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, 103 (11), 4281-6

G Protein Betagamma-Subunits Activated by Serotonin Mediate Presynaptic Inhibition by Regulating Vesicle Fusion Properties

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G Protein Betagamma-Subunits Activated by Serotonin Mediate Presynaptic Inhibition by Regulating Vesicle Fusion Properties

Huzefa Photowala et al. Proc Natl Acad Sci U S A.

Abstract

Neurotransmitters are thought to be released as quanta, where synaptic vesicles deliver packets of neurotransmitter to the synaptic cleft by fusion with the plasma membrane. However, synaptic vesicles may undergo incomplete fusion. We provide evidence that G protein-coupled receptors inhibit release by causing such incomplete fusion. 5-hydroxytryptamine (5-HT) receptor signaling potently inhibits excitatory postsynaptic currents (EPSCs) between lamprey reticulospinal axons and their postsynaptic targets by a direct action on the vesicle fusion machinery. We show that 5-HT receptor-mediated presynaptic inhibition, at this synapse, involves a reduction in EPSC quantal size. Quantal size was measured directly by comparing unitary quantal amplitudes of paired EPSCs before and during 5-HT application and indirectly by determining the effect of 5-HT on the relationship between mean-evoked EPSC amplitude and variance. Results from FM dye-labeling experiments indicate that 5-HT prevents full fusion of vesicles. 5-HT reduces FM1-43 staining of vesicles with a similar efficacy to its effect on the EPSC. However, destaining of FM1-43-labeled vesicles is abolished by lower concentrations of 5-HT that leave a substantial EPSC. The use of a water-soluble membrane impermeant quenching agent in the extracellular space reduced FM1-43 fluorescence during stimulation in 5-HT. Thus vesicles contact the extracellular space during inhibition of synaptic transmission by 5-HT. We conclude that 5-HT, via free Gbetagamma, prevents the collapse of synaptic vesicles into the presynaptic membrane.

Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.
Fig. 1.
5-HT inhibits synaptic transmission by reducing quantal amplitude. (A) Paired recordings between axons and postsynaptic neurons. Presynaptic action potentials (Upper) evoke EPSCs (Lower). (B) 5-HT (300 nM) reduced the EPSC amplitude. (C) Electrode access resistance (Ra) remained stable. Means of 20 responses in control and at the experiment end. Stimuli preceded by a 5 mV voltage step. Ra estimated by fitting an exponential to the current decay. (D) Amplitude plot of EPSCs. After 200 stimuli, 5-HT (300 nM) inhibited EPSCs and abolished the distinction between failures and the unitary amplitude. (E) Amplitude histograms of data in D. Smooth curves centered at 0 pA are Gaussian distributions fitted to postsynaptic noise, measured before (E) and in 5-HT (F) with no statistical difference. The remaining curves are multiple Gaussians fitted to the data (Materials and Methods). (E) A significant difference is seen between failures and unitary events. No significant difference is seen between failures and noise. (F) In 5-HT, the unitary event amplitude is lost. (G and H) Amplitude histogram from five analyzed pairs before (G) and in 5-HT (H). Amplitudes normalized such that the unitary amplitude was 1 for each pair before averaging. The amplitude bins (gray) between failures and unitary events in control are significantly different in 5-HT.
Fig. 2.
Fig. 2.
Six hundred nanomolar 5-HT does not alter release probability. (Aa) Paired recording with no failures. (Ab) 5-HT (600 nM) reduced EPSC amplitudes but did not cause failures. NB in control and 5-HT, the electrical component, is present and invariant. The amplitude of the chemical component varies markedly. (B) From the example in A, EPSC amplitudes plotted before and in 5-HT. (C) Mean EPSC amplitudes and variances from data in A before and in 5-HT. 5-HT left the electrical component unaltered. (Da) Plot of M22 against mean in four pairs. Release probability was altered by raising extracellular Mg2+ by 1 mM (squares) or paired pulse stimulation (circles). Data normalized to the events with larger mean amplitude. Decrease in mean amplitude is coincident with a decrease in M22. (Db) Data are similar to Da but with five paired recordings before and in 5-HT (600 nM). Data are normalized to control. A decrease in mean amplitude accompanied by only a small decrease in M22 represents a change in the unitary amplitude. (Dc) Slopes of M22 vs. mean with time. Data from three sequential epochs of 50 responses in 5-HT. Mean slope is calculated for all five pairs by using the control value of M22 vs. mean as a reference. Data are errors ± SEM.
Fig. 3.
Fig. 3.
5-HT reduces FM 1-43 staining of the presynaptic terminal. (A) Schematic of FM1-43 staining protocol. Reticulospinal axon was recorded with a microelectrode and stimulated (1 Hz; 1,000 action potentials) in FM1-43 (4 μM) perfused over the spinal cord. Excess dye was cleared from the tissue with Advasep 7, and single confocal sections were imaged. (Ba) Image of an axon after labeling with FM1-43 in control. (Bb) After 2,000 stimuli to destain the axon, it was again stimulated with FM 1-43 (1 Hz; 1,000 action potentials) as in Ba but now in 5-HT (30 μM). An image was taken at the same location as Ba. No staining was resolved. (Bc) Bar graph comparing the control staining with the staining in 30 μM 5-HT (n = 20 synapses in four preparations). (Ca) Axon was labeled with FM1-43 as in Ba and imaged. (Cb) Same axon was destained (1,000 stimuli) and restained with FM1-43 in 5-HT (600 nM; 1 Hz; 1,000 action potentials). Staining was apparent at the same locations as Cb, but at reduced intensity. (Cc) Bar graph comparing control staining with staining detected in 600 nM 5-HT (15 synapses in three preparations).
Fig. 4.
Fig. 4.
5-HT prevents FM1-43 destaining. (A) FM1-43 was loaded into synaptic vesicles in lamprey giant axons. Images show after dye loading (Upper), and after destaining with 5,000 stimuli (Lower). (B) As in A, another axon was stained with FM1-43 (Upper). The axon was then stimulated 5,000 times in 5-HT (600 nM) (Lower). Destaining was blocked (n = 9 synapses, 3 preparations). (C) Destaining quantified in control (•), during inhibition of synaptic release with Mg2+ (4 mM, ○), or in 5-HT (600 nM, □). (D) 5-HT (600 nM; a) and Mg2+ (4 mM, b) inhibition of paired EPSCs. (E) Comparison of the inhibition of EPSCs or FM1-43 destaining by 5-HT (a) and Mg2+ (b). 5-HT more profoundly inhibited FM1-43 destaining than the EPSC, whereas Mg2+ showed similar inhibition of both.
Fig. 5.
Fig. 5.
Sulforhodamine quenches FM1-43 trapped in vesicles by 5-HT. (A) Extracellular sulforhodamine does not enter axons. Orthogonal views of the ventro-medial spinal cord: sagittal volume (Left) and volume from dorsal surface (Right). Sulforhodamine (25 μM) was superfused over the spinal cord (red). An axon was labeled with Alexa phalloidin 488 (green) to mark presynaptic terminals and to demonstrate how it penetrates the intracellular void unstained by sulforhodamine. (B) Sulforhodamine (25 μM) had no effect on the EPSC. 5-HT (10 μM) in sulforhodamine inhibited the EPSC as in control. Traces are the means of 10 sequential responses. (C) Sulforhodamine absorption overlaps FM1-43 emission. We excited FM1-43 at 488 nm and detected fluorescence with a bandpass filter (505–560 nm). Sulforhodamine is not excited directly at these wavelengths and does not emit at the detected wavelengths. (D) Stained axon destained in 600 nM 5-HT and sulforhodamine after 2,000 and 3,000 stimuli. (E) 5-HT (600 nM) prevents destaining. In sulforhodamine (25 μM) and 5-HT, destaining was close to complete. (F) Model showing loss of FM1-43 (green) from vesicles during control stimulation. (G) Model showing sulforhodamine (red) quenching FM1-43 trapped in the vesicle during stimulation in 5-HT.

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