Spatial distribution of pre- and postsynaptic sites of axon terminals in the dorsal horn of the frog spinal cord

Axon terminals which could be interpreted as dorsal root boutons, were photographed from a series of 98 ultrathin sections with a Jeol 100B electron microscope. A total of 13 boutons were recovered for computer reconstruction. Two of them were terminal boutons, eight en passant boutons and three boutons were only partially recovered. All boutons contained multiple synaptic sites (maximum 33 and minimum seven) at which axodendritic and axoaxonic synapses were established. Axodendritic synapses were of the asymmetric type and they were directed toward adjacent dendrites. In axoaxonic synapses, which were of the symmetric type, the boutons were invariably on the postsynaptic side. Among the presynaptic profiles axons with spherical and pleomorphic vesicles and dendrites with flattened vesicles could be discerned. On average, each 2.67-microns2 bouton surface area contained one presynaptic site at which an axodendritic synapse was established, and each 7-microns2 surface area contained one postsynaptic site for an axoaxonic (or dendroaxonic) contact. A tendency of grouping of synaptic sites was observed. Distance measurements between the closest neighbours of all synaptic sites were made in four combinations in boutons with the original and with a random distribution of synaptic sites. The arithmetic mean of distances measured between the presynaptic and the closest postsynaptic sites was almost twice as big as that measured in the reverse direction. The difference between these values became greatly reduced in the case of random distribution. The arithmetic mean of distances between the closest neighbours of presynaptic sites was about the same as that between the closest neighbours of postsynaptic sites. This latter value was considerably increased with randomly distributed synaptic sites. The results suggest a non-random distribution of synaptic sites on the surface of boutons. The analysis of cluster formation of synaptic sites performed with a numerical taxonomy technique revealed that the majority of the 153 synaptic sites were comprised in 27 clusters containing both pre- and postsynaptic sites within the 1-micron similarity level. All postsynaptic sites were within 1 micron of one or more presynaptic sites. On the basis of the assumption that the postsynaptic sites are occupied by inhibitory axoaxonic synapses, it is suggested that the transmitter release from the presynaptic sites can be individually controlled in this structural arrangement. A probable mechanism of this function may be the passive invasion of the bouton by the impulse propagating actively along the dorsal root fibre.     

Membrane structure of parallel-fibre synaptic terminals in the cerebellum of the jaundiced Gunn rat: freeze-fracture and E-PTA study

Summary Parallel-fibre synaptic membranes were examined by freeze-fracture and ethanolic-phosphotungstic acid methods in the cerebellum of homozygous (j/j) Gunn rats with hereditary jaundice. Parallel-fibre synapses with dendritic spines of Purkinje cell were severely affected since many Purkinje cells degenerated during the early postnatal period. Some parallel-fibre synaptic terminals lacked their postsynaptic partners and faced astrocytic processes from 18 days of age to the adult stage. These parallel-fibre terminals contained clusters of synaptic vesicles adjacent to synaptic membranes, and synaptic membranes and synaptic cleft materials were identical to those of parallel fibres with postsynaptic partners, as visualized by conventional electron microscopy with osmium tetroxide postfixation and staining of sections with uranyl acetate and lead citrate. In freeze-fractured specimens, the presynaptic membrane of parallel fibres had diffusely distributed large particles and tiny pits on the P-face and protuberances on the E-face, together representing synaptic vesicle attachment sites. Such vesicle attachment sites were present on the presynaptic membranes of parallel fibres without postsynaptic partners from day 18 to the adult stage. After ethanolic-phosphotungstic acid staining, parallel-fibre terminals displayed presynaptic dense projections, intercleft materials and postsynaptic thickening, but some parallel fibres lacked postsynaptic thickening. These observations suggest that the presynaptic membrane structure of the parallel fibre is preserved, even in the absence of a postsynaptic partner, in j/j cerebella. A mechanism for persistence of presynaptic membrane structures without postsynaptic partners in j/j cerebella is discussed.     

Dying-back of Purkinje cell dendrites with synapse loss in aging rats

Qualitative and quantitative changes were found in the cerebellar circuitry of old as compared to young rats. The old group had a reduced number of synapses (at least 30%), however, there was an increase in the size of remaining synaptic components (13.5% for spine head volume, 66% for bouton volume, and 17% for the area of synaptic contact zones). Furthermore, there were pronounced morphological changes in the older group appearing as: 1) prominent lipofuscin bodies in Purkinje cell somata, 2) numerous myelinated fibers in the lower part of the molecular layer, 3) tortuous Purkinje cell dendrites in a thinned molecular layer, and 4) abundant vacuolar profiles and membrane swirls in small and intermediate-sized dendrites. Our findings suggest that Purkinje cell dendrites are dying-back reducing the target field for granule cells and that remaining synaptic sites compensate by increasing synaptic contact area as well as the size of pre- and postsynaptic structures.     

Different kinds of axon terminals forming symmetric synapses with the cell bodies and initial axon segments of layer II/III pyramidal cells. II. Synaptic junctions

Summary Four different types of axon terminals form symmetric synapses with the cell bodies and initial axon segments of pyramidal cells in layer II/III of rat visual cortex. One type belongs to chandelier cells, and the other three kinds of terminals have origins that have not been established yet. These latter are referred to as large, medium-sized and dense terminals. The purpose of the present study was to examine the synaptic junctions formed by all four types of terminal. The synapses formed by the chandelier cell terminals are readily recognized in thin sections because of the characteristic features of both the terminals and the initial axon segments, which are the neuronal elements postsynaptic to them. In en face views of these axo-axonal synapses the junctions can be seen to have presynaptic dense projections that form a grid in which they are triagonally spaced, and have an average centre-to-centre spacing of 84 nm. As an ensemble the projections form the presynaptic grid, which usually has an oval or round outline, but may be notched on one side where projections are absent. The synaptic junctions of the large, medium-sized and dense terminals were examined by making reconstructions of the terminals from serial thin sections. It was found that at the interfaces between the axon terminals and the cell bodies of pyramidal cells, several separate synaptic junctions may be present, in addition to a number of puncta adhaerentia. Thus, there may be as many as five separate synaptic junctions and as few as one. It was also found that while the proportion of the area of the synaptic interface occupied by synaptic junctions was between 12% and 26% for dense terminals, for medium sized terminals it was 10–15%, and for the one large terminal reconstructed it was only 8%. Thus, there can be multiple synaptic junctions between each of these types of axon terminals and a pyramidal cell, and because many of the terminals forming symmetric junctions are boutons en passant, a number of vesicle release sites exist between the presynaptic axon and its postsynaptic partner. The axon terminals forming symmetric synapses in the cerebral cortex are assumed to be inhibitory, and consequently it is suggested that this arrangement of multiple release sites is designed to ensure that stimulation of the presynaptic axon results in an effective level of hyperpolarization of the postsynaptic neuron.     

Large dense-core vesicle exocytosis and membrane recycling in the mossy fibre synapses of the rabbit hippocampus during epileptiform seizures

Summary The ultrastructure of the hippocampal mossy fibre layer was studied in ultrathin sections and freeze-fracture preparations of rabbits under deep Nembutal anaesthesia, after recovery from ether anaesthesia, and 40 min after a single injection of methoxypyridoxine, that is, during the second generalized seizure discharge. The giant mossy fibre boutons contain two types of vesicles: evenly distributed, small round clear vesicles (50 nm) and a few scattered large dense-core vesicles (100 nm). In rare instances fusion of dense-core vesicles with the presynaptic membrane was observed. No differences in the morphology of the mossy fibre synapses were found between anaesthetized and unanaesthetized animals. During epileptiform seizures, however, the size and shape of clear and dense-core vesicles varied greatly. The active synaptic zones were covered with large, core-containing omega profiles or bumps and indentations. Only dense-core vesicles seem to undergo exocytosis. A fusion of clear vesicles with the presynaptic membrane was not observed.Various explanations for the fact that only dense-core vesicles seem to undergo exocytosis are discussed. The hypothesis is put forward that in the mossy fibre bouton two morphologically and functionally distinct populations of synaptic vesicles exist and that only one of them undergoes visible irreversible exocytosis, whereas the majority, that is, the small vesicles discharge their transmitter by reversible fusion.After MP injection features of membrane retrieval were also prominent. Frequently, at the borders of the active synaptic zones coated membrane convolutes of both pre- and postsynaptic membranes had invaded the terminals as well as the postsynaptic spine. Thus, in contrast to electrical stimulation, the self-sustained seizures allow energy-expensive processes such as extensive membrane internalization to take place during the interictal pauses.     

Non-primary afferents to the principal cells of the rostral anteroventral cochlear nucleus of the guinea pig

The non-primary input to the soma of the principal cells of the rostral anteroventral cochlear nucleus (AVCN) of the guinea pig was defined by correlating data from freeze-fractured and thin-sectioned specimens. The principal cells receive three types of non-primary terminals in addition to a single end bulb from the ascending branch of the auditory nerve. The first, the O-boutons are irregularly shaped, medium-sized boutons, containing oval synaptic vesicles. These cover about 13% of the soma and each makes from one to three crescent-shaped axosomatic junctions. Paramembranous dense material is distributed symmetrically between the pre- and postsynaptic junctional membranes. The second, the F-bouton, is a small, ovate bouton which occurs either singly or in small clusters and covers about 5% of the soma. This bouton, containing flattened synaptic vesicles, has a single, oval synaptic junction lined by thin, paramembranous densities distributed symmetrically between the pre-and postsynaptic membranes. The third, the SR-bouton, is a medium-sized bouton containing small, round synaptic vesicles. These boutons cover about 4% of the soma, but more frequently contact dendrites in the neuropil. The junctional region of the bouton is poorly defined. Small tufts of dense material symmetrically line the cytoplasmic surfaces of the pre- and postsynaptic membranes along scattered, narrow undulations of the apposition. In freeze-fracture replicas, several narrow troughs on the cytoplasmic leaflet frequently have a higher density of large intramembranous particles than adjacent membrane and may represent a diffusely organized active zone. No specializations are found on either leaflet of the postsynaptic plasmalemma opposite the non-primary boutons. Correlation of the measurements from thin-sectioned and freeze-fractured material suggests that the oval, flattened, and small round vesiclecontaining boutons occur on the somata of the principal cells in a ratio of 6:3:1.     

Dense core vesicles resemble active-zone transport vesicles and are diminished following synaptogenesis in mature hippocampal slices

Large dense core vesicles (approximately 100 nm) contain neuroactive peptides and other co-transmitters. Smaller dense core vesicles (approximately 80 nm) are known to contain components of the presynaptic active zone and thought to transport and deliver these components during developmental synaptogenesis. It is not known whether excitatory axons in area CA1 contain such dense core vesicles, and whether they contribute to synaptic plasticity of mature hippocampus. Serial section electron microscopy was used to identify dense core vesicles in presynaptic axons in s. radiatum of area CA1 in adult rat hippocampus. Comparisons were made among perfusion-fixed hippocampus and hippocampal slices that undergo synaptogenesis during recovery in vitro. Dense core vesicles occurred in 26.1+/-3.6% of axonal boutons in perfusion fixed hippocampus, and in only 17.6+/-4.5% of axonal boutons in hippocampal slices (P<0.01). Most of the dense core vesicle positive boutons contained only one dense core vesicle, and no reconstructed axonal bouton had more than a total of 10 dense core vesicles in either condition. Overall the dense core vesicles had average diameters of 79+/-11 nm. These small dense core vesicles were usually located near nonsynaptic membranes and rarely occurred near the edge of a presynaptic active zone. Their size, low frequency, locations, and decrease following recuperative synaptogenesis in slices are novel findings that merit further study with respect to small dense core vesicle content and possible contributions to synapse assembly and plasticity in the mature hippocampus.     

Synaptic plasticity in the oedematous human cerebral cortex

Synaptic plastic changes are fundamental events which occur spontaneously during development, maturity and aging processes or can be induced by injury or trauma. To examine lesion-induced synaptic plasticity, cortical biopsies were taken from the frontal, parietal, temporal and occipital cortex of living patients during neurosurgical treatment of brain trauma, brain tumours and vascular malformations, and processed for transmission electron microscopy. Enlargement of both pre- and postsynaptic endings, irregularly shaped, lobulated, stellate and bifurcated presynaptic endings and conformational changes of dendritic spines were observed. Numerous flat, curved and invaginated axodendritic and axospinous asymmetric synapses were distinguished and a smaller proportion of axodendritic and axosomatic symmetric synapses. Activated or sensitized synapses showed numerous frontline spheroid synaptic vesicles, prominent dense presynaptic dense projections and increased length of synaptic membrane complex. Perforated synapses, multiple synapses and serial synapses were also found evincing synaptic splitting and formation of new synaptic connections. The overall images suggest increased number of excitatory circuits, which were correlated with the tonico-clonic convulsion or post-traumatic seizures observed in some patients. Numerous coated vesicles were observed in pre- and postsynaptic structures. Increased number of polyribosomes were found in the dendritic shafts. The dilated spine apparatus, the coated vesicles and the increased number of polyribosomes seem to represent a system for synthesis, transport and storage of synaptic proteins for the formation of new synapses. Coexisting synaptic plasticity and synaptic degeneration were observed in the patients under study. Dendritic and astrocyte synapse-like junctions were also characterized.     

Morphological studies of synapses and varicosities in dissociated cell cultures of sympathetic neurons

Neurons dissociated from the superior cervical ganglia of newborn rats can be grown under conditions which support either adrenergic or cholinergic differentiation. In both cases, the neurons form numerous morphologically specialized synaptic terminals or synapses as well as relatively unspecialized varicosities. The ultrastructure of both types of terminal was compared in mature neuronal cultures and the effects of growth conditions on terminal morphology examined. After aldehyde-osmium fixation, synapses in cultures grown under adrenergic or cholinergic conditions were characterized by asymmetrical membrane specializations comparable to type I or asymmetric synapses; bismuth iodide and ethanolic phosphotungstic acid impregnation of neuronal cultures revealed the presence of characteristic synaptic membrane specializations: a presynaptic grid of dense projections and a wide postsynaptic dense band of uniform thickness. No membrane specializations were apparent in varicosities after aldehyde-osmium fixations or with these stains. Intramembranous particle distributions were examined in freeze-fracture replicas of neurons. Aggregates of large, 10-12 nm particles were found on P-face membrane leaflets of cell bodies and large diameter processes; this distribution is the same as that of synapses in thin-sectioned preparations. These particle aggregates may represent postsynaptic membrane specializations or acetylcholine receptors. The cytoplasmic leaflet of boutons contained large, 12-14 nm particles, which appeared to be concentrated at the region of synaptic contact at putative synapses, but were diffusely distributed in varicosity membranes. Similar large particles were also seen at a much lower density in the membrane E-face. None of these ultrastructural characteristics appeared to vary with transmitter identity or growth conditions. Synaptic vesicle shape, however, did vary in glutaraldehyde-fixed cultures. At all ages examined, neurons grown on monolayers of heart cells contained predominantly round vesicles, whereas neurons grown in the virtual absence of non-neuronal cells possessed pleiomorphic synaptic vesicles. This difference in vesicle shape appeared to be correlated more closely with growth in the presence of non-neuronal cells than with the transmitter present at the time of fixation.     

Sequential events of degeneration and synaptic remodelling in the viper optic tectum following retinal ablation. A degeneration, radioautographic and immunocytochemical study

The ultrastructural changes taking place in the retino-recipient layers of the viper optic tectum were examined between 5 and 122 days after retinal ablation. The initial degeneration of retinotectal terminals proceeds at widely different rates and is characterized by a marked degree of polymorphism in which a number of different patterns can be discerned. In the final stages of degeneration, either both the degenerating bouton and the distal portion of the postsynaptic element are engulfed by reactive glia, or, more frequently, only the degenerating terminal is eliminated and the postsynaptic differentiation remains. The free postsynaptic differentiations are reoccupied predominantly by boutons containing pleiomorphic vesicles and which are for the most part gamma-aminobutyric acid (GABA)ergic, thus forming heterologous synapses; less frequently these sites are occupied by boutons of the ipsilateral visual contingent to form homologous synapses. These two processes, both of which depend on terminal axonal sprouting, take place within the first 3 postoperative months. They are followed by a decrease in the number of heterologous synapses and a concurrent increase in the number of homologous synapses newly formed by optic boutons generated by collateral preterminal sprouting of ipsilateral retinotectal fibres. The data suggest that partial deafferentation of the optic tectum induces a transitory GABAergic innervation of free postsynaptic sites prior to the restoration of new retinal synaptic contacts.     

An ultrastructural study of 5-hydroxytryptamine-, thyrotropin-releasing hormone- and substance P-immunoreactive axonal boutons in the motor nucleus of spinal cord segments L7-S1 in the adult cat

The distribution and fine structure of 5-hydroxytryptamine-, thyrotropin-releasing hormone- and substance P-immunoreactive synaptic boutons and varicosities were studied in the motor nucleus of the spinal cord segments L7-S1 in the cat, using the peroxidase-antiperoxidase immunohistochemical technique and analysis of ultrathin serial sections. The 5-hydroxytryptamine-, thyrotropin-releasing hormone- and substance P-immunoreactive boutons had a similar ultrastructural appearance as judged from serial section analysis. The boutons could be classified into two types on the basis of their vesicular content, with one type containing a large number of small agranular vesicles together with only a few, if any large granular vesicles, while the other type contained a large number of large granular vesicles in addition to small agranular vesicles. The vesicles were spherical or spherical-to-pleomorphic. Postsynaptic dense bodies (Taxi bodies) were occasionally observed in relation to all three types of immunoreactive boutons, which almost invariably formed synaptic junctions with dendrites. Judged by the calibre of the postsynaptic dendrites, the boutons were preferentially distributed to the proximal dendritic domains of motoneurons. In one case, a substance P-immunoreactive bouton formed an axosomatic synaptic contact. In addition to synaptic boutons, 5-hydroxytryptamine-, thyrotropin-releasing hormone- and substance P-immunoreactive axonal varicosities containing a large number of large granular and small agranular vesicles but lacking any form of conventional synaptic contact were observed. Such varicosities were either directly apposing surrounding neuronal elements or separated from the neurons by thin glial processes. The origin of the immunoreactive boutons was not traced, but it was thought likely that the main source of the boutons was neurons with their cell bodies located in the medullary raphe nuclei.     

Single-bouton-mediated synaptic transmission: postsynaptic conductance changes in their relationship with presynaptic calcium signals

Most central neurons contact their dendritic targets at several sites. However, it is not known whether all synapses formed by a single parent axon make the same contribution to the postsynaptic response. In order to answer this question it is necessary to isolate the synaptic currents generated by individual axon terminals. This paper describes a method that was designed to activate transmitter release from solitary synaptic boutons in culture. Neurons from the embryonic rat superior colliculus were grown at low density and double-loaded with a fluorescent marker of synaptic vesicles (FM1-43 or RH414) and a fluorescent Ca2+ indicator (Fura-2, Mag-fura-2, Oregon Green BAPTA-1 or Oregon Green BAPTA-5N). Action potential generation was blocked by tetrodotoxin. Appropriate synaptic boutons were selected under phase-contrast and fluorescence illumination at a magnification of 1000. They were activated by short electrical pulses via a fine-tipped glass pipette filled with bath solution. Presynaptic Ca2+ transients were measured in a region delineated by the FM1-43/RH414 fluorescence. By simultaneous presynaptic Ca2+ imaging and whole-cell recording of postsynaptic responses to single depolarizing pulses, the quantitative relationships between pre- and postsynaptic parameters of synaptic strength in a small synapse of central origin could, for the first time, be analysed. The experiments showed that the average postsynaptic currents depend strongly on the size of the presynaptic Ca2+ transients. However, at any level of presynaptic Ca2+ concentration postsynaptic responses fluctuated in amplitude.     

大白鼠下丘脑腹内侧核突触的电镜观察

本文报告大白鼠下丘脑腹内侧核内突触的类型和突触亚显微结构的形态特征。共观察了1005个突触,其中轴-树突触占96.6%;轴-体突触占2.8%;轴-轴突触占0.5%。在下丘脑腹内侧核中还观察到1例嵴突触,以往未见报道。嵴突触的突触后成分来自树突,呈杵指状突起。突触后膜明显增厚,具有突触下致密小体。两个内含圆形清亮小泡的轴突并列于嵴的两侧壁上,构成嵴突触。在轴-体、轴-树突触中尚观察到并联突触(包括突触复合体)、切线突触及串联突触等复杂的联接形式。本文对某些复合突触的机能也作了讨论。     

Parvalbumin-like immunostaining in the cat inferior colliculus. Light and electron microscopic investigation

The presence of the calcium-binding protein parvalbumin (PV) was studied in neuronal elements of the cat's inferior colliculus (IC) by means of light and electron microscopic immunocytochemistry. Immunostaining of PV was detected in all three main parts of the IC. Several subtypes of large neurons that differed in size and shape were immunostained, comprising approx. 15% of the total number of PV-containing neurons. Approx. half of the labeled neurons were medium sized. Two types of small neurons were found to be PV synthesizing, and comprised approx. 35% of the total PV-containing population. Ultrastructurally, many dendrites were heavily immunolabeled, and the reaction product was present in dendritic spines as well. Several types of synaptic boutons contained reaction product, and terminated on both labeled and unlabeled postsynaptic targets forming asymmetric and symmetric synapses. Approx. 70% of all PV-immunolabeled terminals contained round synaptic vesicles and formed asymmetric synapses. The majority of these boutons were of the "large round" type and corresponded to the terminals of cochlear nuclei. A lower number were of the "small round" type, and were probably corticotectal terminals. The remaining 30% of PV-containing terminals contained pleomorphic or elongated vesicles and formed symmetric synapses. These terminals corresponded with "P" and "F1" bouton types. Part of these boutons appeared to arise from nuclei of the lateral lemniscus and the superior olive, and a certain percentage likely represented endings of inhibitory interneurons.     

Vulnerability of cerebellar development in malnutrition-II. Intrinsic determination of total synaptic area on purkinje cell spines

In a previous investigation, we demonstrated a greater reduction in the ratio of granule cells to Purkinje cells in malnourished female rats than male rats that were reared under the same conditions (Hillman &; Chen, 1981). In this study, the synaptic relationships between these two types of cells were analyzed to determine how this difference in ratio was accommodated in cerebellar circuitry. Control diets (25% casein) and experimental diets (8% casein), having the same calories, were administered pregestationally and through lactation with continuation of the respective diets to offspring until death at 60 days. Molecular layer volume and synaptic density were determined and the relative number of synapses on each Purkinje cell was estimated from the number of synapses in the molecular layer and total number of Purkinje cells. The average length of synaptic profiles on Purkinje cell spines was measured and the average synaptic contact area on each Purkinje cell was calculated. We found that the average number of synapses on individual Purkinje cells decreased in the deficient female group but not in the deficient male group and there was an increase in the average length of synaptic profiles in the malnourished female group but not in the deficient male group.In some cerebella of females, elongated synaptic profiles on giant spines were seen on Purkinje cells. Comparison of the average number of synapses on each Purkinje cell with average length of synaptic profiles revealed an inverse relationship between size and number of synapses for two control and two experimental groups and a subgroup of females with giant spines. After conversion of average profile length to average contact area and taking into account the number of synapses on each Purkinje cell, we found that Purkinje cells for all five groups had the same total synaptic area. This inverse relationship was believed to reflect a constant total contact area for parallel fiber synapses on Purkinje cells.It is suggested that a constancy in total synaptic area means that the amounts of macromolecules that are incorporated into postsynaptic membrane specializations are determined intrinsically by the Purkinje cell. However, the distribution of these molecules to synaptic sites on dendritic trees can be shifted by interaction of parallel fiber synapses with the Purkinje cell.     

Ventral horn synaptology in the rat

Summary The synaptology of the normal ventral horn of the rat was studied. Presynaptic boutons were classified as S (spherical vesicles), F (flattened vesicles), and G (predominance of 700–1200 Å granular vesicles). In addition, Cf, Cs, M, and T synaptic complexes were defined and quantitated. Synaptology was studied on -motoneuron somata, -motoneuron primary dendrites, peripheral dendrites and interneuron somata. In addition, organelles were quantified for the pre- and postsynaptic members of the synaptic complex. All counts were made on coded material and these data were analyzed statistically.Motoneuron somata had significantly more (P < 0.01) F (58%) than S (33%) boutons. This was also the case for the motoneuron primary dendrite (P < 0.01; F, 61%; S, 37%). The small dendrites had more (P < 0.05) S (56%) than F (44%) boutons. More Cf bulbs (P < 0.01) were found on motoneuron somata (9%) than on motoneuron primary dendrites (2%) or interneuron somata (3%). The C complex presynaptic bouton contained spherical (Cs) or flattened (Cf) synaptic vesicles which were attributed to the fixation employed. Cf bulbs were not observed on small dendrites. G bulbs were observed (< 1%) only on small dendrites. M bulbs were not observed on any postsynaptic structure.The boutons of the motoneuron primary dendrites (15% of total afferents) and peripheral dendrites (14% of total afferents) were frequently branched whereas there was significantly (P < 0.01) less branching of boutons on motoneuron and interneuron somata. Small postsynaptic subsurface cisterns were associated with boutons of both the S and F type on all structures. In addition, these cisterns were observed in motoneuron somata (4%) and interneuron somata (2%) without an accompanying bouton. C postsynaptic organelles were observed in motoneuron somata (3%) and primary dendrites (1%) with an overlying neuroglial cell process and no presynaptic bouton.The synaptology of the rat ventral horn is comparable to that in the cat and monkey. However, M (R) and P bulbs were not observed in the rat. This could be due to the sampling method which indicated that synapses with less than 1% occurrence fall at the level of statistical resolution in quantitative electron microscopy. The presence of postsynaptic specialization usually associated with presynaptic boutons with no presynaptic component may be a reflection of the dynamics of normal bouton renewal in the rat ventral horn.     

Light microscopic and ultrastructural localization of immunoreactive substance P in the dorsal horn of monkey spinal cord

Light- and electron-microscopic localization of substance P in the monkey spinal cord was studied by the peroxidase anti-peroxidase technique with the particular aim of examining types of interactions made by substance P-positive boutons with other neuronal elements in the dorsal horn. By light-microscopy dense labeling for immunoreactive substance P was found in laminae I, II (outer zone) and V (lateral region), consistent with findings in other mammalian species. By electron-microscopy, substance P-positive staining was mostly in unmyelinated and in some thinly myelinated small diameter fibers. Substance P-positive terminals contained both large granular vesicles (80-120 nm diameter), which were filled with reaction product, and clear round vesicles (40-60 nm). Substance P-positive large granular vesicles were sometimes observed near presynaptic sites and in contact with dense projection there. Immunoreactive substance P boutons were small to large in size (1-4 micron), formed synapses with somata and large dendrites and were the central axons of synaptic glomeruli where they were in synaptic contact with numerous small dendrites and spines. Substance P-labeled axons frequently formed synapses with dorsal horn neurons which were also postsynaptic to other types of axons. Substance P-positive profiles participated in numerous puncta adhaerentia with unlabeled cell bodies, dendrites and axons. Only rarely, some suggestive evidence was obtained indicating that axons might synapse onto substance P-containing boutons. Biochemical analysis of monkey spinal cord tissue extracts, undertaken to characterize more precisely the immunoreactive substances, indicated that only substance P and its oxide derivative were detected with the antiserum used in the immunocytochemistry. These morphological findings show that substance P is contained within a class of axon terminals, many of which have been shown previously in the monkey to originate from the dorsal root. The results suggest that modulation of substance P primary afferents terminating in the outer dorsal laminae of the monkey spinal cord occurs in part via axonal inputs onto dorsal horn neurons postsynaptic to the primary afferent.     

Quantitative ultrastructure of synapses on functionally identified primary afferent neurons in the cat trigeminal mesencephalic nucleus

Though a number of studies have reported the presence of synapses on neurons in the trigeminal mesencephalic nucleus (Vmes), there have been no quantitative studies of either the density of innervation, or the ultrastructure, of the synapses on single, physiologically identified neurons in this nucleus. In this study we recorded from single neurons in the Vmes, identified them as being either muscle spindle afferents (MS) or periodontal ligament mechanoreceptor afferents (PL), and then labeled the neurons by intra-axonal injection of horseradish peroxidase (HRP). The material was first processed to reveal the HRP activity, following which ultrathin sections through the labeled somata were cut and examined under the electron microscope. Complete serial reconstructions were made through the soma of one MS neuron and one PL neuron, and the contacts on the neurons reconstructed. Boutons were found on the soma, spines, appendages and the axon hillock and the initial segment of the axon. The numbers of boutons terminating on the two neurons were 198 (PL) and 424 (MS), giving a packing density of 4.4 and 10.7 boutons respectively (i.e., number of boutons/100 micron 2 of the postsynaptic membrane). Boutons could be separated into two types on the basis of their vesicles: those containing clear, round vesicles (i.e., S-type) and those containing a mixture of round, oval and flattened vesicles (P-type). Ninety-five (PL neuron) and 99% (MS neuron) of terminals on the two neurons were P-type. All the S-type boutons and 80% of the P-type boutons formed asymmetric synaptic contacts while 10% of the P-type boutons made symmetric contacts. Quantitative measurements of the P-type boutons on the labeled neurons, in which the data of MS and PL neurons were pooled, revealed that bouton volume was highly correlated with bouton surface area, active zone number, total active zone area, vesicle number, and mitochondrial volume. However, comparing the quantitative measurements of the P-type boutons with those of previously reported vibrissa afferent terminals and their associated axon terminals revealed that all the parameters were smaller for the P-type boutons (on Vmes neurons) than those of the vibrissa afferent terminals but similar to those of axon terminals presynaptic to the vibrissa afferents. Taken together, our results emphasize the wide scope for synaptic interactions in the Vmes and suggest that it may be more fruitful to view the Vmes as an integrating center.     

Ultrastructure and synaptic connectivity of main and accessory olfactory bulb efferent projections terminating in the rat anterior piriform cortex and medial amygdala

Neurons in the main olfactory bulb relay peripheral odorant signals to the anterior piriform cortex (aPir), whereas neurons of the accessory olfactory bulb relay pheromone signals to the medial amygdala (MeA), suggesting that they belong to two functionally distinct systems. To help understand how odorant and pheromone signals are further processed in the brain, we investigated the synaptic connectivity of identified axon terminals of these neurons in layer Ia of the aPir and posterodorsal part of the MeA, using anterograde tracing with horseradish peroxidase, quantitative ultrastructural analysis of serial thin sections, and immunogold staining. All identified boutons contained round vesicles and some also contained many large dense core vesicles. The number of postsynaptic dendrites per labeled bouton was significantly higher in the aPir than in the MeA, suggesting higher synaptic divergence at a single bouton level. While a large fraction of identified boutons (29 %) in the aPir contacted 2–4 postsynaptic dendrites, only 7 % of the identified boutons in the MeA contacted multiple postsynaptic dendrites. In addition, the majority of the identified boutons in the aPir (95 %) contacted dendritic spines, whereas most identified boutons in the MeA (64 %) contacted dendritic shafts. Identified boutons and many of the postsynaptic dendrites showed glutamate immunoreactivity. These findings suggest that odorant and pheromone signals are processed differently in the brain centers of the main and accessory olfactory systems.     

Synaptic connections of enkephalin-immunoreactive nerve terminals in the neostriatum: a correlated light and electron microscopic study

Summary Two different antisera to leucine-enkephalin were used to study the localization of enkephalin-like immunoreactive material in the neostriatum and globus pallidus of the rat, by means of the unlabelled antibody-enzyme method. Thin immunoreactive varicose fibres are scattered throughout the neostriatum. In the ventral striatum, fibres come together and follow a relatively straight course for several micrometers, forming tube-like structures which can be traced to cell bodies; these cell bodies are completely surrounded by immunoreactive fibres. Occasional immunoreactive varicose fibres are also found close to another type of neuron throughout the whole neostriatum.Examination by electron microscopy of immunoreactive structures that had been identified first in the light microscope, showed that each of the nearly 200 varicosities examined was a vesicle-containing bouton that formed a synaptic contact. Rarely were asymmetrical synaptic contacts found between immunoreactive boutons and dendritic spines. All other synapses formed by enkephalin-immunoreactive boutons were symmetrical. Two types of postsynaptic neuron were identified; the first type was a medium-sized neuron with the ultrastructural features of a typical striatal spiny neuron. The second type had a larger perikaryon surrounded by numerous immunoreactive varicosities that were found to be boutons forming symmetrical synapses. The long dendrites of this second type of neuron likewise received a dense input of immunoreactive boutons forming symmetrical synapses; such ensheathed dendrites were found to be the tube-like structures seen in the light microscope. The ultrastructural features of these neurons, notably a highly indented nucleus, were those of a rare type of striatonigral neuron. In the globus pallidus, all the enkaphalin-immunoreactive boutons studied formed symmetrical synapses with ensheathed dendrites and perikarya that were similar to the latter type of postsynaptic neuron in the neostriatum. Axo-axonic synapses involving immunoreactive boutons were not seen in our material.The results are consistent with the view that enkephalin-like substances may be synaptic transmitters in the neostriatum and that they may have different actions according to the nature of the postsynaptic target. The finding that one type of neostriatal neuron, and a very similar neuron in the globus pallidus, receives multiple enkephalin-immunoreactive boutons all over its perikaryon and along its dendrites indicates a potentially important role of enkephalin in the convergence of information within the neostriatum and pallidum on to output neurons.