Regulation of neurotransmitter release by synapsin III

Abstract

Synapsin III is the most recently identified member of the synapsin family, a group of synaptic vesicle proteins that play essential roles in neurotransmitter release and neurite outgrowth. Here, through the generation and analysis of synapsin III knock-out mice, we demonstrate that synapsin III regulates neurotransmitter release in a manner that is distinct from that of synapsin I or synapsin II. In mice lacking synapsin III, the size of the recycling pool of synaptic vesicles was increased, and synaptic depression was reduced. The number of vesicles that fuse per action potential was similar between synapsin III knock-out and wild-type mice, and there was no change in the quantal content of EPSCs; however, IPSCs were greatly reduced in synapsin III-deficient neurons. The density and distribution of synaptic vesicles in presynaptic terminals did not appear to be different in synapsin III knock-out mice in comparison to wild-type littermates. In addition to the changes in neurotransmitter release, we observed a specific delay in axon outgrowth in cultured hippocampal neurons from synapsin III knock-out mice. Our data indicate that synapsin III plays unique roles both in early axon outgrowth and in the regulation of synaptic vesicle trafficking.

Fig. 1.

Generation of synapsin III knock-out mice.A, Synapsin III targeting vector consisted of a 6.0 and a 4.7 kb homologous region flanking the neo^Rgene to replace the 2.0 kb fragment containing exon 1 (top line). Restriction map, location of exon 1, and the external probe are shown in the middle line. Structure of the synapsin III locus after homologous recombination is shown in thebottom line. Restriction enzyme sites are as indicated:H, HindIII; E,EcoRI; P, PstI;Xh, XhoI; Ss,SstI. Sizes of the EcoRI fragments in the wild-type and targeted alleles are shown. B, Southern blot analysis of EcoRI-digested tail DNA from wild-type (+/+), heterozygous (+/−), and homozygous (−/−) littermates using the external probe (left). The same blot was stripped and reprobed with the neo probe (right).C, Western blot analysis of expression levels of synapsin III and a few other synaptic vesicle proteins in brain homogenate from wild-type (+/+), heterozygous (+/−), and homozygous (−/−) littermates. KO, Knock-out; WT, wild type.

Fig. 2.

Synaptic vesicle recycling profiles in wild-type and synapsin III-deficient terminals. A, Size of the recycling pool of synaptic vesicles as measured by FM1–43 uptake in response to action potential stimuli. B, Release kinetics as measured by the time course for the depletion of FM1–43 staining. Time constants (τ) for wild-type and synapsin III-deficient boutons are shown. C, Vesicle repriming rates for wild-type and synapsin III-deficient terminals.

Fig. 3.

Evoked neurotransmitter release in wild-type and synapsin III-deficient neurons. A, Representative EPSC traces from wild-type (WT) and synapsin III-deficient neurons (KO). B, EPSC amplitude in neurons from wild-type (WT) or synapsin III knock-out mice (KO) (n= 71 and 69, respectively). C, Representative IPSC traces from wild-type and synapsin III-deficient neurons.D, IPSC amplitude in neurons from wild-type or synapsin III knock-out mice (n = 15 and 28, respectively).

Fig. 4.

Spontaneous neurotransmitter release in wild-type and synapsin III-deficient neurons. A, Representative mEPSC traces from wild-type (WT) and synapsin III-deficient neurons (KO). B, mEPSC amplitude in neurons from wild-type or synapsin III knock-out mice.C, mEPSC frequency in neurons from wild-type or synapsin III knock-out mice. D, Quantal content of EPSCs in neurons from wild-type (n = 29) or synapsin III knock-out (n = 32) mice.

Fig. 5.

Paired-pulse facilitation and synaptic depression at wild-type and synapsin III-deficient synapses. A, Paired-pulse facilitation was measured with the second pulse applied 20 msec after the first one. B, Synaptic depression was evoked by stimulating the neurons with a 20 Hz train. EPSCs were normalized to the initial response to measure the extent of synaptic depression.

Fig. 6.

Effect of synapsin III deletion on the ultrastructure of MFTs in the stratum lucidum of the dorsal hippocampal CA3 region. A, Representative MFT (mf) of a wild-type mouse perforated with two giant spines (Sp) of CA3 proximal apical dendrites. The terminal was densely packed with synaptic vesicles and some electron-dense mitochondrial profiles. B, Representative MFTs of a synapsin III knock-out mouse perforated with three giant spines (Sp). mf, Mossy fiber terminal;Sp, spine. Scale bar, 1 μm.

Fig. 7.

Effect of synapsin III deletion on the ultrastructure of presynaptic terminals within the stratum radiatum of the hippocampal CA1 region. A, Tracing of a representative axon terminal and adjacent postsynaptic spine forming an excitatory synapse. The drawing illustrates the postsynaptic density (1) and the areas where the number of vesicles was estimated: within 50 μm of the presynaptic membrane associated with the active zone (2) and between 50 and 200 μm of the presynaptic membrane (3). Docked vesicles were those physically attached to the presynaptic membrane. B, Representative excitatory synapse of a wild-type mouse. C, Representative excitatory synapse of a synapsin III knock-out mouse. Ax, Axon terminal;Sp, spine. Scale bar, 0.1 μm.

Fig. 8.

Delayed axon outgrowth in synapsin III-deficient neurons. A, Wild-type neurons were cultured for 24 hr and stained with anti-α-tubulin. B, Synapsin III^−/− neurons were cultured for 24 hr and stained with anti-α-tubulin. mp, Minor process;ax, axon. C, Distribution of wild-type and synapsin III^−/− neurons at different stages of development.