Nonsynaptic Release Sites Research Articles

Conditional deletion of neurexins dysregulates neurotransmission from dopamine neurons.

Midbrain dopamine (DA) neurons are key regulators of basal ganglia functions. The axonal domain of these neurons is highly complex, with a large subset of non-synaptic release sites and a smaller subset of synaptic terminals from which in addition to DA, glutamate or GABA are also released. The molecular mechanisms regulating the connectivity of DA neurons and their neurochemical identity are unknown. An emerging literature suggests that neuroligins, trans-synaptic cell adhesion molecules, regulate both DA connectivity and neurotransmission. However, the contribution of their major interaction partners, neurexins (Nrxns) is unexplored. Here we tested the hypothesis that Nrxns regulate DA neuron neurotransmission. Mice with conditional deletion of all Nrxns in DA neurons (DAT::Nrxns KO) exhibited normal basic motor functions. However, they showed an impaired locomotor response to the psychostimulant amphetamine. In line with an alteration in DA neurotransmission, decreased levels of the membrane DA transporter (DAT) and increased levels of the vesicular monoamine transporter (VMAT2) were detected in the striatum of DAT::Nrxns KO mice, along with reduced activity-dependent DA release. Strikingly, electrophysiological recordings revealed an increase of GABA co-release from DA neuron axons in the striatum of these mice. Together, these findings suggest that Nrxns act as regulators of the functional connectivity of DA neurons.

Non‐synaptic receptors and transporters involved in brain functions and targets of drug treatment

Beyond direct synaptic communication, neurons are able to talk to each other without making synapses. They are able to send chemical messages by means of diffusion to target cells via the extracellular space, provided that the target neurons are equipped with high-affinity receptors. While synaptic transmission is responsible for the 'what' of brain function, the 'how' of brain function (mood, attention, level of arousal, general excitability, etc.) is mainly controlled non-synaptically using the extracellular space as communication channel. It is principally the 'how' that can be modulated by medicine. In this paper, we discuss different forms of non-synaptic transmission, localized spillover of synaptic transmitters, local presynaptic modulation and tonic influence of ambient transmitter levels on the activity of vast neuronal populations. We consider different aspects of non-synaptic transmission, such as synaptic-extrasynaptic receptor trafficking, neuron-glia communication and retrograde signalling. We review structural and functional aspects of non-synaptic transmission, including (i) anatomical arrangement of non-synaptic release sites, receptors and transporters, (ii) intravesicular, intra- and extracellular concentrations of neurotransmitters, as well as the spatiotemporal pattern of transmitter diffusion. We propose that an effective general strategy for efficient pharmacological intervention could include the identification of specific non-synaptic targets and the subsequent development of selective pharmacological tools to influence them.

The locus ceruleus norepinephrine system

The locus ceruleus (LC) contains norepinephrine (NE)-synthesizing neurons that send diffuse projections throughout the CNS. The LC-NE system has a major role in arousal, attention, and stress response. In the brain, NE may also contribute to long-term synaptic plasticity, pain modulation, motor control, energy homeostasis, and control of local blood blow. The LC is severely affected in neurodegenerative disorders such as Alzheimer disease (AD) and Parkinson disease (PD). Dysregulation of LC-NE system has been implicated in sleep and arousal disorders, attention deficit hyperactivity disorder, and posttraumatic stress disorder and constitutes a target for pharmacologic treatment of these conditions. The neurobiology of the LC–noradrenergic system has been the subject of several excellent reviews.1–9 The LC is a cluster of NE-containing neurons located in the upper dorsolateral pontine tegmentum (figure 1). These neurons have extensively branched axons that project throughout the neuraxis and provide the sole source of NE to the neocortex, hippocampus, cerebellum, and most of the thalamus.1,2 Despite its widespread distribution, noradrenergic innervation shows regional specificity. For example, brain areas involved in spatial attention (such as the prefrontal and parietal cortices) receive particularly dense LC-NE inputs. In general, individual LC neurons send axon collaterals to multiple targets that process the same sensory information. Norepinephrine is released both at typical synapses and at nonsynaptic release sites; extrasynaptic NE mediates paracrine effects on neurons, glial cells, and microvessels.1–4,8 Figure 1 Anatomic organization of the locus ceruleus–norepinephrine system The norepinephrine (NE) neurons of the locus ceruleus (LC) are located in the upper dorsolateral pontine tegmentum and can be identified by their immunoreactivity for tyrosine hydroxylase, the rate-limiting enzyme for catecholamine synthesis. These neurons have extensively branched axons that project throughout the neuraxis and provide the sole source of NE to the neocortex, hippocampus, cerebellum, and most of the thalamus. Modified, …

Ultrastructural study of the Central Nervous System of Endemic Geocentrophora(Prorhynchida, Platyhelminthes) from Lake Baikal

AbstractThe ultrastructure of the central nervous system of four species of the Geocentrophoragroup (Prorhynchida, Lecithoepitheliata) has been studied with Geocentrophora waginias a model organism. This is the first ultrastructural study of the nervous system in flatworms of the endemic Geocentrophoragroup from Lake Baikal.The neurons are characterized by invaginations of the plasma membrane with extracellular material (ECM) extending deep into the neuronal cytoplasm. Two types of neuron are distinguished on the basis of the character of the cytoplasm and the content of neuronal vesicles. Several more vesicle types are observed in the nerve processes in the neuropile.The following kinds of neuronal release sites are distinguished: single synapses, shared synapses, nonsynaptic release sites characterized by omega profiles and neuromuscular contacts. Special features of the synapses in the species of Geocentrophoraare pronounced local thickenings of the presynaptic membrane connected to paramembraneous densities. The ECM‐filled invaginations and the local presynaptic thickenings are features that distinguish the neurons of Geocentrophoraspp. from previously described turbellarian neurons.

Dense-core vesicles and non-synaptic exocytosis in the central body of the crayfish brain

The central body in the median protocerebrum of the brain of the crayfish Cherax destructor is a distinctive area of dense neuropile, the nerve fibres of which contain three main types of vesicles: electronlucent vesicles (diameter 35 nm), dense-core vesicles (diameter 64 nm), and large structured dense-core vesicles (diameter 98 nm, maximum 170 nm). Different vesicle types were found together in the same neurons. Electronlucent vesicles were seen at presynaptic sites and rarely observed in the state of exocytosis. Exocytosis of densecore and structured dense-core vesicles was a regular feature on non-synaptic release sites either close to, or at some distance from pre- and subsynaptic sites. Non-synaptic exocytotic sites are more often observed than chemical synapses. Different forms of exocytosis seen at non-synaptic sites included the release of single densecore vesicles, packets of dense-core vesicles, and rows of dense-core vesicles lined up along cell membranes and around fibre invaginations. Swelling and the enhanced electron density of extracellular non-synaptic spaces may mark the positions of prior exocytotic events. In vitro treatment of the brain with tannic acid buffer solution followed by conventional double fixation resulted in the augmentation of non-synaptic exocytosis. Electron microscopy of proctolin- and serotonin-immunoreactive nerve fibres shows them to contain dense-core and electron-lucent vesicles and to be surrounded by many unlabelled profiles similarly laden with dense-core vesicles and electron-lucent vesicles, indicating the presence of other, not yet identified, neuroactive compounds.

Secretory activity and postembryonic development of the tentacle sensory system controlling growth hormone-producing neurons in Lymnaea stagnalis

The cerebral neuroendocrine peptidergic light green cells (LGC) of the freshwater snail Lymnaea stagnalis regulate body growth. The LGC are controlled by a tentacle sensory system that consists of two types (S1 and S2) of primary sensory neuron located at the base of each tentacle. Sensory (S2) axons make synaptic contacts (type A synapse-like structures) with the somata and axons of the LGC, where they release the contents of secretory granules, by exocytosis (demonstrated with the ultrastructural tannic acid-Ringer incubation method). Ultracytochemistry indicates that the granule contents are glycoproteinaceous. Furthermore, the S2 axons release secretory material in a nonsynaptic fashion into the interneuronal space of the central nervous system (CNS), at the level of the neuropiles of the cerebral ganglia and of the cerebral commissure. This release occurs by exocytosis from nonsynaptic release sites. It is proposed that the tentacle sensory system not only (synaptically) controls LGC activity but also influences other, remote neuronal targets in the CNS in a nonsynaptic (“at long distance,” “paracrine,” “hormone-like”) fashion. Already in newly hatched snails (with a shell height of 1 mm) S2 axons show a fair rate of exocytotic activity, in both synaptic and nonsynaptic respects. During postembryonic development the secretory capacity of the S2 sensory neurons increases markedly, by increases in (1) the number of axons, (2) the size of the secretory granules, and (3) exocytosis activity. This increased capacity may meet a growing demand of the developing CNS, including the LGC, for neurochemical input from the tentacle sensory system.

Ultrastructural demonstration of exocytosis in the supraependymal plexus of the rat

Fibres of the supraependymal plexus of the rat were studied ultrastructurally along the aqueduct through the mesencephalic central grey substance. Thin unmyelinated varicose fibres were found supraependymally in intimate contact with the ventricular surface of the ependymal lining. The varicosities contained electron-lucent clear vesicles (diameter approx, 50 nm) and dense-core secretory granules (diameter approx, 75 nm). All varicosities were immunoreactive for serotonin. None of the varicosities showed any ultrastructural characteristics of a synaptic contact. Exocytoses were frequently found after tannic acid treatment. The results strongly suggest that serotonin is released into the ventricular system from varicose non-synaptic release sites. The significance of such release is discussed.

Developmental and comparative aspects of nonsynaptic release by the egg-laying controlling caudodorsal cells of basommatophoran snails

In an immunoelectron microscope study the postembryonic development of the cerebral caudodorsal cells (CDC) in the freshwater snail Lymnaea stagnalis was studied as well as the development of similar neurons in other basommatophoran families. The CDC of adult L. stagnalis control egg-laying and associated behaviors by releasing various peptides, including the ovulation hormone CDCH. The CDC release peptides from neurohemal axon terminals and from nonsynaptic release sites of axon collaterals. During postembryonic development the collateral system develops synchronously with the neurohemal area. The first collaterals appear in the cerebral commissure of juvenile snails (10 mm shell height; S = 10). Up to S = 30 they gradually increase in size and length and eventually run through the entire inner compartment. Secretory granules in both collaterals and neurohemal axon terminals increase in size as well. Immunoelectron microscopy combined with the TARI-method for the demonstration of exocytosis indicates that CDCH-release from collaterals and neurohemal terminals occurs already in S = 10; exocytosis of immunoreactive granule contents takes place from nonsynaptic release sites, unspecialized areas of the axolemma of the collaterals. Release activity in the collaterals gradually increases up to S ≥ 20. Neurohemal release activity shows a similar picture except for a steep increase in adult snails. A distinct glial sheath, separating the neurohemal area from the collateral system, appears around S = 15. Representatives of three families of Basommatophora, viz. the lymnaeid L. ovata, the planorbid Planorbis planorbis, and the bulinid Bulinus truncatus possess a well-developed collateral system showing many signs of exocytosis. A glial sheath separates the collaterals from the neurohemal area. Secretory granules of the CDC in L. ovata stain weakly positive with the anti-CDCH antiserum. Since the other Basommatophora did not show immunoreactivity, the chemical structure of egg laying peptides in Basommatophora seems to be genus specific. Apparently the secretory activity of both the neurohemal area and the collateral system is not only important in the sexually mature animal, being involved in the control of egg laying and egg-laying behavior, but also in the juvenile snail. The finding of a collateral system in representatives of three basommatophoran families strongly indicates the importance of the system for the control of reproduction in basommatophoran snails in general.

Quantitative immunoelectron microscopy and tannic acid study of dynamics of neurohaemal and non-synaptic peptide release by the caudodorsal cells of Lymnaea stagnalis

The caudodorsal cells (CDCs) of the freshwater snail Lymnaea stagnalis control egg laying and egg-laying associated behaviours by releasing various peptides including the ovulation hormone CDCH. Previously it has been shown that release occurs (1) into the haemolymph from neurohaemal axon terminals in the outer compartment of the cerebral commissure, and (2) into the intercellular space of the central nervous system from non-synaptic release sites of axon collaterals in the inner compartment of the commissure. Outer and inner compartments are separated by a sheath of glial cells. In the present study the secretory dynamics of neurohaemal and collateral release have been studied. Immunoelectron microscopy with an antibody against a synthesized fragment of the egg-laying hormone [CDCH (21–36)] indicates that CDCH is released by exocytosis from both sites: positive immunoreaction was found for the contents of secretory granules and contents that underwent exocytosis, and furthermore in the intercellular spaces of the inner and outer compartments. Quantitative (immunogold) electron microscopy combined with either the tannic acid-glutaraldehyde-osmium tetroxide (TAGO) method or the tannic acid-Ringer incubation (TARI) method for the visualization and quantification of exocytosis of CDCH, shows different dynamics of neurohaemal and collateral CDCH release. Neurohaemal release is strongly increased during electrical activity of the CDCs (active state). This increase does not only appear from an increased number of (immunopositive) exocytoses (3×) but also from increases in (1) the percentages of all stationary and all exocytosing granule contents that are immunopositive (both increase from 70% to 85%), (2) the degree of immunopositivity per exteriorized granule content (2×) and (3) the degree of immunopositivity in the intercellular space of the neurohaemal area (5×). Collaterals show a different picture: CDCH release particularly occurs during electrical silence (resting and inhibited states). No effect was noted of the electrical state of the CDCs on the percentages of CDCH-immunoreactive stationary or exteriorized granule contents, nor on the degree of immunopositivity of the exteriorized contents. Furthermore, the degree of immunopositivity in the intercellular space of the inner compartment is drastically decreased (8×). Finally, both in the resting and the active state, the percentage of CDCH-positive exocytosing contents in the collaterals is smaller than that of CDCH-positive stationary contents, whereas in the neurohaemal area these percentages do not differ. It is concluded that the CDCs establish dual secretory dynamics in the neurohaemal and collateral system, not only with respect to the intensity of exocytosis but also with respect to the amount of CDCH released per secretory granule. Possible cellular mechanisms underlying these dynamics are discussed. The dual secretory capacity may be crucial for the CDCs in timing and coordinating the activities of reproductive organs (by peptide release from the neurohaemal area) and of central neurons that control egg-laying-associated behaviours (by peptides released non-synaptically from the collaterals).

Structural aspects, potassium stimulation and calcium dependence of nonsynaptic neuropeptide release by the egg laying controlling caudodorsal cells of Lymnaea stagnalis

The cerebral peptidergic caudodorsal cells of the freshwater snail Lymnaea stagnalis control egg laying and egg-laying behaviour by releasing peptides into (1) the haemolymph, from neurohaemal axon terminals in the periphery of the cerebral commissure and (2) the intercellular space of the central nervous system, from collaterals in the inner compartment of this commissure. Recently, it was shown that collateral release occurs from nonsynaptic release sites, which lack the morphological specializations that are characteristic of classical synapses. Probably, these sites enable the caudodorsal cells to communicate with central neurons in a nonsynaptic (“paracrine”, “diffuse”, “hormone-like”) fashion. The structural and ionic bases of nonsynaptic release were studied using the tannic acid-Ringer incubation-method for the detection of exocytotic release of secretory granule contents in vitro. Elevation of the extracellular potassium concentration strongly stimulates exocytotic activity in the collaterals. No stimulation was found in the absence of extracellular calcium ions. Similar results have been obtained for the neurohaemal axon terminals. Electron-dense material occurs apposed at the cytoplasmic side of the axolemma of collaterals (ethanolic phosphotungstic acid method). This material appears homologous with the presynaptic dense projections forming the “vesicular grid” in classical synapses. Such projections are also present in the neurohaemal axon terminals. It is concluded that secretion from nonsynaptic release sites in caudodorsal cell collaterals shares fundamental characteristics with secretion from conventional neuronal release sites (neurohaemal axon terminals and classical synapses); release occurs by exocytosis of secretory granules, is associated with a vesicular grid, is stimulated by membrane depolarization, and depends on the presence of extracellular calcium ions. The results underline the importance of nonsynaptic release sites for interneuronal communication within the central nervous system.

Visualization of non-synaptic release sites in the myenteric plexus of the snail Helix pomatia

Following conventional glutaraldehyde osmium tetroxidc fixation, a rich mycnteric plexus was detected in the gastrointestinal tract of the snail Helix pomatia. Although hundreds of nerve processes were observed in the extensive myoneural neuropil, true synaptic specializations were not recognized in them. In the absence of synaptic specializations, tannic acid Ringer incubation was applied to visualize the non-synaptic release sites in the enteric nerve plexus. After incubation for I h. a great number of exocytosis profiles were recorded at nerve muscle contacts, at axoglial connections and in the myoneural neuropil.The frequency of occurrence of exocytosis profiles was the same for adrenergic and peptidergic libres. In some gut wall areas, a dense staining of both basal lamina and collagen Ohres was observed. Invagination of dense basal lamina into the omega-shaped profiles of the axolemma led to ȁfalse exocytosisȁ profiles. A detailed morphological analysis is needed to distinguish false exocytosis profiles from the true transmitter-releasing loci.

Ultrastructural characterization of exocytotic release sites in different layers of the median eminence of the rat.

The release of neuronal secretory products by exocytosis in different layers of the median eminence of the rat was investigated ultrastructurally after perfusion with Ringer solution containing tannic acid. Exocytotic images were observed in all layers studied. Neurohaemal release sites were found in the pars externa of the palisade layer, where they occurred not only against the basal lamina of the pericapillary space, but also opposite, adjacent to neuronal and glial elements. In the lateral portion of the pars externa of the palisade layer most release sites were separated from the pericapillary space or the pial surface by ependymal or glial processes. In the pars interna of the palisade layer, and in the reticular, fibre and subependymal layers, release was observed in different types of axonal processes without morphological synaptic specializations. We suggest that products released in the pars externa of the palisade layer are destined to reach the capillaries of the primary portal plexus. Although the non-vascular release sites may serve a similar hormonal function, they may alternatively represent the morphological correlate of axo-axonal contacts or of paracrine, non-synaptic release sites.

Morphological basis for nonsynaptic communication within the central nervous system by exocytotic release of secretory material from the egg-laying stimulating neuroendocrine caudodorsal cells of Lymnaea stagnalis

The fine structure of the axons of the cerebral, egg-laying stimulating caudodorsal cells of the snail Lymnaea stagnalis has been studied with various light and electron microscope techniques. Special attention was paid to exocytotic release of secretory material (demonstrated with the tanic acid method) from nonsynaptic release sites in the cerebral commissure. This phenomenon has been compared with neurohaemal release. The commissure consists of two morphological compartments, separated by a sheath of glial cells. The outer compartment is formed by the neurohaemal area of the caudodorsal cells, the inner consists of thousands of, mainly unidentified, axons. Furthermore, ventral caudodorsal cells send axons through the inner compartment. These give rise to collaterals, which divide into smaller collaterals, forming an extensive network (“collateral system”) throughout the inner compartment. Eventually, collaterals end blindly within the inner compartment. They contain the same three morphological types of secretory granule as the neurohaemal axon terminals. The collaterals never form synaptic contacts; exocytotic release of the contents of secretory granules takes place at nonsynaptic release sites. These sites occur rather dispersed and do not face one particular type of neighbouring neural element. As in the neurohaemal area, both single and multiple exocytoses occur. Widened intercellular spaces, filled with flocculent, electron-dense material, occur near highly active nonsynaptic release sites. The spaces are often bordered by glial cells and may facilitate diffusion of released secretory material through the inner compartment. Apparently, a ventral caudodorsal cell releases secretory material in two fashions: (1) from neurohaemal axon terminals into the haemolymph and (2) nonsynaptically, from the collaterals into the intercellular space of the central nervous system. Possible functions of the glial sheath between the neurohaemal area and the inner compartment are proposed. Most likely, the collateral system enables the caudodorsal cells to communicate with targets within the central nervous system in a nonsynaptic fashion. A possible target is the cerebral Ring Neuron, which sends an axon branch through the inner compartment and, as was previously shown neurophysiologically, is controlled by the caudodorsal cells in a nonsynaptic fashion.

On the Role of Glial Cells in Dual Secretory Dynamics of the Caudodorsal Cells of l yMnaea Stagnalis

The Caudodorsal cells (CDC) of Lymnaea stagnalis are neurons that control egg laying and egg-laying behaviour by releasing various peptides, including the ovulation hormone CDCH. Release occurs in a dual fashion, viz. 1. into the haemolymph, from neurohacmal axon terminals in the outer compartment of the cerebral commissure and 2. into the intercellular space of the central nervous system, from nonsynaptic release sites of axon collaterals in the inner compartment of the commissure. Previous studies have shown that neurohaemal CDCH-release is maximal during electrical activity of the CDC (active state) whereas CDCH-release from the collaterals particularly occurs during electrical silence (resting and inhibition states). Inner and outer compartment are separated by a continuous sheath of glial cells. Immunoelectron microscopy with an antibody against a synthesized fragment of CDCH (CDCH20-36) including the use of the TAGO-method for the visualization of CDCH-release, shows that in the active state CDCH-immunoreactivity of the intercellular space is much higher (x5) in the outer than in the inner compartment. Apparently, the glial sheath prevents displacement of CDCH from one compartment to the other. In the cells of the sheath many of the immunogold particles are located over small, electron-lucent vesicles, suggesting endocytotic uptake of CDCH. Furthermore, the sheath cells appear able to block and to take up protein A and trypan blue; other substances like tannic acid are not prevented from crossing the sheath. It is concluded that the glial sheath acts as a selective barrier, preventing only particular substances like CDCH from passing it. By blocking and, possibly, by ingesting CDCH (and other CDC-peptides), the sheath may contribute to the control of peripheral and central targets, exerted by CDCH secreted from the neurohaemal area and from the collateral system, respectively.

Ultrastructural demonstration of nonsynaptic release sites in the central nervous system of the snail lymnaea stagnalis, the insect periplaneta americana, and the rat

Release of neuronal secretory products by exocytosis was studied ultrastructurally in the central nervous systems of three different species (the snail Lymnaea stagnalis, the cockroach Periplaneta americana and the rat). Tissues were fixed with: (1) a mixture of glutaraldehyde and osmium tetroxide, (2) the tannic acid-glutaraldehyde-osmium tetroxide (TAGO) method, and (3) the tannic acid-Ringer incubation (TARI) method. Especially after TARI-treatment, release of the contents of the secretory vesicles by exocytosis could be clearly demonstrated in: (1) synapses, (2) neurohaemal axon terminals ( L. stagnalis), and (3) neuronal processes without morphological synaptic specializations (nonsynaptic release sites). Release from nonsynaptic release sites occurs in most cases over a large area of the plasma membrane of a neuronal process facing several neural elements. On the basis of the differences in morphology of the secretory vesicles at nonsynaptic release sites, it is proposed that various types of (peptidic) messenger are released from such sites. In some neurones of L. stagnalis nonsynaptic release sites have been found together with synapses, or with neurohaemal axon terminals (caudodorsal cells, light green cells and light yellow cells). The possibility that nonsynaptic release sites represent the morphological correlates of nonsynaptic communication in the central nervous system has been discussed.