Synapsins differentially control dopamine and serotonin release

Abstract

Synapsins are a family of synaptic vesicle proteins that are important for neurotransmitter release. Here we have used triple knock-out (TKO) mice lacking all three synapsin genes to determine the roles of synapsins in the release of two monoamine neurotransmitters, dopamine and serotonin. Serotonin release evoked by electrical stimulation was identical in substantia nigra pars reticulata slices prepared from TKO and wild-type mice. In contrast, release of dopamine in response to electrical stimulation was approximately doubled in striatum of TKO mice, both in vivo and in striatal slices, in comparison to wild-type controls. This was due to loss of synapsin III, because deletion of synapsin III alone was sufficient to increase dopamine release. Deletion of synapsins also increased the sensitivity of dopamine release to extracellular calcium ions. Although cocaine did not affect the release of serotonin from nigral tissue, this drug did enhance dopamine release. Cocaine-induced facilitation of dopamine release was a function of external calcium, an effect that was reduced in TKO mice. We conclude that synapsins play different roles in the control of release of dopamine and serotonin, with release of dopamine being negatively regulated by synapsins, specifically synapsin III, while serotonin release appears to be relatively independent of synapsins. These results provide further support for the concept that synapsin function in presynaptic terminals varies according to the neurotransmitter being released.

Figure 1.

Representative traces of 5-HT release in coronal brain slices containing the SNr from WT and TKO mice evoked by a 100 Hz stimulation train (20 pulses, black line). Inset, The cyclic voltammogram is used for identification of 5-HT.

Figure 2.

DA release evoked by a single stimulation pulse (vertical black line) in striatal brain slices from WT or TKO animals. A, Representative responses of stimulated DA release; the time of stimulation is shown by the vertical bar. Inset, The cyclic voltammogram identifies the released neurotransmitter as DA. B, Mean [DA]_max from multiple locations of each genotype show an approximate twofold difference in striatal DA release (**p < 0.01, n = 8 TKO, n = 6 WT). C, Representative traces of DA release evoked by a single stimulus pulse in WT or S3KO animals. Time of stimulus is shown as a vertical black line. D, Measurements of DA release at multiple locations of each genotype indicate that S3KO slices have approximately double the amount of DA release measured in WT slices (*p < 0.05, n = 5 S3KO, n = 5 WT).

Figure 3.

The effects of repetitive stimuli on DA release. Single stimuli were applied at intervals of 0.3 s, 3 min, and 5 min, and the amount of DA released in response to each stimulus was normalized to the amount of DA released in response to the first stimulus.

Figure 4.

Electrically stimulated DA release in the striatum measured in vivo. A, Average release profiles measured in WT and TKO mice (n = 8 for each) in response to 0.5 s stimulus train (300 μA, 20 Hz; ***p < 0.001). B, [DA]_max values exhibit similar frequency dependencies but release is uniformly greater in TKO animals. C, [DA]_p measured from the frequency-dependent release shown in B (*p < 0.05). D, Although similar responses to stimulus intensity (60 Hz stimulations) are found in both genotypes, DA release is greater in TKO animals.

Figure 5.

Calcium dependence of DA release in striatal slices. A, Relative [DA]_max for TKO and WT animals at 0.5–5 mm extracellular [Ca²⁺]. TKO animals release significantly more DA at all [Ca²⁺]. B, Log-plot used to determine stoichiometric relationship between DA release and [Ca²⁺] using a fit to the Hill equation.

Figure 6.

Effects of cocaine on stimulated 5-HT and DA release and uptake in brain slices. Left traces, Representative responses of 5-HT release in SNr from WT (upper) and TKO (lower) mice in the presence and absence of 10 μm cocaine for 35 min. Right traces, Representative responses of DA release in striatal tissue from WT (upper) and TKO (lower) mice measured before and 35 min after the introduction of 2 μm cocaine.

Figure 7.

The effect of 2 μm cocaine on the Ca²⁺ dependence of DA release in striatal slices from WT and TKO mice. A, Relative [DA]_max in TKO and WT animals as a function of extracellular [Ca²⁺] in the presence of 2 μm cocaine. Data are normalized relative to the amount of DA release measured in WT slices at 2.4 mm Ca²⁺. TKO slices release more DA at all [Ca²⁺], although little difference in the Ca²⁺ sensitivity of release is seen between the genotypes when cocaine is present. B, Ratio of release in the presence of cocaine to that in its absence as a function of external [Ca²⁺] for WT and TKO animals (n = 6, each genotype); measured release was normalized to release at 2.4 mm Ca²⁺ for each genotype. In slices from WT mice, cocaine significantly increased electrically stimulated DA release at all [Ca²⁺] below 2.4 mm; however, TKO animals are only facilitated at low levels of [Ca²⁺]. ***p < 0.001 indicates significant difference from 2.4 mm Ca²⁺.