Synaptic Competition Sculpts the Development of GABAergic Axo-Dendritic but Not Perisomatic Synapses

The neurotransmitter GABA regulates many aspects of inhibitory synapse development. We tested the hypothesis that GABA_A receptors (GABA_ARs) work together with the synaptic adhesion molecule neuroligin 2 (NL2) to regulate synapse formation in different subcellular compartments. We investigated mice (“γ2 knockdown mice”) with an engineered allele of the GABA_AR γ2 subunit gene which produced a mosaic expression of synaptic GABA_ARs in neighboring neurons, causing a strong imbalance in synaptic inhibition. Deletion of the γ2 subunit did not abolish synapse formation or the targeting of NL2 to distinct types of perisomatic and axo-dendritic contacts. Thus synaptic localization of NL2 does not require synaptic GABA_ARs. However, loss of the γ2 subunit caused a selective decrease in the number of axo-dendritic synapses on cerebellar Purkinje cells and cortical pyramidal neurons, whereas perisomatic synapses were not significantly affected. Notably, γ2-positive cells had increased axo-dendritic innervation compared with both γ2-negative and wild-type counterparts. Moreover heterologous synapses on spines, that are found after total deletion of GABA_ARs from all Purkinje cells, were rare in cerebella of γ2 knockdown mice. These findings reveal a selective role of γ2 subunit-containing GABA_ARs in regulating synapse development in distinct subcellular compartments, and support the hypothesis that the refinement of axo-dendritic synapses is regulated by activity-dependent competition between neighboring neurons.     

Synaptic interactions between ghrelin- and neuropeptide Y-containing neurons in the rat arcuate nucleus

Morphological relationships between neuropeptide Y- (NPY) like and ghrelin-like immunoreactive neurons in the arcuate nucleus (ARC) were examined using light and electron microscopy techniques. At the light microscope level, both neuron types were found distributed in the ARC and could be observed making contact with each other. Using a preembedding double immunostaining technique, some NPY-immunoreactive axon terminals were observed at the electron microscope level to make synapses on ghrelin-immunoreactive cell bodies and dendrites. While the axo-somatic synapses were mostly symmetric in nature, the axo-dendritic synapses were both symmetric and asymmetric. In contrast, ghrelin-like immunoreactive (ghrelin-LI) axon terminals were found to make synapses on NPY-like immunoreactive (NPY-LI) dendrites although no NPY-like immunoreactive perikarya were identified receiving synapses from ghrelin-LI axon terminals. NPY-like axon terminals were also found making synapses on NPY-like neurons. Axo-axonic synapses were also identified between NPY- and ghrelin-like axon terminals. The present study shows that NPY- and ghrelin-LI neurons could influence each other by synaptic transmission through axo-somatic, axo-dendritic and even axo-axonic synapses, and suggests that they participate in a common effort to regulate the food-intake behavior through complex synaptic relationships.     

离体培养的小鼠脊髓固有神经元的突触构筑

运用电子显微镜观察分析原代分离培养鼠胚脊髓的固有神经元的突触构筑。培养中主要可见中、小型神经元,彼此之间可形成大量的突触,以非对称性突触占多数,有轴-树突触和轴-体突触,树-树突触为少见。根据以前学者分类标准将终扣分成S、F、M、和G四型。超微结构有利于提示固有神经元经过简单突触从脊髓固有神经纤维接受突触传入,表示它们的冲动只是突触后机制控制信息传递。     

离体培养的小鼠脊髓固有神经元的突触构筑

An electron microscopic analysis of the synaptic architecture in propriospinal neurons of cultured fetal mouse spinal cord has been undertaken. The size of the perikarya in the cultured spinal cord represents a range from small- to medium sized neurons, which form many synapses each other. There are many axo-dendritic and axo-somatic synapses in the culture but direct dendro-dendritic apposition is rarely seen. Four morphological types of synaptic boutons, S, F, M and G are classified according to criteria used by previous investigators. The ultrastructural details available suggest that the propriospinal neurons receive synaptic input from propriospinal fibers through simple synapses. It may indicate that their impulses can be controlled only postsynaptically.     

“Self” versus “Non-Self” Connectivity Dictates Properties of Synaptic Transmission and Plasticity

Autapses are connections between a neuron and itself. These connections are morphologically similar to “normal” synapses between two different neurons, and thus were long thought to have similar properties of synaptic transmission. However, this has not been directly tested. Here, using a micro-island culture assay in which we can define the number of interconnected cells, we directly compared synaptic transmission in excitatory autapses and in two-neuron micronetworks consisting of two excitatory neurons, in which a neuron is connected to one other neuron and to itself. We discovered that autaptic synapses are optimized for maximal transmission, and exhibited enhanced EPSC amplitude, charge, and RRP size compared to interneuronal synapses. However, autapses are deficient in several aspects of synaptic plasticity. Short-term potentiation only became apparent when a neuron was connected to another neuron. This acquisition of plasticity only required reciprocal innervation with one other neuron; micronetworks consisting of just two interconnected neurons exhibited enhanced short-term plasticity in terms of paired pulse ratio (PPR) and release probability (Pr), compared to autapses. Interestingly, when a neuron was connected to another neuron, not only interneuronal synapses, but also the autaptic synapses on itself exhibited a trend toward enhanced short-term plasticity in terms of PPR and Pr. Thus neurons can distinguish whether they are connected via “self” or “non-self” synapses and have the ability to adjust their plasticity parameters when connected to other neurons.     

Agrin plays an organizing role in the formation of sympathetic synapses

Agrin is a nerve-derived factor that directs neuromuscular synapse formation, however its role in regulating interneuronal synaptogenesis is less clear. Here, we examine agrin's role in synapse formation between cholinergic preganglionic axons and sympathetic neurons in the superior cervical ganglion (SCG) using agrin-deficient mice. In dissociated cultures of SCG neurons, we found a significant decrease in the number of synapses with aggregates of presynaptic synaptophysin and postsynaptic neuronal acetylcholine receptor among agrin-deficient neurons as compared to wild-type neurons. Moreover, the levels of pre- and postsynaptic markers at the residual synapses in agrin-deficient SCG cultures were also reduced, and these defects were rescued by adding recombinant neural agrin to the cultures. Similarly, we observed a decreased matching of pre- and postsynaptic markers in SCG of agrin-deficient embryos, reflecting a decrease in the number of differentiated synapses in vivo. Finally, in electrophysiological experiments, we found that paired-pulse depression was more pronounced and posttetanic potentiation was significantly greater in agrin-deficient ganglia, indicating that synaptic transmission is also defective. Together, these findings indicate that neural agrin plays an organizing role in the formation and/or differentiation of interneuronal, cholinergic synapses.     

Fine structure of the area postrema of the rabbit brain

Summary The area postrema of the rabbit, which was perfused with glutaraldehyde and postfixed in osmium tetroxide, was observed under the electron microscope. This area showed neuronal and neuroglial structures similar to those of ordinary cerebral tissue, except for rich blood capillaries, which were surrounded by conspicuous perivascular spaces. Parenchymal cells included a moderate number of small neurons and large numbers of specific astrocyte-like cells. The neuropil consisted of a small number of thin myelinated and many non-myelinated nerve fibers of varying calibers, axo-dendritic synapses, and neuroglial cell processes, leaving no spaces between them. The axons and synaptic terminals contained moderate amounts of granular vesicles, which were similar in size to those found in the hypothalamus and were supposed to contain catecholamine. Glycogen paticles were demonstrated mainly in the cytoplasm of the astrocyte-like cells.     

Nature of the distribution of synapses in the neurons of the reticular formation and some afferent nuclei]

Under study was the morphology of synaptic terminations of the brain reticular formation in cats and dogs as well as that of the afferent nuclei of the cat's posterior columns. The neurons of the Goll's and Burdach's nuclei have a richly ramified dendritic network. In this connection the main mass of synapses is disposed on the dendrites and their different branchings. The dendrites of the multipolar cells of the reticular formation have the main type of branching, but the cells are distributed from the nerve cell body at a considerable distance (up to 50 mu and more). The synapses are observed at the total length of the dendrite, so the axo-dendritic contacts quantitatively prevail over axo-somatic ones. The morphological data are compared with physiological axo-somatic and axo-dendritic concepts of the role of synapses in conducting the nerve impulse.     

Transmission Efficacy and Plasticity in Glutamatergic Synapses Formed by Excitatory Interneurons of the Substantia Gelatinosa in the Rat Spinal Cord

Background

Substantia gelatinosa (SG, lamina II) is a spinal cord region where most unmyelinated primary afferents terminate and the central nociceptive processing begins. The glutamatergic excitatory interneurons (EINs) form the majority of the SG neuron population, but little is known about the mechanisms of signal processing in their synapses.

Methodology

To describe the functional organization and properties of excitatory synapses formed by SG EINs, we did non-invasive recordings from 183 pairs of monosynaptically connected neurons. An intact presynaptic SG EIN was specifically stimulated through the cell-attached pipette while the evoked EPSCs/EPSPs were recorded through perforated-patch from a postsynaptic neuron (laminae I-III).

Principal Findings

We found that the axon of an SG EIN forms multiple functional synapses on the dendrites of a postsynaptic neuron. In many cases, EPSPs evoked by stimulating an SG EIN were sufficient to elicit spikes in a postsynaptic neuron. EPSCs were carried through both Ca²⁺-permeable (CP) and Ca²⁺-impermeable (CI) AMPA receptors (AMPARs) and showed diverse forms of functional plasticity. The synaptic efficacy could be enhanced through both activation of silent synapses and strengthening of already active synapses. We have also found that a high input resistance (R_IN, >0.5 GΩ) of the postsynaptic neuron is necessary for resolving distal dendritic EPSCs/EPSPs and correct estimation of their efficacy.

Conclusions/Significance

We conclude that the multiple synapses formed by an SG EIN on a postsynaptic neuron increase synaptic excitation and provide basis for diverse forms of plasticity. This functional organization can be important for sensory, i.e. nociceptive, processing in the spinal cord.     

Structure of Excitatory Synapses and GABAA Receptor Localization at Inhibitory Synapses Are Regulated by Neuroplastin-65

Formation, maintenance, and activity of excitatory and inhibitory synapses are essential for neuronal network function. Cell adhesion molecules (CAMs) are crucially involved in these processes. The CAM neuroplastin-65 (Np65) highly expressed during periods of synapse formation and stabilization is present at the pre- and postsynaptic membranes. Np65 can translocate into synapses in response to electrical stimulation and it interacts with subtypes of GABA_A receptors in inhibitory synapses. Here, we report that in the murine hippocampus and in hippocampal primary culture, neurons of the CA1 region and the dentate gyrus (DG) express high Np65 levels, whereas expression in CA3 neurons is lower. In neuroplastin-deficient (Np^−/−) mice the number of excitatory synapses in CA1 and DG, but not CA3 regions is reduced. Notably this picture is mirrored in mature Np^−/− hippocampal cultures or in mature CA1 and DG wild-type (Np^+/+) neurons treated with a function-blocking recombinant Np65-Fc extracellular fragment. Although the number of GABAergic synapses was unchanged in Np^−/− neurons or in mature Np65-Fc-treated Np^+/+ neurons, the ratio of excitatory to inhibitory synapses was significantly lower in Np^−/− cultures. Furthermore, GABA_A receptor composition was altered at inhibitory synapses in Np^−/− neurons as the α1 to α2 GABA_A receptor subunit ratio was increased. Changes of excitatory and inhibitory synaptic function in Np^−/− neurons were confirmed evaluating the presynaptic release function and using patch clamp recording. These data demonstrate that Np65 is an important regulator of the number and function of synapses in the hippocampus.     

Light and electron microscopic autoradiography of substantia nigra of rat after intraventricular administration of tritium labelled norepinephrine,dopamine, serotonin and the precursors

Summary In autoradiographies of substantia nigra in rat, it has been observed that after intraventricular injections of ³H-dopamine and ³H-norepinephrine respectively the silvergrains are accumulated in nigra neurons and their dendritic branches. The incorporation was more pronounced in the case of ³H-norepinephrine than ³H-dopamine. This seems to indicate that exogenous norepinephrine may have stronger affinity to nigra neurons and their dendrites than exogenous dopamine. In addition, some ³H-dopamine and ³H-norepinephrine labelled nerve terminals were observed in axo-dendritic synapses. In contrast to these data, ³H-5HTP and ³H-5HT administration showed almost all silver grains accumulated in the neuropil when observed in light microscopic autoradiography. Electron micrographs further reveal that the incorporation of ³H-5HTP and ³H-5HT was mostly within axo-dendritic boutons with more frequent dense core vesicles. These data again strongly suggest that substantia nigra receives a large number of serotoninergic fibres forming axo-dendritic synapses which may play an important role in modulation of substantia nigra function.Dr. Parizek was on leave of absence from the Charles University, Faculty of Medicine, Hradec Králové, Czechoslovakia.     

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons. The neurons are large (up to 1 mm in diameter) and identifiable, with distinct sizes, shapes, positions and pigmentations, and the cell bodies are externally exposed in five paired ganglia distributed throughout the body of the animal. These properties have allowed investigators to delineate the circuitry underlying specific behaviors in the animal¹. The monosynaptic connection between sensory and motor neurons is a central component of the gill-withdrawal reflex in the animal, a simple defensive reflex in which the animal withdraws its gill in response to tactile stimulation of the siphon. This reflex undergoes forms of non-associative and associative learning, including sensitization, habituation and classical conditioning. Of particular benefit to the study of synaptic plasticity, the sensory-motor synapse can be reconstituted in culture, where well-characterized stimuli elicit forms of plasticity that have direct correlates in the behavior of the animal^2,3. Specifically, application of serotonin produces a synaptic strengthening that, depending on the application protocol, lasts for minutes (short-term facilitation), hours (intermediate-term facilitation) or days (long-term facilitation). In contrast, application of the peptide transmitter FMRFamide produces a synaptic weakening or depression that, depending on the application protocol, can last from minutes to days (long-term depression). The large size of the neurons allows for repeated sharp electrode recording of synaptic strength over periods of days together with microinjection of expression vectors, siRNAs and other compounds to target specific signaling cascades and molecules and thereby identify the molecular and cell biological steps that underlie the changes in synaptic efficacy.An additional advantage of the Aplysia culture system comes from the fact that the neurons demonstrate synapse-specificity in culture^4,5. Thus, sensory neurons do not form synapses with themselves (autapses) or with other sensory neurons, nor do they form synapses with non-target identified motor neurons in culture. The varicosities, sites of synaptic contact between sensory and motor neurons, are large enough (2-7 microns in diameter) to allow synapse formation (as well as changes in synaptic morphology) with target motor neurons to be studied at the light microscopic level.In this video, we demonstrate each step of preparing sensory-motor neuron cultures, including anesthetizing adult and juvenile Aplysia, dissecting their ganglia, protease digestion of the ganglia, removal of the connective tissue by microdissection, identification of both sensory and motor neurons and removal of each cell type by microdissection, plating of the motor neuron, addition of the sensory neuron and manipulation of the sensory neurite to form contact with the cultured motor neuron.Open in a separate windowClick here to view.^{(105M, flv)}     

Synaptic interactions of luteinizing hormone-releasing hormone (LHRH) neurons in the guinea pig preoptic area

Previous studies from many laboratories have failed to demonstrate a significant synaptic input to luteinizing hormone-releasing hormone (LHRH) neurons in the rodent or primate hypothalamus/preoptic area. Having now developed immunocytochemical procedures that result in excellent ultrastructural preservation as well as in retention of antigenicity (Silverman AJ: J Comp Neurol 227:452, 1984), we have reinvestigated the question of the organization of the synaptic arrangements of LHRH neurons in the medial preoptic area of the guinea pig. Afferent inputs to these LHRH neurons include several varieties of axo-somatic and axo-dendritic synapses. Presynaptic terminals contain either round clear vesicles or a mixture of round and flattened vesicles. Most of these terminals, especially when serial sections are examined, contain dense-core granules. Well-defined synaptic clefts are evident and postsynaptic densities can be identified for asymmetrical connections. However, the presence of reaction product in the postsynaptic structure makes it difficult to categorize symmetrical terminals. In addition to these classical inputs, LHRH neurons also enter into complex heterodox synaptic relationships with their neighbors, including somato-dendritic and dendro-dendritic synapses in which the LHRH neuron can be either the pre- or postsynaptic element. These results suggest that complex synaptic relationships might account for the multiple levels of regulation of neurohormone release.     

Some observations on the fine structure of the corpus striatum of the rat brain

Summary The rat corpus striatum was perfused vitally with glutaraldehyde, immersed in OsO₄ and then observed under an electron microscope.Numerous small cells in the neostriatum show a simple cytoplasmic structure, while the large cells possess a complicated fine structure. These are differentiated under the elctron microscope into two kinds, which seem to have functional differences. The large pallidal cells containing much pale cytoplasm are covered with many varied axonal boutons from the cell body to the dendritic terminal making numerous axo-somatic or axo-dendritic trunk synapses. Numerous axo-dendritic, or spine synapses are recognized in the neostriatal neuropil.These numerous axon terminals, which belong to striatal nerve cells or other nuclei of the brain, are classified morphologically into several types. At least five types of synaptic vesicles are distinguished by their size or by the presence of fine dense granules on their membranes, and seem to be specific to the neostriatum.Many myelin interruptions and several kinds of glial cells in the corpus striatum are observed. Moreover, the ventricular wall of the caudate nucleus, namely, the ependyma, and two kinds of subependymal cells are described and discussed with reference to the subependymal layer.     

Synaptic transmission is impaired at neuronal autonomic synapses in agrin-null mice

Neuronal synapse formation is a multistep process regulated by several pre- and postsynaptic adhesion and signaling proteins. Recently, we found that agrin acts as one such synaptogenic factor at neuronal synapses in the PNS by demonstrating that structural synapse formation is impaired in the superior cervical ganglia (SCG) of z+ agrin-deficient mice and in SCG cultures derived from those animals. Here, we tested whether synaptic function is defective in agrin-null (AGD-/-) ganglia and began to define agrin's mechanism of action. Our electrophysiological recordings of compound action potentials showed that presynaptic stimulation evoked action potentials in approximately 40% of AGD-/- ganglionic neurons compared to 90% of wild-type neurons; moreover, transmission could not be potentiated as in wild-type or z+ agrin-deficient ganglia. Intracellular recordings also showed that nerve-evoked excitatory postsynaptic potentials in AGD-/- neurons were only 1/3 the size of those in wild-type neurons and mostly subthreshold. Consistent with these defects in transmission, we found an approximately 40-50% decrease in synapse number in AGD-/- ganglia and cultures, and decreased levels of differentiation at the residual synapses in culture. Furthermore, surface levels of acetylcholine receptors (AChRs) were equivalent in cultured AGD-/- and wild-type neurons, and depolarization reduced the synaptic localization of AChRs in AGD-/- but not wild-type neurons. These findings provide the first direct demonstration that agrin is required for proper structural and functional development of an interneuronal synapse in vivo. Moreover, they suggest a novel role for agrin, in stabilizing the postsynaptic density of nAChR at nascent neuronal synapses.     

Ciliated neurons and different types of synapses in anterior hypothalamic nuclei of reptiles

Summary The magnocellular paraventricular and supraoptic nuclei and the parvocellular preoptic and periventricular nuclei have been studied by light and electron microscopy in Emys orbicularis, Lacerta agilis and Elaphe longissima. The ultrastructure of cerebrospinal fluid (CSF)-contacting neurons was described in the preoptic and periventricular nuclei of Emys and Lacerta species. Single 9×2+0 cilia similar to those of the CSF-contacting dendritic terminals were found on perikarya of non CSF-contacting nerve cells, in all four investigated nuclei. The cilia project from funnel-like invaginations of the perikarya into the intercellular space. In the neurons of the nuclei studied, granular vesicles were found, their size being mainly 1,600 Å in the paraventricular nucleus, about 1,800 Å in the supraoptic nucleus, 1,100 Å in the periventricular nucleus and 800 Å, or up to 1,250 Å in the preoptic nucleus. In general, the neurons possess synapses of the axo-somatic, axo-somatic spine, axo-dendritic and axo-dendritic spine types. In the supraoptic nucleus, multiple interdigitated synapses were observed. Presynaptically, either synaptic vesicles only, or synaptic vesicles and dense core vesicles of different sizes (600 to 800 Å, about 1,100 Å, 1250 Å, and up to 2,000 Å) were found. It is discussed whether the above described 9×2+0 cilia may represent some kind of hypothalamic sensory structure that earlier physiological studies postulated to exist. The ciliated hypothalamic perikarya are considered by the authors to be a more differentiated form of the CSF-contacting neurons. The different types of synapses indicate multilateral connections of the nerve cells of the nuclei studied.Dedicated to Prof. Dr. Berta Scharrer on the occasion of her 70th birthday     

Morphology of the pineal organ in the salamander Ensatina eschscholtzi

The pineal organ of Ensatina eschscholtzi, a terrestrial and secretive species of salamander of the family Plethodontidae, is a photoreceptive structure lying on the dorsal surface of the diencephalon. The pineal is flattened with a broad lumen and consists of three cell types: photoreceptors, supportive cells, and neurons. Pineal photoreceptors are typical vertebrate photoreceptors and possess outer segment formations which, however, are frequently contorted and disorganized. Sloughing of apical portions of outer segments and vesiculation along the lateral edges of outer segment membrane disks are consistently observed and presumed to represent mechanisms of outer segment membrane recycling. Photoreceptors have basal processes which synapse with neural dendrites. Synapses between photoreceptor basal processes are occasionally observed. All synapses are characterized by synaptic ribbon structures of variable number, size, and configuration. Dense-core vesicles are occasionally observed mingled with clear synaptic vesicles within photoreceptor basal processes. Supportive cells within the pineal function in phagocytosis and recycling of shed outer segment membrane material, and neurons are localized at the lateral margins of the organ. The latter send axons into the ipsilateral side of the dorsal diencephalon. The pineal organ of Ensatina shows marked variation in overall size (cell total), cell type proportions, absolute neuron number, and ratio of photoreceptor number to neuron number for individual pineals. None of these morphological parameters is correlated with body size, sex, or season, and it is assumed that such variability represents significant variation in photosensory capabilities. It is suggested that the pineal organ of Ensatina is a partially degenerate photoreceptive structure.     

The organisation of invertebrate brains: cells,synapses and circuits

Meinertzhagen, I.A. 2010. The organisation of invertebrate brains: cells, synapses and circuits. —Acta Zoologica (Stockholm) 91 : 64–71 Invertebrate brains are structurally diverse. Neuron numbers range from ～10² to 10⁸ in different groups, compared with larger numbers in vertebrate brains, ～10⁷ to 10¹⁴. The underpopulated brains of invertebrates are noted in their extreme cases for having few cells, and neurons that can be identified from animal to animal, many known in great detail. Although few in number, invertebrate neurons nevertheless comprise many classes. Correlated with the paucity of their number they are sparsely connected, many having ～50 synapses or fewer. Synaptic densities, roughly 1 per μm³ of neuropile, differ little from those for much larger vertebrate neurons. Invertebrate neurons differ from their vertebrate counterparts in the position of their soma, generally in a cortex surrounding the neuropile that consequently occupies a relatively small volume. Their axons typically lack myelin and, supporting a range of conduction velocities, have diameters that differ over a wide range, from 10³ to 10^?1μm. Nerves with thousands of axons differ from neuropile fascicles, which typically have 20 or less. Unlike most vertebrate synapses, but like those of the vertebrate retina, synapses in many invertebrate groups – probably all ecdysozoans and possibly some lophotrochozoans – have synaptic contacts with multiple postsynaptic elements, dyads, triads and so on.     

Structure of peripheral synapses: autonomic ganglia

Final motor neurons in sympathetic and parasympathetic ganglia receive synaptic inputs from preganglionic neurons. Quantitative ultrastructural analyses have shown that the spatial distribution of these synapses is mostly sparse and random. Typically, only about 1%-2% of the neuronal surface is covered with synapses, with the rest of the neuronal surface being closely enclosed by Schwann cell processes. The number of synaptic inputs is correlated with the dendritic complexity of the target neuron, and the total number of synaptic contacts is related to the surface area of the post-synaptic neuron. Overall, most neurons receive fewer than 150 synaptic contacts, with individual preganglionic inputs providing between 10 and 50 synaptic contacts. This variation is probably one determinant of synaptic strength in autonomic ganglia. Many neurons in prevertebral sympathetic ganglia receive additional convergent synaptic inputs from intestinofugal neurons located in the enteric plexuses. The neurons support these additional inputs via larger dendritic arborisations together with a higher overall synaptic density. There is considerable neurochemical heterogeneity in presynaptic boutons. Some synapses apparently lack most of the proteins normally required for fast transmitter release and probably do not take part in conventional ganglionic transmission. Furthermore, most preganglionic boutons in the ganglionic neuropil do not form direct synaptic contacts with any neurons. Nevertheless, these boutons may well contribute to slow transmission processes that need not require conventional synaptic structures.