Francisco F. De-Miguel

Synaptic and extrasynaptic secretion of serotonin

AbstractSerotonin is a major modulator of behavior in vertebrates and invertebrates and deficiencies in the serotonergic system account for several behavioral disorders in humans.The small numbers of serotonergic central neurons of vertebrates and invertebrates produce their effects by use of two modes of secretion: from synaptic terminals, acting locally in “hard wired” circuits, and from extrasynaptic axonal and somatodendritic release sites in the absence of postsynaptic targets, producing paracrine effects.In this paper, we review the evidence of synaptic and extrasynaptic release of serotonin and the mechanisms underlying each secretion mode by combining evidence from vertebrates and invertebrates. Particular emphasis is given to somatic secretion of serotonin by central neurons.Most of the mechanisms of serotonin release have been elucidated in cultured synapses made by Retzius neurons from the central nervous system of the leech. Serotonin release from synaptic terminals occurs from clear and dense core vesicles at active zones upon depolarization. In general, synaptic serotonin release is similar to release of acetylcholine in the neuromuscular junction.The soma of Retzius neurons releases serotonin from clusters of dense core vesicles in the absence of active zones. This type of secretion is dependent of the stimulation frequency, on L-type calcium channel activation and on calcium-induced calcium release.The characteristics of somatic secretion of serotonin in Retzius neurons are similar to those of somatic secretion of dopamine and peptides by other neuron types. In general, somatic secretion by neurons is different from transmitter release from clear vesicles at synapses and similar to secretion by excitable endocrine cells.

Extrasynaptic exocytosis and its mechanisms: a source of molecules mediating volume transmission in the nervous system

We review the evidence of exocytosis from extrasynaptic sites in the soma, dendrites, and axonal varicosities of central and peripheral neurons of vertebrates and invertebrates, with emphasis on somatic exocytosis, and how it contributes to signaling in the nervous system. The finding of secretory vesicles in extrasynaptic sites of neurons, the presence of signaling molecules (namely transmitters or peptides) in the extracellular space outside synaptic clefts, and the mismatch between exocytosis sites and the location of receptors for these molecules in neurons and glial cells, have long suggested that in addition to synaptic communication, transmitters are released, and act extrasynaptically. The catalog of these molecules includes low molecular weight transmitters such as monoamines, acetylcholine, glutamate, gama-aminobutiric acid (GABA), adenosine-5-triphosphate (ATP), and a list of peptides including substance P, brain-derived neurotrophic factor (BDNF), and oxytocin. By comparing the mechanisms of extrasynaptic exocytosis of different signaling molecules by various neuron types we show that it is a widespread mechanism for communication in the nervous system that uses certain common mechanisms, which are different from those of synaptic exocytosis but similar to those of exocytosis from excitable endocrine cells. Somatic exocytosis has been measured directly in different neuron types. It starts after high-frequency electrical activity or long experimental depolarizations and may continue for several minutes after the end of stimulation. Activation of L-type calcium channels, calcium release from intracellular stores and vesicle transport towards the plasma membrane couple excitation and exocytosis from small clear or large dense core vesicles in release sites lacking postsynaptic counterparts. The presence of synaptic and extrasynaptic exocytosis endows individual neurons with a wide variety of time- and space-dependent communication possibilities. Extrasynaptic exocytosis may be the major source of signaling molecules producing volume transmission and by doing so may be part of a long duration signaling mode in the nervous system.

Somatic exocytosis of serotonin mediated by L-type calcium channels in cultured leech neurones

We studied somatic exocytosis of serotonin and its mediation by L‐type calcium (Ca2+) channels in cultured Retzius neurones of the leech. Exocytosis was induced by trains of impulses at different frequencies or by depolarisation with 40 mm potassium (K+), and was quantified by use of the fluorescent dye FM 1–43. Stimulation increased the membrane fluorescence and produced a pattern of FM 1–43 fluorescent spots of 1.28 ± 0.01 μm in diameter, provided that Ca2+ was present in the bathing fluid. Individual spots lost their stain during depolarisation with 40 mm K+. Electron micrographs showed clusters of dense core vesicles, some of which were in contact with the cell membrane. Presynaptic structures with clear vesicles were absent from the soma. The number of fluorescent spots per soma, but not their diameter or their fluorescence intensity, depended on the frequency of stimulation. Trains at 1 Hz produced 19.5 ± 5 spots per soma, 77.9 ± 13.9 spots per soma were produced at 10 Hz and 91.5 ± 16.9 spots per soma at 20 Hz. Staining patterns were similar for neurones in culture and in situ. In the presence of the L‐type Ca2+ channel blocker nimodipine (10 μm), a 20 Hz train produced only 22.9 ± 6.4 spots per soma, representing a 75 % reduction compared to control cells (P < 0.05). Subsequent incubation with 10 mm caffeine to induce Ca2+ release from intracellular stores increased the number of spots to 73.22 ± 12.5. Blockers of N‐, P‐, Q‐ or invertebrate Ca2+ channels did not affect somatic exocytosis. Our results suggest that somatic exocytosis by neurones shares common mechanisms with excitable endocrine cells.

Regulation of Synaptic Vesicle Docking by Different Classes of Macromolecules in Active Zone Material

The docking of synaptic vesicles at active zones on the presynaptic plasma membrane of axon terminals is essential for their fusion with the membrane and exocytosis of their neurotransmitter to mediate synaptic impulse transmission. Dense networks of macromolecules, called active zone material, (AZM) are attached to the presynaptic membrane next to docked vesicles. Electron tomography has shown that some AZM macromolecules are connected to docked vesicles, leading to the suggestion that AZM is somehow involved in the docking process. We used electron tomography on the simply arranged active zones at frog neuromuscular junctions to characterize the connections of AZM to docked synaptic vesicles and to search for the establishment of such connections during vesicle docking. We show that each docked vesicle is connected to 10–15 AZM macromolecules, which fall into four classes based on several criteria including their position relative to the presynaptic membrane. In activated axon terminals fixed during replacement of docked vesicles by previously undocked vesicles, undocked vesicles near vacated docking sites on the presynaptic membrane have connections to the same classes of AZM macromolecules that are connected to docked vesicles in resting terminals. The number of classes and the total number of macromolecules to which the undocked vesicles are connected are inversely proportional to the vesicles’ distance from the presynaptic membrane. We conclude that vesicle movement toward and maintenance at docking sites on the presynaptic membrane are directed by an orderly succession of stable interactions between the vesicles and distinct classes of AZM macromolecules positioned at different distances from the membrane. Establishing the number, arrangement and sequence of association of AZM macromolecules involved in vesicle docking provides an anatomical basis for testing and extending concepts of docking mechanisms provided by biochemistry.

Extrasynaptic neurotransmission as a way of modulating neuronal functions.

Extrasynaptic neurotransmission allows neuronal communication at variable distances, based on the release and diffusion of chemical substances from the soma, axon, or dendrites of neurons, or after transmitter leak-out from the synaptic cleft (De-Miguel and Trueta, 2005; Fuxe et al., 2007). Extrasynaptic neurotransmission modulates the input–output relationships of whole circuits by changing the electrical properties of neuronal populations, their chemical and electrical connectivities and also by activating the transport and release of transmitters by glial cells. The term synaptic transmission immediately refers to a confined process in which the arrival of an impulse at a presynaptic ending evokes a rapid burst of exocytosis that triggers an electrical reaction in the postsynaptic cell, within millisecond time and micrometer distance scales. By contrast, extrasynaptic exocytosis involves diverse ultrastructural and mechanistic principles that, when active, expand the timing and volume possibilities of nerve cell communication (Trueta et al., 2003, 2004; De-Miguel and Trueta, 2005; Agnati et al., 2010). Extrasynaptic sites release transmitters into the extracellular fluid in the absence of identifiable postsynaptic counterparts, producing the activation mainly of extrasynaptic receptors in neighboring neurons and/or glial cells. Although extrasynaptic exocytosis is triggered by electrical activity, it may start after a lag (asynchronous transmission) and may continue for seconds or even minutes once the depolarization has ended, thus expanding the timing of neuronal signaling. Extrasynaptic exocytosis exists in central and peripheral neurons of vertebrates and invertebrates and involves many different types of substances including low molecular weight transmitters, such as acetylcholine, GABA, glutamate, ATP, and biogenic amines, and peptides such as substance P, vasopressin, oxytocin, beta-endorphin, and also proteins (De-Miguel and Trueta, 2005). Diffusible modulators such as nitric oxide (Joca et al., 2007) and endocannabinoids (Yuan and Burrell, 2010) produced by postsynaptic targets also act on neighboring groups of neurons and glial cells. The intracellular mechanisms of extrasynaptic exocytosis vary from one neuronal compartment to another in the same neuron and also from one neuron type to another (De-Miguel and Trueta, 2005). For example, in axonal varicosities, clear vesicles may be apposed to the resting plasma membrane as in canonical presynaptic active zones or may rest at a certain distance from the plasma membrane, but these endings lack morphologically-defined postsynaptic counterparts, therefore being asynaptic and sources of extrasynaptic release. Transmitters and/or peptides may also be packed onto axonal and somatic dense core vesicles, most of which rest at a distance from the plasma membrane and in response to electrical activity move towards it and fuse, as in excitable endocrine cells. A single neuron may use several of these exocytosis mechanisms in separate compartments and it is also common that more than one mechanism may coexist in the same compartment. For example, while the soma of leech serotonergic Retzius neurons releases serotonin exclusively from dense core vesicles (Trueta et al., 2003), synaptic clear vesicles in serotonergic neurons of leech or mammals are surrounded by serotonin-containing dense core vesicles that release their contents extrasynaptically (De-Miguel and Trueta, 2005). Electrical stimulation of serotonergic leech or mammalian neurons increases the serotonin concentration in the extracellular fluid microns away from the release sites, and serotonin release modulates the activity of neurons (Perrier and Cotel, 2008) and in addition inhibits the electrical activity of serotonergic neurons (Cercos et al., 2009). Increasing evidence shows that in response to extrasynaptic exocytosis, glial cells may take up and afterwards release transmitters, and by doing so may modulate neuronal activity. Extrasynaptic transmission represents a subtype of volume (diffuse) transmission, namely the short-distance transmission of signaling molecules (inter alia transmitters, neuropeptides, and diffusible modulators) taking place in the extracellular fluid of local circuits (Fuxe and Agnati, 1991a,b; Descarries and Mechawar, 2000; Fuxe et al., 2007, 2010). Even the classical synaptic transmitter glutamate can be released to reach the extracellular fluid and activate extrasynaptic glutamate receptors located close to active glutamate synapses, as shown in brain slices, leading to increases in astroglial glutamate release (Del Arco et al., 2003). Glutamate as an astroglial derived volume transmission signal can then contribute to metabolic and trophic adjustments in the neuron-astrocytic unit in response to the activity of the glutamate synapses. Volume transmission also includes long distance diffusion in the extracellular fluid and the cerebrospinal fluid mainly mediated via neuropeptides like beta-endorphin and their fragments as well as proteins like, e.g., prolactin and Interleukin 1beta released from neurons and/or from glial cells (Agnati et al., 1986). This diversity of mechanisms and their effects adds complexity to the nervous system and raises many questions that still wait for answers. From the physiological point of view, one may ask how the neuronal firing pattern determines whether to evoke synaptic and/or extrasynaptic exocytosis in a given neuron; how activation of different synaptic inputs induces release from different release compartments, and by doing so affect different targets (Velazquez-Ulloa et al., 2003). From the behavioral point of view it becomes highly interesting to explore how the timing of circuits and therefore behaviors are modulated by extrasynaptic transmission. Some psychiatric and neurological dysfunctions, such as depression may be related to deficiencies in extrasynaptic transmission (Fuxe et al., 1991). A new research field is exploring whether antidepressant drugs used to reduce the symptoms of depression and/or of post-traumatic stress syndrome exert their effects by increasing the extracellular fluid levels of transmitters such as serotonin and thus volume transmission and by doing that restore the activity of distinct 5-HT receptor subtypes (Jansson et al., 2002), and in parallel activate neurogenesis, thus favoring mood elevation on one hand and the recovery of damaged brain areas on the other. This may occur at least in part through activating neurogenesis. Developmental and evolutionary biologists may also find the topic inspiring for future research, since some of the exocytosis mechanisms involved in extrasynaptic transmission evolved before chemical synapses. For a similar reason, one may expect that during development, the extracellular levels of transmitters, modulators, and peptides may contribute to axonal pathfinding and electrogenesis before massive synaptogenesis takes place. Extrasynaptic transmission expands our view about how the nervous system works and also imposes new challenges to the way we plan our research. New technological developments are focused on imaging transmitters (Kaushalya et al., 2008a,b) and new computational tools are being developed to analyze transmitter mobilizations or long-term changes of neuronal circuit activity. New definitions and mechanisms may become visible. Meanwhile, this is a good moment for a first common effort to analyze and discuss extrasynaptic transmission in different systems and from different perspectives. We hope the readers of this issue will find the articles included of significant interest and helpful clarifying the field of extrasynaptic transmission.

Synaptic Integration in Electrically Coupled Neurons

Interactions among chemical and electrical synapses regulate the patterns of electrical activity of vertebrate and invertebrate neurons. In this investigation we studied how electrical coupling influences the integration of excitatory postsynaptic potentials (EPSPs). Pairs of Retzius neurons of the leech are coupled by a nonrectifying electrical synapse by which chemically induced synaptic currents flow from one neuron to the other. Results from electrophysiology and modeling suggest that chemical synaptic inputs are located on the coupled neurites, at 7.5 microm from the electrical synapses. We also showed that the space constant of the coupled neurites was 100 microm, approximately twice their length, allowing the efficient spread of synaptic currents all along both coupled neurites. Based on this cytoarchitecture, our main finding was that the degree of electrical coupling modulates the amplitude of EPSPs in the driving neurite by regulating the leak of synaptic current to the coupled neurite, so that the amplitude of EPSPs in the driving neurite was proportional to the value of the coupling resistance. In contrast, synaptic currents arriving at the coupled neurite through the electrical synapse produced EPSPs of constant amplitude. This was because the coupling resistance value had inverse effects on the amount of current arriving and on the impedance of the neurite. We propose that by modulating the amplitude of EPSPs, electrical synapses could regulate the firing frequency of neurons.

NeuronGrowth, a software for automatic quantification of neurite and filopodial dynamics from time-lapse sequences of digital images.

We developed NeuronGrowth, a software for the automatic quantification of extension and retraction of neurites and filopodia, from time‐lapse sequences of two‐dimensional digital micrographs. NeuronGrowth requires a semiautomatic characterization of individual neurites in a reference frame, which is then used for automatic tracking and measurement of every neurite over the whole image sequence. Modules for sequence alignment, background subtraction, flat field correction, light normalization, and cropping have been integrated to improve the quality of the analysis. Moreover, NeuronGrowth incorporates a deconvolution filter that corrects the shadow‐cast effect of differential interference contrast (DIC) images. NeuronGrowth was tested by analyzing the formation of outgrowth patterns by individual leech neurons cultured under two different conditions. Phase contrast images were obtained from neurons plated on CNS homogenates and DIC images were obtained from similar neurons plated on ganglion capsules as substrates. Filopodia were measured from fluorescent growth‐cones of chick dorsal root ganglion cells. Quantitative data of neurite extension and retraction obtained by three different users applying NeuronGrowth and two other manually operated software packages were similar. However, NeuronGrowth required less user participation and had a better time performance when compared with the other software packages. NeuronGrowth may be used in general to quantify the dynamics of tubular structures such as blood vessels. NeuronGrowth is a free plug‐in for the free software ImageJ and can be downloaded along with a user manual, a troubleshooting section and other information required for its use from http://www.ifc.unam.mx or http://www.ifc.unam.mx/ffm/index.html.

Exocytosis of serotonin from the neuronal soma is sustained by a serotonin and calcium-dependent feedback loop

The soma of many neurons releases large amounts of transmitter molecules through an exocytosis process that continues for hundreds of seconds after the end of the triggering stimulus. Transmitters released in this way modulate the activity of neurons, glia and blood vessels over vast volumes of the nervous system. Here we studied how somatic exocytosis is maintained for such long periods in the absence of electrical stimulation and transmembrane Ca2+ entry. Somatic exocytosis of serotonin from dense core vesicles could be triggered by a train of 10 action potentials at 20 Hz in Retzius neurons of the leech. However, the same number of action potentials produced at 1 Hz failed to evoke any exocytosis. The 20-Hz train evoked exocytosis through a sequence of intracellular Ca2+ transients, with each transient having a different origin, timing and intracellular distribution. Upon electrical stimulation, transmembrane Ca2+ entry through L-type channels activated Ca2+-induced Ca2+ release. A resulting fast Ca2+ transient evoked an early exocytosis of serotonin from sparse vesicles resting close to the plasma membrane. This Ca2+ transient also triggered the transport of distant clusters of vesicles toward the plasma membrane. Upon exocytosis, the released serotonin activated autoreceptors coupled to phospholipase C, which in turn produced an intracellular Ca2+ increase in the submembrane shell. This localized Ca2+ increase evoked new exocytosis as the vesicles in the clusters arrived gradually at the plasma membrane. In this way, the extracellular serotonin elevated the intracellular Ca2+ and this Ca2+ evoked more exocytosis. The resulting positive feedback loop maintained exocytosis for the following hundreds of seconds until the last vesicles in the clusters fused. Since somatic exocytosis displays similar kinetics in neurons releasing different types of transmitters, the data presented here contributes to understand the cellular basis of paracrine neurotransmission.

Different determinants on growth and synapse formation in cultured neurons.

THE influence of substrate and target on growth and synapse formation was investigated in identified leech neurons in culture. Nociceptive neurons and annulus erector motoneurons were cultured on ganglion capsules, a substrate they encounter in the leech. Within a few hours, single nociceptive cells sprouted profusely, whereas annulus erector cells failed to grow. When annulus erector neurons were plated near a nociceptive cell on the same capsule both neurons grew, made contact and formed an electrical synapse, different from the chemical synapse they form in the ganglion, but identical to that formed when plated on Concanavalin A. These results suggest that substrate and target have complementary effects regulating growth, but fail to define the type of synapse formed.

Cycling of Dense Core Vesicles Involved in Somatic Exocytosis of Serotonin by Leech Neurons

We studied the cycling of dense core vesicles producing somatic exocytosis of serotonin. Our experiments were made using electron microscopy and vesicle staining with fluorescent dye FM1-43 in Retzius neurons of the leech, which secrete serotonin from clusters of dense core vesicles in a frequency-dependent manner. Electron micrographs of neurons at rest or after 1 Hz stimulation showed two pools of dense core vesicles. A perinuclear pool near Golgi apparatuses, from which vesicles apparently form, and a peripheral pool with vesicle clusters at a distance from the plasma membrane. By contrast, after 20 Hz electrical stimulation 47% of the vesicle clusters were apposed to the plasma membrane, with some omega exocytosis structures. Dense core and small clear vesicles apparently originating from endocytosis were incorporated in multivesicular bodies. In another series of experiments, neurons were stimulated at 20 Hz while bathed in a solution containing peroxidase. Electron micrographs of these neurons contained gold particles coupled to anti-peroxidase antibodies in dense core vesicles and multivesicular bodies located near the plasma membrane. Cultured neurons depolarized with high potassium in the presence of FM1-43 displayed superficial fluorescent spots, each reflecting a vesicle cluster. A partial bleaching of the spots followed by another depolarization in the presence of FM1-43 produced restaining of some spots, other spots disappeared, some remained without restaining and new spots were formed. Several hours after electrical stimulation the FM1-43 spots accumulated at the center of the somata. This correlated with electron micrographs of multivesicular bodies releasing their contents near Golgi apparatuses. Our results suggest that dense core vesicle cycling related to somatic serotonin release involves two steps: the production of clear vesicles and multivesicular bodies after exocytosis, and the formation of new dense core vesicles in the perinuclear region.