Acetylcholine as a trigger of the somatic exocytosis in Retzius neurons

The redistribution of vesicles containing serotonin in leech neurons was studied using the fluorescent, scanning ion-conductance, and laser phase microscopy methods. During acetylcholine receptor (AChR) activation in Retzius neurons, the changes of Ca2+ desorption, cellular stiffness and the cell optical phase difference (OPD) were established. It was found that the amplitude of OPD changes in the near-membrane area (membrane and near-membrane of the cytoplasm layers) increases upon AChR activation and this is, possibly, associated with the neurons vesicle redistribution. The decrease in the cell stiffness upon AChR activation suggests the crucial role of cytoskeleton for vesicle transport and release. Ca2+ rise in the cytoplasm during AChR activation may regulate the mitochondrial recruitment to regions with high energy demand for vesicle trafficking.


Introduction
The neurilemma depolarization of neuron plasma membrane is accompanied with the generation of action potential (AP) and increase in Na + -and Ca 2+ -influx into the cell.
The neuron AP generation with different frequencies (rhythmic activity, RA) is based on an activation of the voltage-gated and ligand-operated channels and the long lasting neuron plasmalemma changes (surface membrane potential and membrane viscosity, reorganization of cytoplasm structures). The spontaneous electrical activity of neurons in leech ganglia can be modified by the certain neurotransmitters [1][2][3]. However, the regulatory mechanisms of RA modulation in neurons are still poorly understood.
The Retzius cells in the leech (Rz-neurons) produce and release serotonin (5-HT) that plays an important neuromodulator role and may regulate the RA [4][5][6]. Serotonin may act in three different ways in the nervous system: as a transmitter at the synapses; as a paracrine modulator upon diffusion at a distance from its release sites, and as a hormonal modulator by circulating in the blood stream [7]. The three modes can affect a single neuronal circuit. The secretion of serotonin can occur synaptically or extrasynaptically.
The modulation of neural circuits requires the large amounts of signalling molecules that are released not only in the synapses but also from extrasynaptic sites in the soma, dendrites and axons. This secretion maintains transmitter concentrations in the extracellular spaces of not only near, but also distant neurons, glial cells and blood vessels [8]. The volume transmission in response to transmitter release from extrasynaptic sites, in the soma, dendrites and axons recently attracted more attention and was termed "extrasynaptic communication" [9]. Serotonin is stored in dense core vesicles of the leech Retzius cells. Electrical stimulation of the cells or a long-lasting increase of intracellular Ca 2+ may trigger the serotonin exocytosis [10]. The serotonin release from 3 the Retzius cells depends on the membrane potential shifts, the RA frequency, on L-type calcium channel activation and on calcium-induced calcium release [7]. However, there are no direct evidence how other neurotransmitters of the extracellular "broth" may modulate the serotonin exocytosis. What are the natural intrinsic stimuli that modify neuronal rhythmic activity to trigger the extrasynaptic release of serotonin?
In the leech ganglion there is a basal level of acetylcholine (ACh) presence that is counteracted by endogenous acetylcholinesterase (AChE) activity to regulate the membrane potential and support a sustained firing rate of Retzius neurons [11]. We propose that the basal ACh release may influence the output of these neuromodulatory serotonergic neurons.
A complex response to acetylcholine (ACh) is displayed by the Retzius cells bearing receptors with different nicotinic pharmacological profiles. The activation of nicotinic acetylcholine receptors (AChR) can cause the membrane potential changes and modification of Retzius cells RA [2,[12][13][14] . But there is no information in the literature that ACh action can evoke the extrasynaptic exocytosis of serotonin in the Retzius cells.
There are scarсe references about the interplay between acetylcholine and serotonin action on Retzius cells. The extracellular acetylcholine application to the bath exerts a short-term increase in the frequency of the spontaneous impulse activity (RA). On the contrary, presence of serotonin in the extracellular solution leads to a temporary deceleration of the impulse activity [2,13].
As revealed with electron microscopy significant ultrastructure changes occur during AchR action on Retzius cells: the increase of the number of electron-transparent neurosecretory granules, the swelling of the mitochondria and the endoplasmic reticulum, partial elimination of ribosomes [9]. But in case of serotonin application the neuron ultrastructure can hardly be distinguished from the normal. Investigation of the responses mediated by nicotinic receptors in well-characterized serotonergic neurons in situ is likely 4 to provide more direct information on the physiological role of AChR activation in the regulation of RA and extrasynaptic communication.
In this study, we address the functional role(s) of the nicotinic receptors in the central nervous system of invertebrates in connection to extrasynaptic release of transmitter substances. For the first time we revealed that acetylcholine can cause serotonin release in Retzius cells. The 5-HT exocytosis molecular mechanisms during AChR activation in Retzius cell were investigated: membrane Ca 2+ -desorption, cytoskeleton rearrangements (cellular stiffness) and the neuron cytoplasm optic phase profile (OPD)) dynamics. We propose that acetylcholine and serotonin present in the ganglion may cooperate to sustain the proper functioning of neuronal circuits.

Statistics
The results were statistically processed using the GraphPad Prism software,

Retzius cell optic phase difference
The OPD of the Retzius cell is determined the neuron cytoplasm optical density and volume. The optic phase difference correlates with the neuron membrane or/and intracellular processes: (1). this phase shift may be caused by the processes that occur in the cytoplasm, such as cyclosis, exo-and endocytosis. The transport of different vesicles, volume changes of mitochondria and nucleus influence the optical density changes; (2). the optical density changes correlate with the near membrane volume and membrane fluidity. In these experiments, a distribution of OPDs of the Retzius cell was investigated (Fig.1A). The optical heterogeneity of neuron topography is associated with the different cytoplasm subcellular organelles and vesicles. In addition, the OPDs changes were found both in the cell and in the near-membrane region (a resolution of 10 nm) (Fig. 1B). It has been established that in control conditions (without AChR activation) cytoplasm of Retzius neurons had the small OPD changes in the perinuclear and the neurolemma regions (Fig.1B). Probably, the OPD changes in the near-membrane region are associated with the vesicle redistribution during Retzius neurons spontaneous RA [9]. There are two clusters of serotonin vesicles in the Retzius cell: the membrane and the perinuclear, located close to the Golgi apparatus. During neuron electrostimulation the release of near-membrane vesicles occurs in the first place; further stimulation causes the release of perinuclear vesicles [19]. It is likely that the AChR activation initiated both, but primary the near membrane vesicles movement. In addition, the maximal OPD changes in the nucleus region might be associated with the mitochondria redistribution, which are localized around nucleus or its volume changes. Note, in the nonserotoninergic neuron the ACh action did not lead to significant OPD changes (Fig.2B).
So, the neuron OPD changes during the AChR activation are, probably, associated with the vesicles' movement and subsequent serotonin exocytosis.

Retzius cellular stiffness during the AChR activation
The inspection of cellular stiffness was performed by means of scanning ion conductance microscopy. This approach provides information about cytoskeleton structure and its rearrangements [18]. During AChR activation in Retzius neurons the cell stiffness decreased twice after several minutes (Fig.3).

Retzius cell membrane-bound calcium and the mitochondria inner membrane potential shift during the AChR activation
The neuron plasma membrane proteins and the mobility of cytoskeleton proteins are important factor for the vesicle redistribution during AchR activation [19][20]. It was found, that ACh receptor activation (not serotonin action) can change the level of membrane-bound calcium (Fig. 4).  The mitochondrial potential changes during RA are associated with the Ca 2+ accumulation [25]. In this regard, the changes of inner mitochondria membrane potential in Retzius neuron during AChR activation was investigated upon reduction of extracellular Ca 2+ concentration. The absence of the extracellular Ca 2+ does not completely block the development of the inner mitochondrial membrane depolarization during AChR activation. Our results suggest that the mitochondria depolarization is depended on Ca 2+influx from the extracellular medium and occurs, partly, due to the Ca 2+ -release from the intracellular compartments or plasma membrane desorption.

Discussion
It is known that in the Retzius cell, the localization of the clusters of vesicles with serotonin is associated with sites of the plasma membrane maximal depolarizations and electrostimulation caused a heterogeneous vesicles distribution in the different plasma membrane region [7]. So, the release of the vesicles with serotonin does not occur evenly throughout the neuron membrane, but there are areas for the formation of clusters of vesicles with serotonin and these areas correspond to the areas with the membrane potential local changes. Possible, the maximum OPD changes, localized in the nearmembrane region of the Retzius neuron, where maximal changes in the membrane potential are observed and reflect the accumulation and the vesicles redistribution during AChR activation ( Fig. 2A). Possible, that without AChR activation, the vesicle clusters and mitochondria are distant from the plasma membrane. The AChR activation evokes Ca 2+ entry through L-type channels or membrane binding Ca 2+ desorption (Fig. 4)

Conflicts of Interest:
The authors declare no conflict of interest.