Action potential-independent and pharmacologically unique vesicular serotonin release from dendrites

Abstract

Serotonin released within the dorsal raphe nucleus (DR) induces feedback inhibition of serotonin neuron activity and consequently regulates mood-controlling serotonin release throughout the forebrain. Serotonin packaged in vesicles is released in response to action potentials by the serotonin neuron soma and terminals, but the potential for release by dendrites is unknown. Here, three-photon microscopy imaging of endogenous serotonin in living rat brain slice, immunofluorescence, and immunogold electron microscopy detection of VMAT2 (vesicular monoamine transporter 2) establish the presence of vesicular serotonin within DR dendrites. Furthermore, activation of glutamate receptors is shown to induce vesicular serotonin release from dendrites. However, unlike release from the soma and terminals, dendritic serotonin release is independent of action potentials, relies on L-type Ca(2+) channels, is induced preferentially by NMDA, and displays distinct sensitivity to the selective serotonin reuptake inhibitor (SSRI) antidepressant fluoxetine. The unique control of dendritic serotonin release has important implications for DR physiology and the antidepressant action of SSRIs, dihydropyridines, and NMDA receptor antagonists.

Figure 1.

Fluoxetine-sensitive depletion of puncta by pCA. A, Top, Summed fluorescence from 3P image stack using serotonin optics in a DR brain slice before and after a 20 min bath application of 20 μm pCA. Bottom, Single image planes from the boxed areas in A. Scale bars: Top, 20 μm; bottom, 2 μm. B, Quantification of puncta depletion by aCSF (CTL, n = 13), pCA (n = 16), 10 μm fluoxetine (n = 7), or fluoxetine and pCA (n = 6). pCA was different from each of the other conditions: ***p < 0.001. Error bars indicate SEM.

Figure 2.

VMAT2 is present in serotonin neuron dendrites. Top, Montage of single optical sections of the dorsal raphe nucleus immunolabeled for serotonin (5-HT, green), VMAT2 (red), and the dendritic marker MAP2 (blue). Scale bar, 10 μm. 5-HT and VMAT2 immunolabeling are present in somata (arrowheads in top panel), laterally traversing dendrites (A), dendrites cut in cross section (B), and axon-like processes (C). A, Merged image (left) and each fluorophore visualized independently (right). 5-HT has a diffuse, cytoplasmic distribution within dendrites and is also detected as small hot spots of immunolabeling (arrowhead). These often coincide with VMAT2 immunolabeling in structures identified as dendrites due to the presence of MAP2 (arrowheads). B, 5-HT immunolabeling in dendrites (2–3 μm in diameter; arrowhead and arrow). The arrowhead points to coincident 5-HT, VMAT2, and MAP2. C, 5-HT axon with varicosities also immunolabeled for VMAT2 are morphologically distinguished by their smaller size (<1 μm) and beaded morphology, and correspondingly lack MAP2 immunolabeling. Note that fine 5-HT-immunolabeled axon (arrowhead) is double labeled for VMAT2 but lacks corresponding MAP2 immunolabeling. Scale bar: Boxed region insets, 5 μm.

Figure 3.

Dendritic distribution of immunogold labeling for VMAT2 in the DR. A, At the ultrastructural level, dendritic profiles [Den, identified by their large cross section (outlined in yellow) and microtubules] contain immunogold labeling for VMAT2. Ax, Adjacent axons. B, Higher magnification of boxed area in A reveals immunogold labeling is associated with a cluster of clear vesicles (arrows) as well as a large dense-core vesicle (arrowheads). C, Dendrite cut in cross section contains a cluster of clear vesicles decorated with immunogold labeling for VMAT2 (arrows). D, In some cases, VMAT2 immunolabeling appeared associated with vesicular structures reminiscent of early endosomes and tubule vesicles (tv). A dense-core vesicle is also visible (arrowhead) in this dendrite.

Figure 4.

Dendritic localization and glutamate receptor-dependent depletion of puncta. A, Summed fluorescence from a stack of images of a sulforhodamine B-filled serotonin neuron (red). Scale bar, 10 μm. At this gain, the limited volume of axons precludes their detection by soluble sulforhodamine B fluorescence, but large tapering dendrites are evident. Inset, Single optical section showing overlay of serotonin (green) and sulforhodamine B (red) from a dendrite. B, Left, Pseudocolored puncta before (Pre) and after (Post) electrical field stimulation (5 Hz, 2 min, 10 mA). Scale bar, 2 μm. Right, Quantification of fluorescence loss [ΔF% = (1 − F_Post/F_Pre) * 100] in puncta upon stimulation (Stim, n = 18), in zero Ca²⁺ (Stim in 0 Ca²⁺, n = 10), and in the presence of GluRIs (50 μm APV and 10 μm NBQX, n = 25). C, Serotonin release by 1 min bath application of control aCSF (CTL), NMDA (50 μm), AMPA (10 μm), or agonists in zero Ca²⁺ aCSF (0 Ca²⁺, n = 8). **p < 0.01, ***p < 0.001. Error bars indicate SEM.

Figure 5.

Localized puncta depletion by glutamate puffs. A, 3P serotonin image in the brain slice with a puffer pipette indicated in white. The boxed regions i and ii are positioned in front and to the side of the pipette, respectively. Scale bar, 20 μm. B, Boxed regions i and ii. Scale bar, 5 μm. C, Quantification of responses evoked by puffing vehicle (CTL, n = 7) or glutamate (Glu) analyzed in front of the pipette (i, n = 13) or to the side of the pipette (ii, n = 11). i-Glu was different from CTL and ii-Glu: *p < 0.05. Error bars indicate SEM.

Figure 6.

VMAT2 is required for dendritic release. A, Top, Image stack showing a dHT-labeled raphe neuron. Bottom, Pseudocolored dHT signal in the boxed region of the dendrite before (CTL) and after AMPA. Scale bars: Top, 20 μm; bottom, 2 μm. B, Dendritic dHT fluorescence response to vehicle (CTL, n = 9), AMPA in 0 Ca²⁺ (n = 9), AMPA (10 μm; n = 5), and AMPA in slices in which dHT was applied in the presence of reserpine (500 nm; n = 13). AMPA was different from other experimental conditions: *p < 0.05. Error bars indicate SEM.

Figure 7.

Dendritic serotonin release does not require voltage-gated sodium channels. A, Pseudocolored summed z-projection of serotonin signal in soma (scale bar, 10 μm) and single optical section of dendritic serotonin puncta (scale bar, 2 μm) before (CTL) and after AMPA in the presence of TTX (1 μm). B, Quantification of somatic and dendritic serotonin release response in the absence and presence of TTX (n ≥ 8). ****p < 0.0001. Error bars indicate SEM.

Figure 8.

Dendritic serotonin release is independent of somatic action potentials. A, Recorded somatic APs evoked by 0.2 nA current pulse injection at 10 Hz for 30 s. Calibration: Vertical, 25 mV; horizontal, 2 s. Inset, Expanded timescale of 0.5 s region indicated by black bar. B, Serotonin release in the soma and dendritic puncta evoked by somatic APs (n ≥ 4). Dendritic puncta ranged in distance from soma (∼25–125 μm) with no apparent trend in release with distance. **p < 0.01. Error bars indicate SEM.

Figure 9.

Dendritic release requires L-type Ca²⁺ channels. A, Dendritic release responses to AMPA or NMDA in the presence and absence of IEM 1460 (100 μm) or nimodipine (3 μm). n ≥ 5. ****p < 0.0001. B, Somatic release response to AMPA or NMDA in the presence and absence of nimodipine (3 μm). n ≥ 5. Nimodipine did not have a significant effect in the soma. Error bars indicate SEM.

Figure 10.

Distinct sensitivity of dendritic serotonin release to the SSRI fluoxetine. A, Somatic and dendritic AMPA-evoked serotonin release responses in the presence of fluoxetine (Fluox, 10 μm) and WAY 100635 (WAY, 100 nm). n ≥ 7. **p < 0.01; ***p < 0.001. B, Fractional inhibition of AMPA-evoked release by fluoxetine in the dendrites (red; IC₅₀ = 1.5 μm) and soma (black; IC₅₀ = 0.3 μm); n ≥ 7. C, Fractional inhibition of AMPA-evoked release by DPAT; n ≥ 8. Error bars indicate SEM.