotted versus external calcium concentration for neurons expressing R233Q (open circles). These data were best fit by a calcium dependence curve whereKCa = 0.45 mm (dashed line). Note the rightward shift of the R233Q curve relative to the matched wild-type control curve. A third Ca2+-dependence curve whereKCa = 0.88 mm (dotted line) is shown for R233Q rescue of Syt I null neurons (open triangles). The R233Q rescue curve shows an even stronger rightward shift relative to the wild-type curve. The number of neurons in each group is indicated, and plotted values represent the mean ± SEM. The error bars at some points are smaller than the symbols. [Ca]ext, External calcium concentration.
Figure 4.
Figure 4.
Paired-pulse ratio is not altered by acute upregulation of Syt IV but is increased by expression of Syt I R233Q.A, Top, Representative EPSCs recorded in paired-pulse mode from a wild-type autaptic neuron in 1, 2.5, and 10 mm external calcium ([Ca]ext). External magnesium concentration is held constant at 1.5 mm. The membrane potential was clamped at –60 mV, and EPSCs were evoked by a pair of 1 ms depolarizing steps delivered 45 ms apart. The paired-pulse ratio is defined as the peak amplitude of the second EPSC relative to the first EPSC. Bottom, PPR plotted versus external calcium concentration.B, Bar graph of PPRs in 2.5 mm external calcium. The number of neurons in each group is indicated, and plotted values represent the mean ± SEM. **p < 0.01 (unpaired Student'st test). n.s., Not significant.
Figure 5.
Figure 5.
Rate of depression during 20 Hz stimulation is not altered by acute upregulation of Syt IV but is reduced by expression of SytIR233Q.A, Example EPSCs in response to a stimulus train of 25 pulses at 20 Hz. Data were collected at a rate of one train every 40 s to allow full recovery between stimuli.B, The peak amplitude of each EPSC in the train was normalized (Norm) to the peak amplitude of the first EPSC and plotted versus time to show the rate of depression. The number of neurons in each group is indicated, and plotted values represent the mean ± SEM.
Figure 6.
Figure 6.
Kinetics of neurotransmitter release are not altered by acute upregulation of Syt IV.A, Degree of EPSC block (percentage) during application of 0.25 μm γ-DGG. The degree of block is measured as the reduction in EPSC peak amplitude relative to the average of predrug and washout EPSC peak amplitude. Inset, Example EPSC traces of predrug baseline, γ-DGG application, and EPSC recovery after drug washout for an uninfected wild-type neuron.B, EPSC peak amplitudes measured during application of 50 μm CTZ and normalized to predrug EPSC size (dotted line). Inset, Example EPSC traces of predrug baseline, CTZ application, and EPSC recovery after drug washout for a Syt IV-expressing neuron. The number of neurons in each group is indicated, and plotted values represent the mean ± SEM.
Figure 7.
Figure 7.
Rate of FM 4-64 destaining is not altered by acute upregulation of Syt IV.A, Time course of FM 4-64 fluorescence changes during electrical field stimulation at 10 Hz for 1 min. At each time point, the fluorescence of the release sites was normalized to the fluorescence value obtained immediately preceding stimulation. Images were collected at a rate of 1 Hz.B, Average fluorescence values of release sites obtained from the first image acquired during destaining experiments.C, Average percentage of the total fluorescence lost during the 10 Hz stimulation period. Values were calculated from five GFP and five Syt IV experiments with 20 release sites from each experiment, for a total of 100 release sites analyzed per group. Plotted values represent the mean ± SEM. a.u., Arbitrary units.
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References

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