Ultrastructural correlates of interneuronal function in the abdominal ganglion of Aplysia californica

The synaptic inputs and outputs of the major interneuron L10 of the abdominal ganglion of Aplysia were studied using an intracellular staining technique for the electron microscope. The sites of both the chemical synaptic input and output of L10 are localized to the dendritic arborizations that arise from the axon in the ganglion neuropil. Thus, the interneuronal functions are mediated at the dendritic processes and could occur in the absence of spiking in the axon and cell body. The sites of L10 synaptic output are presumed to be at. aggregations of vesicles and mitochondria in the dendrites. The synaptic vesicle content of L10, a cholinergic neuron, with many large dense vesicles resembles that described for serotonergic cells in Aplysia, making distinction of synaptic pharmacology by ultrastructure difficult. Focal membrane specializations with a clear synaptic cleft were not observed between L10 and its large population of postsynaptic cells. In contrast, clear focal input sites were frequently found on L10. Gap junctions, sites of probable electrical coupling between L10 and other neurons, were also found. These observations are discussed as evidence that many synapses do not have focal specializations.     

Ultrastructure of calcitonin gene-related peptide-immunoreactive nerve fibres in guinea-pig peribronchial ganglia.

Calcitonin gene-related peptide-immunoreactive (CGRP-IR) nerves within guinea-pig peribronchial ganglia were studied at ultrastructural level using pre-embedding immunohistochemistry. Preterminal CGRP-IR axons were unmyelinated and contained singular immunoreactive dense core vesicles. CGRP-IR axon terminals were filled with numerous non-reactive small clear vesicles and few immunoreactive dense core vesicles. Some of these terminals were presynaptic to large neuronal processes emerging from local ganglion cells. Another population of presynaptic varicosities lack CGRP-IR. Within CGRP-IR terminals, non-reactive clear vesicles were clustered at the presynaptic membrane whereas CGRP-IR large vesicles remained in some distance from the synaptic cleft. The present observations indicate that: (1) at least two neurochemically different types of synaptic input exist to guinea-pig peribronchial ganglia. (2) CGRP-IR presynaptic terminals probably utilize a non-peptide transmitter for fast synaptic transmission, whilst the peptides are likely to be released parasynaptically and may act in a modulatory fashion.     

BDNF increases synapse density in dendrites of developing tectal neurons in vivo

Neuronal connections are established through a series of developmental events that involve close communication between pre- and postsynaptic neurons. In the visual system, BDNF modulates the development of neuronal connectivity by influencing presynaptic retinal ganglion cell (RGC) axons. Increasing BDNF levels in the optic tectum of Xenopus tadpoles significantly increases both axon arborization and synapse density per axon terminal within a few hours of treatment. Here, we have further explored the mechanisms by which BDNF shapes synaptic connectivity by imaging tectal neurons, the postsynaptic partners of RGCs. Individual neurons were co-labeled with DsRed2 and a GFP-tagged postsynaptic density protein (PSD95-GFP) to visualize dendritic morphology and postsynaptic specializations simultaneously in vivo. Immunoelectron microscopy confirmed that PSD95-GFP predominantly localized to ultrastructurally identified synapses. Time-lapse confocal microscopy of individual, double-labeled neurons revealed a coincident, activity-dependent mechanism of synaptogenesis and axon and dendritic arbor growth, which is differentially modulated by BDNF. Microinjection of BDNF into the optic tectum significantly increased synapse number in tectal neuron dendritic arbors within 24 hours, without significantly influencing arbor morphology. BDNF function-blocking antibodies had opposite effects. The BDNF-elicited increase in synapse number complements the previously observed increase in presynaptic sites on RGC axons. These results, together with the timescale of the response by tectal neurons, suggest that the effects of BDNF on dendritic synaptic connectivity are secondary to its effects on presynaptic RGCs. Thus, BDNF influences synaptic connectivity in multiple ways: it enhances axon arbor complexity expanding the synaptic territory of the axon, while simultaneously coordinating synapse formation and stabilization with individual postsynaptic cells.     

Synapses in the rat substantia nigra

The composition and organization of the input to the rat substantia nigra were studied with the electron microscope. Four distinct types of synaptic boutons were described. The first contained small (381 A), clear synaptic vesicles. The second type contained the small, clear vesicles and several large, dense-core vesicles. The third ending contained large, dense-core vesicles and larger (581 A) clear vesicles. The fourth ending, found on the axon hillock and other terminal boutons, contained slightly elongated, clear synaptic vesicles. The presence of these four boutons was discussed in light of the known afferent input and neurochemical composition of the substantia nigra.     

Fine structure of a spider joint receptor and associated synapses.

The fine structure of a joint receptor (R10) in a spider leg (Zygiella x-notata) was examined with light and electron microscopy. The R10 receptor consists of a compact ganglion which is situated near the dorsal joint membrane of the femur/patella joint. Each of the ten sensory cells comprising the ganglion sends one branching dendrite into the hypodermis underlying the joint membrane. All dendritic branches together form a sheet-like meshwork 50 microns wide and 1 microns thick, which is traversed obliquely by hypodermis cells. When the joint is stretched shearing forces are apparently transmitted to the receptive dendritic branches via microtubular bundles inside the hypodermis cells. The soma and dendrites of the sensory cells receive numerous synaptic input from presumably efferent fibres. The fine structure of these synapses is described and compared with other peripheral and central spider synapses. All R10 synapses contain small synaptic vesicles (32 nm diameter), whereas motor endplates possess large vesicles (38 nm). Central synapses have two significantly different vesicle populations which are either of the small or large variety. Since synapses with small vesicles are supposedly inhibitory, receptor cells in spiders might be under efferent control. Such a system is unknown in insects or crustaceans, but may be typical for arachnids.     

Ultrastructural studies on the afferent synaptic input to oxytocin-containing neurons in the paraventricular nucleus of the hypothalamus

A diverse afferent synaptic input to immunostained oxytocin magnocellular neurons of the paraventricular nucleus of the rat hypothalamus is described. By electron microscopy, immunoreactive material is present within cell bodies and neuronal processes and it is associated primarily with neurosecretory granules and granular endoplasmic reticulum. Afferent axon terminals synapse on perikarya, dendritic processes, and possibly axonal processes of oxytocin-containing neurons. The presynaptic elements of the synaptic complexes contain clear spherical vesicles, a mixture of clear spherical and ellipsoidal vesicles, or a mixture of clear and dense-centered vesicles. The postsynaptic membranes of oxytocinergic cells frequently show a prominent coating of dense material on the cytoplasmic face which gives the synaptic complex a marked asymmetry.     

Electron microscope investigation of synapses,synapse-like structures,and possible sites of release of neurosecretory material in the buccal ganglia of Helix pomatia (Mollusca)

Summary In the buccal ganglia of Helix pomatia synapses and sites of possible release of neurosecretory material were investigated electron microscopically. There is one chemical synapse and one electrotonic synapse in the neuropile of the ganglion. No synapses could be detected in the buccal nerves, cerebro-buccal connectives, or in the buccal commissure. The synaptic cleft of the chemical synapse is about 25 nm wide and contains electron-dense material whereas the cleft of the electrotonic synapse is only 5 nm wide. The presynaptic fibre of the chemical synapse contains clear vesicles and dense core vesicles. The release sites of neurosecretory material are found at the initial segment of the axons, at perikarya of neurones, and at the perineurium of the ganglion. If the terminals are located at the plasmalemma of a nerve cell, these release sites are called synapse-like structures according to Roubos and Moorer-van Delft (1979). The synapse-like structures show all structural elements of synapses, except the 25 nm cleft containing dense material; the cleft is only 15–20 nm wide here like the normal cleft between neurones and glial cells or between two fibres. If the secretory material is released at the periphery through the perineurium the terminal is called synaptoid according to Scharrer (1970). In all cases, i.e. synapses, synapse-like structures, and synaptoids, clear vesicles were found in the axon terminal. This finding provides further evidence that clear vesicles always accompany the release of substances from axon endings.     

Synaptic input to the axon hillock and initial segment of inhibitory interneurons in the cerebellar cortex of the rat

Summary The axon hillock (AH) and initial segment (IS) of 10 Golgi neurons and 6 basket cells in the cerebellar cortex of the rat were investigated by electron microscopy using serial sections. An average of 10.4 and 11.3 synaptic terminals were observed to establish synaptic contact with the axon hillock region of Golgi and basket cells, respectively. Most of these terminals were identified as the varicosities of the ascending parallel fibers. It is suggested that the focal innervation of AH regions represents an excitatory input pattern which is basically different from the randomly distributed, huge, parallel-fiber input onto the dendritic trees of Golgi and basket cells. In contrast to Golgi and basket neurons, no accumulation of parallel-fiber synapses was observed around the AH of stellate cells. The IS proper of the three neuronal types were devoid of true axo-axonal synapses.     

Immunocytochemical observations of corticotropin-releasing-factor-containing neurons in the rat hypothalamus with special reference to neuronal communication

Synapses between neurons with corticotropin-releasing-factor-(CRF)-like immunoreactivities and other immunonegative neurons in the hypothalamus of colchicine-treated rats, especially in the paraventricular nucleus (PVN) and the supraoptic nucleus (SON) were observed by immunocytochemistry using CRF antiserum. The immunoreactive nerve cell bodies and fibers were numerous in both the PVN and the SON. The CRF-containing neurons had synaptic contacts with immunonegative axon terminals containing a large number of clear synaptic vesicles alone or combined with a few dense-cored vesicles. We also found CRF-like immunoreactive axon terminals making synaptic contacts with other immunonegative neuronal cell bodies and fibers. And since some postsynaptic immunonegative neurons contained many large neurosecretory granules, they are considered to be magnocellular neurosecretory cells. These findings suggest that CRF functions as a neurotransmitter and/or modulator in addition to its function as a hormone.     

The synaptic organization of visual interneurons in the lobula complex of flies

Summary The synaptic organization of three classes of cobalt-filled and silver-intensified visual interneurons in the lobula complex of the blowfly Calliphora (Col A cells, horizontal cells and vertical cells) was studied electron microscopically. The Col A cells are regularly spaced, columnar, small field neurons of the lobula, which constitute a plexus of arborizations at the posterior surface of the neuropil and the axons of which terminate in the ventrolateral protocerebrum. They show postsynaptic specializations in the distal layer of their lobula-arborizations and additional presynaptic sites in a more proximal layer; their axon terminals are presynaptic to large descending neurons projecting into the thoracic ganglion. The horizontal and vertical cells are giant tangential neurons, the arborizations of which cover the anterior and posterior surface of the lobula plate, respectively, and which terminate in the perioesophageal region of the protocerebrum. Both classes of these giant neurons were found to be postsynaptic in the lobula plate and pre- and postsynaptic at their axon terminals and axon collaterals. The significance of these findings with respect to the functional properties of the neurons investigated is discussed.     

Axons containing neuropeptide Y innervate arginine vasopressin-containing neurons in the rat paraventricular nucleus

Summary Synaptic connections between neurons immunoreactive for arginine vasopressin (AVP) and axon terminals immunoreactive for neuropeptide Y (NPY) were found in the magnocellular part of the paraventricular nucleus (PVN) in the rat hypothalamus. In pre-embedding double immunolabeling, NPY axon terminals labeled with diamin-obenzidine (DAB) reaction product established synaptic junctions on the perikarya and neuronal processes of AVP neurons labeled with silver-gold particles. Ultrastructural morphology of the neurons was more suitably preserved by a combination of pre- and post-embedding procedures. The presynaptic NPY terminals contained many small clear vesicles and a few cored vesicles, and DAB chromogen (immunoreaction product) was located on the surface of the vesicular profiles and on the core. The postsynaptic AVP neurons possessed many large secretory granules labeled with gold particles. At the synaptic junctions, small clear vesicles were accumulated at the presynaptic membrane, and the postsynaptic membrane was coated with a dense accumulation of fine electron dense particles. The perikarya also received synapses made by immuno-negative axon terminals containing many small clear vesicles and a few cored vesicles. These terminals were found more frequently than those containing NPY.     

Axons containing neuropeptide Y innervate arginine vasopressin-containing neurons in the rat paraventricular nucleus. Dual electron microscopic immunolabeling

Synaptic connections between neurons immunoreactive for arginine vasopressin (AVP) and axon terminals immunoreactive for neuropeptide Y (NPY) were found in the magnocellular part of the paraventricular nucleus (PVN) in the rat hypothalamus. In pre-embedding double immunolabeling, NPY axon terminals labeled with diaminobenzidine (DAB) reaction product established synaptic junctions on the perikarya and neuronal processes of AVP neurons labeled with silver-gold particles. Ultrastructural morphology of the neurons was more suitably preserved by a combination of pre- and post-embedding procedures. The presynaptic NPY terminals contained many small clear vesicles and a few cored vesicles, and DAB chromogen (immunoreaction product) was located on the surface of the vesicular profiles and on the core. The postsynaptic AVP neurons possessed many large secretory granules labeled with gold particles. At the synaptic junctions, small clear vesicles were accumulated at the presynaptic membrane, and the postsynaptic membrane was coated with a dense accumulation of fine electron dense particles. The perikarya also received synapses made by immuno-negative axon terminals containing many small clear vesicles and a few cored vesicles. These terminals were found more frequently than those containing NPY.     

The presynaptic release apparatus is functional in the absence of dendritic contact and highly mobile within isolated axons

Whether contact of an axon with a dendrite is a necessary inductive signal for the assembly of functional presynaptic machinery is controversial. Combining FM1-43 imaging with retrospective immunocytochemistry, we observe many functional synaptic vesicle (SV) release sites lacking postsynaptic specializations in cultured hippocampal neurons. These "orphan" release sites share the same exocytic machinery and mechanisms of endocytic recycling as mature synaptic sites. Moreover, quantitative analysis of FM1-43 destaining at these orphan release sites reveals similar kinetics with slightly lower release probabilities. Time-lapse imaging of FM1-43 reveals that orphans are generated by complete or partial mobilization of synaptic release sites that retain their functionality in transit. Orphan clusters fuse with existing synaptic release sites or form novel release sites onto dendrites. Mobilization and stabilization of orphan boutons to new sites of dendritic contact may represent a necessary presynaptic counterpart to postsynaptic changes observed during development and plasticity in the CNS.     

Structure of anterior dorsal ventricular ridge in snakes.

Anterior dorsal ventricular ridge (ADVR) is a major subcortical, telencephalic nucleus in snakes. Its structure was studied in Nissl, Golgi, and electron microscopic preparations in several species of snakes. Neurons in ADVR form a homogeneous population. They have large nuclei, scattered cisternae of rough endoplasmic reticulum in their cytoplasm, and bear dendrites from all portions of their somata. The dendrites have a moderate covering of pedunculated spines. Clusters of two to five cells with touching somata can be seen in Nissl, Golgi, and electron microscopic preparations. The area of apposition may contain a series of specialized junctions which resemble gap junctions. Three populations of axons can be identified in rapid Golgi preparations of snake ADVR. Type 1 axons course from the lateral forebrain bundle and bear small varicosities about 1 mu long. Type 2 axons arise from ADVR neurons and bear large varicosities about 5 mu long. The origin of the very thin type 3 axons is not known; they bear small varicosities about 1 mu long. The majority of axon terminals in ADVR are small (1 mu to 2 mu long), contain round synaptic vesicles, and form asymmetric active zones. This type of axon terminates on dendritic spines and shafts and on somata. A small percentage of terminals are large, 5 mu in length, contain round synaptic vesicles, and form asymmetric active zones. This type of axon terminates only on dendritic spines. A small percentage of terminals are small, contain pleomorphic synaptic vesicles, and form symmetric active zones. This type of axon terminates on dendritic shafts and on somata.     

THE FINE STRUCTURE OF MOTOR ENDPLATE MORPHOGENESIS

The fine structure of the developing neuromuscular junction of rat intercostal muscle has been studied from 16 days in utero to 10 days postpartum. At 16 days, neuromuscular relations consist of close membrane apposition between clusters of axons and groups of myotubes. Focal electron-opaque membrane specializations more intimately connect axon and myotube membranes to each other. What relation these focal contacts bear to future motor endplates is undetermined. The presence of a group of axons lying within a depression in a myotube wall and local thickening of myotube membranes with some overlying basal lamina indicates primitive motor endplate differentiation. At 18 days, large myotubes surrounded by new generations of small muscle cells occur in groups. Clusters of terminal axon sprouts mutually innervate large myotubes and adjacent small muscle cells within the groups. Nerve is separated from muscle plasma membranes by synaptic gaps partially filled by basal lamina. The plasma membranes of large myotubes, where innervated, simulate postsynaptic membranes. At birth, intercostal muscle is composed of separate myofibers. Soleplate nuclei arise coincident with the peripheral migration of myofiber nuclei. A possible source of soleplate nuclei from lateral fusion of small cells' neighboring areas of innervation is suspected but not proven. Adjacent large and small myofibers are mutually innervated by terminal axon networks contained within single Schwann cells. Primary and secondary synaptic clefts are rudimentary. By 10 days, some differentiating motor endplates simulate endplates of mature muscle. Processes of Schwann cells cover primary synaptic clefts. Axon sprouts lie within the primary clefts and are separated from each other. Specific neural control over individual myofibers may occur after neural processes are segregated in this manner.     

Synapse Loss and Dendrite Remodeling in a Mouse Model of Glaucoma

It has been hypothesized that synaptic pruning precedes retinal ganglion cell degeneration in glaucoma, causing early dysfunction to retinal ganglion cells. To begin to assess this, we studied the excitatory synaptic inputs to individual ganglion cells in normal mouse retinas and in retinas with ganglion cell degeneration from glaucoma (DBA/2J), or following an optic nerve crush. Excitatory synapses were labeled by AAV2-mediated transfection of ganglion cells with PSD-95-GFP. After both insults the linear density of synaptic inputs to ganglion cells decreased. In parallel, the dendritic arbors lost complexity. We did not observe any cells that had lost dendritic synaptic input while preserving a normal or near-normal morphology. Within the temporal limits of these observations, dendritic remodeling and synapse pruning thus appear to occur near-simultaneously.     

Ultrastructural localization of substance P immunoreactivity in the ventral horn of the rat spinal cord

At the light microscope level, the minute concentrations of substance P (SP) in rat spinal ventral horn can be visualized best by amplification with the double bridge PAP method of Vacca et al. (1975; 1980) in 5 microns paraffin tissue sections. Morphologically, the immunoreactive sites resemble punctate bodies. They occur in close apposition with the large ventral horn cells and their associated neuronal processes. By the Sternberger PAP procedure, we now describe these punctate bodies at the electron microscope level. Ultrastructurally, they appear as tiny boutons (terminal and preterminal) and small unmyelinated processes. The boutons and processes typically contain one to several immunolabeled dense core vesicles among many immunolabeled clear vesicles. They range in size near the limit of resolution of the light microscope (LM), thereby justifying further the use of LM amplification staining by the double bridge method. The immunolabeled boutons often synapse with large smooth dendrites (which may originate from motoneurons) by asymmetrical or symmetrical synaptic densities. Their synaptic densities appear immunostained as well. The data support the view that the electrophysiological action of SP in the ventral horn occurs in part by synaptic action along the processes of the ventral horn cells. Other mechanisms of action are considered for the peptide as well. Additional types of membrane specializations (synaptoid junctions) and SP neural circuits are described below.     

Neuronal compartments and axonal transport of synapsin I

Studies on the transport kinetics and the posttranslational modification of synapsin I in mouse retinal ganglion cells were performed to obtain an insight into the possible factors involved in forming the structural and functional differences between the axon and its terminals. Synapsin I, a neuronal phosphoprotein associated with small synaptic vesicles and cytoskeletal elements at the presynaptic terminals, is thought to be involved in modulating neurotransmitter release. The state of phosphorylation of synapsin I in vitro regulates its interaction with both synaptic vesicles and cytoskeletal components, including microtubules and microfilaments. Here we present the first evidence that in the mouse retinal ganglion cells most synapsin I is transported down the axon, together with the cytomatrix proteins, at the same rate as the slow component b of axonal transport, and is phosphorylated at both the head and tail regions. In addition, our data suggest that, after synapsin I has reached the nerve endings, the relative proportions of variously phosphorylated synapsin I molecules change, and that these changes lead to a decrease in the overall content of phosphorus. These results are consistent with the hypothesis that, in vivo, the phosphorylation of synapsin I along the axon prevents the formation of a dense network that could impair organelle movement. On the other hand, the dephosphorylation of synapsin I at the nerve endings may regulate the clustering of small synaptic vesicles and modulate neurotransmitter release by controlling the availability of small synaptic vesicles for exocytosis.     

The abdominal ganglion of Aplysia brasiliana: a comparative morphological and electrophysiological study, with notes on A. dactylomela.

The ultrastructure and electrophysiological properties of neurons in the abdominal (visceral) ganglion of the marine opisthobranch gastropod Aplysia brasiliana have been investigated to determine whether this preparation compares favorably with the well studied A. californica for neurobiological research. In general, the topography, morphology and physiological characteristics, including synaptic connections, of neurons in this ganglion are quite similar to those of A. californica. There is close correspondence between the two animals in terms of each of the identified cells or neuronal clusters in the ganglion, including the presence of the cell L10 (interneuron I) in A. brasiliana which makes synaptic connections comparable with those in A. californica. New follower cells of this interneuron have been found in A. brasiliana. This species offers some advantages in that the connective tissue surrounding the ganglion is thinner and more transparent, making cell identification and penetration easier. A. brasiliana appears to exhibit the behaviors of A. californica that have been used in previous functional analyses of neural circuits. In addition, this species swims and exhibits a "burrowing" activity less commonly seen in A. californica. The rich repertoire of behaviors and accessibility of large identifiable and functionally interconnected neurons makes this species of Aplysia an excellent model preparation for future neurobiological studies. Similar, less thorough, investigations of the abdominal ganglion of A. dactylomela indicate that this species is also very similar to A. californica in terms of the identified cells in the abdominal ganglion.     

The brain of the horseshoe crab (Limulus polyphemus). III. Cellular and synaptic organization of the corpora pedunculata.

The large, hemispherical mass of the Limulus corpora pedunculata consists of two highly branched lobes, each connected to the protocerebrum by a narrow stalk. About 10(4) afferent fibers enter through the stalks and make diverse, profuse, and often reciprocal contacts with several million Kenyon (intrinsic) cells and one another. The Kenyon cell axonal arborizations converge on a few hundred efferent dendrites. The afferent fiber types can be classified into five types. Type A forms the club-shaped core of glomeruli and circumglomerular annuli, and contains small flat vesicles, suggesting an inhibitory function. Type B terminates with bushy endings in glomeruli and is presynaptic to both Kenyon cells and to Type A terminals. It has clear round vesicles and is the presumptive excitatory input. Type C terminates on other afferents, in glomeruli, and rarely on Kenyon cell bodies, contains angular (neurosecretory) granules and is postulated to impart circadian rhythm. Type D terminates on Kenyon cell somata and the initial neurite segment (but not in glomeruli), and contains dense-cored vesicles. Type E terminates in peduncles on other afferents and Kenyon cell telodendria. It contains dense vesicles. The C, D, and E afferents have reciprocal synaptic connections with Kenyon cell axon terminals. Glomeruli thus receive three different inputs of presumptive inhibitory (A), excitatory (B), and neuromodulatory nature (C). Kenyon cells, increasing in number up to about 1 x 10(8) in the adult, show minor variations in their dendritic pattern and have only one rare variant cell type. Interactions between them occur primarily at their axonal boutons as they crowd around efferent fibers. The latter have large receptive fields, some of their large somata are located within the confines of the corpora pedunculata, and they receive input almost only from Kenyon cells. Numerical and directional details of the circuitry in the corpora pedunculata have been extracted by a combination of light and electron microscopy, serial sectioning, silver staining, and stereology. The corpora pedunculata appear to process primarily the voluminous chemosensory input from the appendages, an assumption that is supported by the major connections of the organ.