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Comparative Study
, 27 (22), 5857-68

Presynaptic G-protein-coupled Receptors Regulate Synaptic Cleft Glutamate via Transient Vesicle Fusion

Affiliations
Comparative Study

Presynaptic G-protein-coupled Receptors Regulate Synaptic Cleft Glutamate via Transient Vesicle Fusion

Eric J Schwartz et al. J Neurosci.

Abstract

When synaptic vesicles fuse with the plasma membrane, they may completely collapse or fuse transiently. Transiently fusing vesicles remain structurally intact and therefore have been proposed to represent a form of rapid vesicle recycling. However, the impact of a transient synaptic vesicle fusion event on neurotransmitter release, and therefore on synaptic transmission, has yet to be determined. Recently, the molecular mechanism by which a serotonergic presynaptic G-protein-coupled receptor (GPCR) regulates synaptic vesicle fusion and inhibits synaptic transmission was identified. By making paired electrophysiological recordings in the presence and absence of low-affinity antagonists, we now demonstrate that activation of this presynaptic GPCR lowers the peak synaptic cleft glutamate concentration independently of the probability of vesicle fusion. Furthermore, this change in cleft glutamate concentration differentially inhibits synaptic NMDA and AMPA receptor-mediated currents. We conclude that a presynaptic GPCR regulates the profile of glutamate in the synaptic cleft through altering the mechanism of vesicle fusion leading to qualitative as well as quantitative changes in neural signaling.

Figures

Figure 1.
Figure 1.
Three-dimensional reconstruction of a reticulospinal synapse. A, Actin clusters in presynaptic active zones of reticulospinal axons (green) are visualized by phalloidin toxin conjugated to Alexa 488 fluoro. Postsynaptic spinal neurons are filled with Alexa 568 fluoro dextran (red). The patch electrode is seen on the bottom right portion of the image. B, Enlargement of A. Dendrites of the spinal neuron (red) oppose presynaptic active zones (green) at several distinct locations along the axon. C, Left, Schematic for recording synaptically paired neurons. The presynaptic axon (green) is recorded with a microelectrode, and the postsynaptic spinal neuron is recorded with a patch electrode (red). Right, Action potentials evoked in the presynaptic axon (green trace) resulted in corresponding EPSCs in the spinal neuron (red traces). D, Enlargement of image in B. The arrowhead denotes the annular fluorescent pattern of phalloidin-labeled actin clusters. Measurements of the diameter (1) and shortest distance between actin clusters (2) were made. E, A concentration of 10 μm GYKI abolished the chemical component of synaptic transmission to reveal the isolated electrical component (gray). F, A low dose of 5-HT (300 nm; gray) partially inhibits the chemical component of the EPSC.
Figure 2.
Figure 2.
5-HT increases the potency of Kyn-mediated inhibition indicative of a reduction in the peak glutamate cleft concentration. A, A concentration of 200 μm Kyn (red) reduced the EPSC to 48% of control (black). A concentration of 300 nm 5-HT (bottom, gray) reduced the EPSC to 46% of control. Application of 200 μm Kyn with 5-HT (Kyn&5-HT; bottom, blue) reduced the EPSC to 14% of the EPSC in 5-HT alone. The pure electrical component was revealed by application of 20 μm CNQX (thin black traces). B, EPSCs for all four conditions. Subtraction of the electrical component of the EPSCs reveals isolated AMPA receptor responses (left). EPSCs recorded in 200 μm Kyn with 300 nm 5-HT (Kyn&5-HT; blue) and 300 nm 5-HT (gray) are scaled (dashed lines) to control (right, black). 5-HT increased the potency of 200 μm Kyn (blue vs red traces). C, FM2-10 dye destaining from synaptic vesicles. Ci, Cii, Image of axon stained with FM2-10 dye before (Ci) and after (Cii) stimulus-evoked destaining of FM1-43 dye from vesicles in control conditions (1 Hz, 5000 action potentials). The graph of normalized florescent intensity versus stimulus number demonstrates Kyn (open circles; 200 μm) does not alter FM2-10 dye destaining from fusing vesicles during synaptic transmission. Error bars indicate SEM.
Figure 3.
Figure 3.
5-HT does not increase the potency of high-affinity noncompetitive AMPA receptor antagonist GYKI-mediated inhibition. A, The high-affinity noncompetitive AMPA receptor antagonist GYKI (3 μm; dark gray) reduced the EPSC to 58% of control (black). 5-HT (300 nm) alone inhibited the EPSC to 48% of control (bottom, gray). Application of 3 μm GYKI with 300 nm 5-HT (light gray) reduced the EPSC to 56% of the EPSC in 5-HT alone. B, EPSCs recorded in control (black and dark gray traces) and 5-HT (gray and light gray traces) are scaled to the same size, and the electrical components are subtracted for clarity. In contrast to Kyn, the inhibitory potency of GYKI is not altered by 300 nm 5-HT (right, compare light gray vs dark gray traces).
Figure 4.
Figure 4.
Inhibiting synaptic transmission by lowering Pr does not alter the peak concentration of synaptic glutamate transients. A, An intermediate dose of Kyn (150 μm; dark gray) inhibited synaptic transmission to 83% of control (black). Raising the concentration of Mg2+ (from 1.8 to 3.8 mm; gray trace) to decrease Pr reduced the EPSC to 66% of control. Simultaneously applying Kyn and raising the concentration of Mg2+ (light gray) reduced the EPSC to 82% of the EPSC in high-Mg2+ alone (gray). B, EPSCs recorded in control (black and dark gray traces) and in high Mg2+ (gray and light gray traces) are scaled (right) to the same size after subtraction of the electrical component. High Mg2+ does not increase the potency of inhibition mediated by Kyn (compare dark gray vs light gray traces).
Figure 5.
Figure 5.
Paired-pulse facilitation. A, The effects of 3.8 mm Mg2+, Kyn, and 5-HT (gray traces) on paired-pulse facilitation were compared with control (black traces). Raising the Mg2+ concentration to 3.8 mm (top) nearly doubles the paired-pulse ratio, whereas neither Kyn (middle) nor 5-HT (bottom) altered the paired-pulse ratio. B, Bar graph depicting the paired-pulse ratios under the conditions of raised Mg2+ concentration (3.8 mm), Kyn (200 μm), and 5-HT (600 nm). The number of pairs recorded for each condition is shown in parentheses. Error bars indicate SEM.
Figure 6.
Figure 6.
5-HT slightly prolongs the decay of the EPSC. Subtraction of the electrical component from the traces reveals the isolated chemical component of the EPSCs. A, Top, Average of 12 consecutive traces in control (black) and 300 nm 5-HT (gray). Bottom, The average response in 300 nm 5-HT (gray) is scaled to control (black). Analysis of 12 individual EPSCs reveals that the decay (τ) of the EPSC is slightly prolonged in 5-HT (bottom). The mean of single exponential fits from 12 individual trials are shown for 5-HT (gray) and control (black). B, Average of 12 consecutive traces in control (black) and 3.8 mm Mg2+ (gray). Responses in 3.8 mm Mg2+ (gray) are scaled to control (black). The decay of the EPSC is unchanged in 3.8 mm Mg2+. Mean exponential fits from 12 individual trials are shown for 3.8 mm Mg2+ (gray) and control (black). C, Plot of values of τ in milliseconds versus amplitude of the EPSCs (in D) recorded in sequential stimuli. D, Individual traces (left) are scaled to the largest trace (right) to directly compare the decay of the EPSC.
Figure 7.
Figure 7.
Probing the relative glutamate affinities of AMPA and NMDA receptors with caged glutamate. A, Neuron recorded with an electrode containing fluorescein (50 μm). The uncaging laser was targeted to the circled point in the bottom left region (contrast expanded to reveal dendrite). B, Experimental setup. Neurons were recorded with an electrode containing fluorescein (50 μm). A double-barreled pipette superfused caged glutamate over the recorded neuron, and NMDA (blue) or AMPA (red) receptor-mediated responses were isolated pharmacologically. C, At the time marked by the arrow and the dotted line, the region marked by the white dot in A was irradiated with 357 nm light (4 ns) at a relative intensity indicated by the numbers, while d-AP-5 and caged glutamate were applied. Increasing intensity revealed an AMPA receptor-mediated response. D, The experiment in C was repeated, but NBQX was substituted for d-AP-5. This evoked an NMDA receptor-mediated response with a lower activation threshold than that of AMPA and which saturated at intensities that only marginally activated AMPA responses. E, Relative flash intensity plotted versus current amplitude for NMDA (blue) and AMPA (red) receptor-mediated currents (n = 3). Error bars indicate SEM.
Figure 8.
Figure 8.
Confirming 5-HT GPCR-mediated reductions in synaptic cleft glutamate concentration at colocalized NMDA receptors. A, Spontaneous EPSCs recorded in control (top) and Mg2+-free external solution. B, Cumulative frequency and peak amplitude data are plotted for the same cell shown in A. No change in event frequency is seen after wash of Mg2+, indicating that no new event population is revealed. In addition, no change in peak amplitude of the data was recorded, consistent with a larger AMPA receptor-mediated event even after removal of Mg2+. C, Averaging the responses reveals the decay is prolonged in the Mg2+-free external solution, consistent with both NMDA and AMPA receptors mediating the responses. D, Overlaid traces of evoked NMDA and AMPA EPSCs (Vh = +50 mV) and AMPA EPSCs (Vh = −70 mV) recorded at the same synapse. E, Average of traces in D. The NMDA receptor component was measured as the average amplitude during the time points indicated by the shaded region, and the AMPA receptor component was measured as the peak (arrow). Red lines represent exponential fits to decay time constants for the AMPA and NMDA EPSCs. F, Plot of the amplitude of the NMDA versus AMPA EPSCs at −70 mV (left) and +50 mV (right). Linear fits demonstrate a strong correlation (right, red line). G, NMDA EPSCs isolated in Mg2+-free Ringer's solution and 20 μm GYKI. The magnitude of inhibition mediated by 300 nm 5-HT on NMDA receptor EPSCs is magnified by the low-affinity NMDA receptor antagonist l-AP-5 (200 μm). A concentration of 200 μm l-AP-5 inhibits the EPSC amplitude to 52.1 ± 10.2% of control (bottom black trace; n = 4). 5-HT and l-AP-5 inhibit the EPSC to 48.6 ± 6.5% of the response in l-AP-5 (top gray trace; n = 4). H, Bar graph depicting the magnified inhibitory effect of 300 nm 5-HT on NMDA EPSCs in the presence of the low-affinity NMDA receptor antagonist l-AP-5. A concentration of 300 nm 5-HT alone inhibits the EPSCs to 88.0 ± 6.6% of control (left; n = 10). The inhibitory effect of the same dose of 5-HT on NMDA EPSCs is significantly greater in 200 μm l-AP-5 (p < 0.05, two-sample equal-variance t test). *p < 0.05, significance. Error bars indicate SEM.
Figure 9.
Figure 9.
Differential effect of 5-HT on NMDA and AMPA receptor components. A, Dose–response curves for isolated AMPA (red) and NMDA (blue) synaptic responses at the reticulospinal synapse. The arrow denotes differential inhibition of NMDA and AMPA responses to 300 nm 5-HT. Error bars indicate SEM. B, Paired recording in which 300 nm 5-HT inhibits AMPA receptor-mediated synaptic currents (red). C, The same dose of 5-HT (300 nm) only modestly inhibits NMDA receptor-mediated synaptic currents (blue). D, Example traces of the effect of 300 nm 5-HT (gray) in control (top, black) Ringer's solution and 300 nm 5-HT (gray) in Mg2+-free Ringer's solution (bottom, black) for the same paired recording. E, Diagram depicting a possible mechanism of 5-HT GPCR-mediated reduction in synaptic cleft glutamate concentration. Activation of a presynaptic 5-HT GPCR promotes transient vesicle fusion and inhibits synaptic transmission through a direct interaction of Gβγ with the release machinery (Gβγ-partial fusion). Switching from complete fusion to Gβγ-partial fusion lowers the peak synaptic cleft glutamate (green) concentration. Because of the different glutamate binding characteristics of AMPA and NMDA receptors, reducing the synaptic cleft glutamate concentration differentially inhibits postsynaptic AMPA receptors (red) relative to NMDA receptors (blue). 5-HTR, 5-HT receptor.

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