Serotonin release from the neuronal cell body and its long-lasting effects on the nervous system

Abstract

Serotonin, a modulator of multiple functions in the nervous system, is released predominantly extrasynaptically from neuronal cell bodies, axons and dendrites. This paper describes how serotonin is released from cell bodies of Retzius neurons in the central nervous system (CNS) of the leech, and how it affects neighbouring glia and neurons. The large Retzius neurons contain serotonin packed in electrodense vesicles. Electrical stimulation with 10 impulses at 1 Hz fails to evoke exocytosis from the cell body, but the same number of impulses at 20 Hz promotes exocytosis via a multistep process. Calcium entry into the neuron triggers calcium-induced calcium release, which activates the transport of vesicle clusters to the plasma membrane. Exocytosis occurs there for several minutes. Serotonin that has been released activates autoreceptors that induce an inositol trisphosphate-dependent calcium increase, which produces further exocytosis. This positive feedback loop subsides when the last vesicles in the cluster fuse and calcium returns to basal levels. Serotonin released from the cell body is taken up by glia and released elsewhere in the CNS. Synchronous bursts of neuronal electrical activity appear minutes later and continue for hours. In this way, a brief train of impulses is translated into a long-term modulation in the nervous system.

Keywords: extrasynaptic; neuron–glia communication; serotonergic modulation; serotonin; somatic exocytosis; transmitter release.

Figure 1.

Ultrastructure of somatic release sites. (a) Electron micrograph of a Retzius neuron in the ganglion that had been fixed after stimulation with 1 Hz trains. The vesicle clusters (vc) remained distant from the plasma membrane (pm), although bound to it through bundles of microtubules (cs). Vesicles appear near mitochondria (m) and endoplasmic reticulum (er). The golgy apparatus (g) and the nucleus (n) are marked on the upper right side. Another population of vesicle clusters and multivesicular bodies (mvb) appear more internally. As explained later in the text, multivesicular bodies are formed after vesicle exocytosis. Retzius neurons are surrounded by layers of a giant glial cell (g). Scale bar, 500 nm. (b) After stimulation with 20 Hz trains, the vesicle clusters appear opposed to the plasma membrane (arrows) and flanked by endoplasmic reticulum and mitochondria. Scale bar, 1 µm. Adapted with permission from De-Miguel et al. [43].

Figure 2.

Timing of the signals that evoke somatic exocytosis. Time course of the stimulation train (black), the fast calcium transient (blue), measured as the fluorescence of Fluo-4, and exocytosis measured from the fluorescence increase of FM1–43 dye in spots produced upon fusion of vesicle clusters. The FM1–43 kinetics were obtained from different neurons. The dF/F values are the fluorescence values at each time point normalized to the baseline fluorescence. Note the logarithmic increases in the timing at each successive step.

Figure 3.

Calcium increases upon electrical stimulation in the soma of a Retzius neuron. A sequence of images made at the onset of stimulation (0 s) and at different times after stimulation show the peak of the fast calcium transient (0.6 s) invading the cell body. This transient is produced by calcium entry through L-type calcium channels and fed by calcium-induced calcium release. The contour of the cell can be inferred by the red colour. Some green and blue signals are due to out of focus light. In the following images the fast calcium transient has disappeared and instead, the peripheral serotonin-dependent calcium transient remains. The end of this transient occurs after the end of exocytosis. Adapted with permission from Leon-Pinzon et al. [54].

Figure 4.

The mechanism for somatic 5-HT exocytosis by Retzius neurons. (a) At rest, vesicle clusters (vc) and mitochondria (m) are distant from the plasma membrane. Both are attached to microtubules (mt) that arrive at the plasma membrane (pm). Endoplasmic reticulum (er) rests between the plasma membrane and the vesicle clusters. (b) A train of impulses evokes transmembrane Ca²⁺ entry through L-type channels (L Cach). Ca²⁺ triggers exocytosis from vesicles that rest close to the plasma membrane and in parallel, activates ryanodine receptors (RyR) and Ca²⁺-induced Ca²⁺ release, presumably from endoplasmic reticulum. The fast Ca²⁺ transient reaches mitochondria (m), which respond by producing ATP. Kinesin motors (km) are activated. (c) Vesicle clusters are transported towards the plasma membrane. The peripheral vesicle clusters and mitochondria receive more Ca²⁺ and ATP than the central clusters. Therefore, they are transported more efficiently towards the plasma membrane. As vesicles arrive at the plasma membrane and fuse, the 5-HT that had been released activates 5HT₂ receptors (5HT₂R) coupled to phospholipase C (PLC). Activation of PLC induces the formation of IP₃, which acts on receptors (IP₃R) and activates Ca²⁺ release from submembrane endoplasmic reticulum (ER). Ca²⁺ evokes further exocytosis, thus closing the local feedback loop. (d) Arrival of vesicle clusters at the plasma membrane produces the large-scale exocytosis. (e) The feedback loop ends when the last vesicles in the cluster fuse. The Ca²⁺ levels return to rest and the system goes back to its off-state (a). Endocytosis of electrodense vesicles produces multivesicular bodies (MVB) that are transported to perinuclear regions of the soma. Image adapted with permission from Leon-Pinzon et al. [54].

Figure 5.

Stimulation of a Retzius neuron synchronizes the electrical activity of multiple neurons. (a) Intracellular stimulation with 20 Hz trains was carried out through an intracellular electrode (yellow) inserted into one of the Retzius neurons. Multiunit electrical activity was collected through suction electrodes from one connective nerve (green) and two nerve roots (red and blue). (b) Before one of the Retzius neurons was stimulated the electrical recordings contained non-correlated spikes produced by different neurons, as inferred from the varying unit sizes. (c) Twenty-five minutes after stimulation of the Retzius neuron with 20 Hz trains the electrical activity displayed a bursting pattern with multiunit synchronization.