Nitrergic neurons make synapses on dual-input dendritic spines of neurons in the cerebral cortex and the striatum of the rat: implication for a postsynaptic action of nitric oxide

Pre-embedding electron microscopic immunocytochemistry was used to examine the ultrastructure of neurons containing nitric oxide synthase and to evaluate their synaptic relationships with target neurons in the striatum and sensorimotor cerebral cortex. Intense nitric oxide synthase immunoreactivity was found by light and electron microscopy in a type of aspiny neuron scattered in these two regions. The intensity of the labeling was uniform in the soma, dendrites and axon terminals of these neurons. In both forebrain regions, nitric oxide synthase-immunoreactive neurons received synaptic contacts from unlabeled terminals, which were mostly apposed to small-caliber dendrites. The unlabeled symmetric contacts were generally about four times as abundant as the unlabeled asymmetric contacts on the nitric oxide synthase-immunoreactive neurons. Terminals labeled for nitric oxide synthase were filled with synaptic vesicles and were observed to contact unlabeled neurons. Only 54% (in the cerebral cortex) and 44.3% (in the striatum) of the nitric oxide synthase-immunoreactive terminals making apposition with the target structures were observed to form synaptic membrane specializations within the plane of the randomly sampled sections. The most common targets of nitric oxide synthase-immunoreactive terminals were thin dendritic shafts (54% of the immunoreactive terminals in the cortex and 75.7% of the immunoreactive terminals in the striatum), while dendritic spines were a common secondary target (42% of the immunoreactive terminals in the cortex and 20.6% of the immunoreactive terminals in the striatum). The spines contacted by nitric oxide synthase-immunoreactive terminals typically also received an asymmetric synaptic contact from an unlabeled axon terminal.These findings suggest that: (i) nitric oxide synthase-immunoreactive neurons in the cortex and striatum preponderantly receive inhibitory input; (ii) nitric oxide synthase-containing terminals commonly make synaptic contact with target structures in the cortex and striatum; (iii) spines targeted by nitric oxide synthase-containing terminals in the cortex and striatum commonly receive an asymmetric contact as well, which may provide a basis for a synaptic interaction of nitric oxide with excitatory input to individual spines.     

Ultrastructure of human cerebral cortical synapses: age-specific aspects

The paper deals with electron microscopic studies of the cerebral cortex in human beings aged 70-80 years. The synapses show a decrease in the number of synaptic vesicles, their impaired distribution in the presynaptic process. There are changes in the mechanisms of interaction of synaptic vesicles and a presynaptic membrane. In the latter, the vesicles loose their discreteness, some of them are replaced by a fine-granular material. The joining of the vesicles and membrane is impaired. A part of active synaptic areas is transformed to desmose-like contacts. The findings are indicative of synaptic dysfunction.     

Synaptogenesis and distribution of presynaptic axonal varicosities in low density primary cultures of neocortex: an immunocytochemical study utilizing synaptic vesicle-specific antibodies, and an electrophysiological examination utilizing whole cell recording

Summary Low-density primary cultures of neocortical neurons were utilized to examine: (i) early interactions of growing neurites with morphological characteristics of axons with other neuronal elements, and (ii) the distribution of presynaptic axonal varicosities closely apposed to MAP-2 immunoreactive, putatively postsynaptic, dendrites. At the light microscopical level axonal varicosites, presumably presynaptic terminals, were identified using immunocytochemistry incorporating antibodies specific for the synaptic vesicle antigens synaptophysin and synapsin. The presence of synaptophysin- and synapsin-immunoreactive swellings along axonal processes was first detected at 5 days post-plating and was also apparent in axons growing in isolation. At 5–7 daysin vitro, immunolabelled axonal varicosities in close apposition to putative postsynaptic dendrites (MAP-2 immunoreactive) dendrites were detected. Electrophysiologically active synaptic contacts can also readily be detected at this stage. After 3 weeksin vitro presynaptic contacts do appear to be distributed heterogeneously along postsynaptic dendrites of many neurons in culture. As the culture matures a higher number of presynaptic profiles can be seen along dendrites, with a centrifugal distribution, e.g. a higher density of presynaptic axonal terminals in close apposition to more distal regions of larger dendrites, putatively considered to be apical dendrites of pyramidal-like neurons. In our cultures, the overall increase in the density and the pattern of distribution of presynaptic axon terminals immunoreactive for synaptic vesicle antigens closely apposed to putative post-synaptic structures mimics the general postnatal increase of synaptic density in the neocortexin vivo. Thus, low density primary cultures of neocortical neurons offer a valuable system to explore and manipulate (i) the molecular and cellular basis of neocortical synaptogenesis, and (ii) the pharmacology of neocortical synaptic transmission.     

Cortical and subcortical patterns of synaptophysinlike immunoreactivity in Alzheimer''s disease.

Quantification of synaptophysinlike immunoreactivity is a valuable method for studying the presynaptic terminals in the normal and damaged nervous system. The present report shows that in the control brain, the predominant pattern of synaptic immunostaining in the neocortex was that of an evenly distributed densely granular immunolabeling of the neuropil, while in the paleocortex and in subcortical areas of the brain most of the presynaptic terminals were distributed along the dendritic arborizations or around the neuronal somata. The immunochemical and the immunohistochemical analysis of the Alzheimer's disease tissue showed that the frontal and parietal cortex presented the most severe and widespread loss, with a 45% loss in synaptophysin immunoreactivity. These areas showed an average 35% loss of large neurons. The visual cortex, hippocampus, entorhinal cortex, nucleus basalis of Meynert, and locus ceruleus displayed some degree of loss, but to a lesser extent. In addition to this loss, the basic patterns of organization of the presynaptic terminals were altered, with the presence of abundant, enlarged synaptophysin-labeled terminals. This study further supports the role of synaptic pathology in Alzheimer's disease.     

Immunohistochemical distribution of the two isoforms of synaphin/complexin involved in neurotransmitter release: localization at the distinct central nervous system regions and synaptic types

The cellular and subcellular localization of the two synaphin isoforms, proteins associated with the docking/fusion complex crucial to neurotransmitter release, was studied in the rat central nervous system by using light microscopic and electron microscopic immunohistochemistry with monoclonal antibodies specific to each isoform. Synaphin 1 (complexin II) was predominantly expressed in neurons of the central nervous system regions such as cerebral cortex (the II, III and VI cortical layers), claustrum, hippocampus, entorhinal cortex, amygdaloid nuclei, substantia nigra pars compacta, superior colliculus, pontine reticulotegmental nucleus and inferior olive, whereas synaphin 2 (complexin I) was in the cerebral cortex (the IV cortical layer), thalamus, locus coeruleus, gigantocellular reticular field, cuneate nucleus and cerebellar basket and stellate cells. In some regions, including the caudate–putamen, globus pallidus, pontine reticular nucleus, cerebellar nuclei and spinal gray matter, synaphin 1 was mainly present in small or medium-sized neurons, while synaphin 2 was in large cells. Medial habenular nucleus and cerebellar granule cells showed both immunoreactivities. In the neuropil of the cerebral cortex and hippocampus, synaphin 1 expression was accentuated in the axon terminals of axospinal and axodendritic synapses, while synaphin 2 was predominant in the axon terminals of axosomatic synapses. In the axon terminals, both immunolabelings were associated with synaptic vesicles and the plasma membrane, being accentuated in the vicinity of synaptic contacts. In the cerebral cortex, both immunoreactivities were also present occasionally in dendrites and dendritic spines, associated with microtubules and the plasma membrane including the postsynaptic densities.These results suggest that the two isoforms of synaphin are involved in synaptic function at the distinct presynaptic regions in the central nervous system, and that some dendrites are another functional site for the proteins.     

An ultrastructural study of the lateral reticular nucleus in the rat

Summary A systematic study of the normal synaptic patterns within the lateral reticular nucleus (LRN) of the rat revealed various synaptic relationships. Two types of axon terminals were identified according to the morphology of the synaptic vesicles contained within them. Axon terminals with round vesicles established asymmetrical synaptic contacts with the somata and all areas of the dendritic trees including somatic and dendritic appendages. Pleomorphic-vesicle terminals established symmetrical synaptic contacts on somata and their appendages and on all sizes of dendrites and their appendages. Both round and pleomorphicvesicle terminals were infrequently seen to synapse upon the somata and proximal dendrites. The round-vesicle terminals outnumbered the pleomorphic-vesicle terminals on the dendritic trees. Terminals of the en passant type were also common throughout the LRN. Both round and pleomorphic-vesicle terminals were observed simultaneously contacting the soma and one or more dendritic profiles, or two different dendritic profiles. Synaptic configurations (glomeruli) were also observed in all three divisions of the nucleus. They consisted of a large, central, round-vesicle terminal contacting a number of small-calibre dendritic processes. This arrangement was surrounded by one or more sheets of glial lamellae. Puncta adherentia were observed on the apposed membranes of adjacent cells, adjacent dendrites and adjacent axon terminals.     

Distribution of synaptic ribbons in the developing organ of Corti

Summary Studies of synaptogenesis in the developing organ of Corti in the intact mouse and in culture indicate that the inner and outer hair cells contain three populations of synaptic ribbons, i.e. ribbons adjacent to nerve fibres, free intracellular ribbons and misplaced ribbons apposed to non-neuronal elements. Ribbons adjacent to nerve fibres can be further classified into: ribbons synaptically engaged, ribbons participating in formation of presynaptic complexes only and ribbons that are not engaged to the hair cell membrane. In the developing innervated cultures the ribbon distributions are similar to those in the normal animal. Inner and outer hair cells differ in distribution of the ribbons. In the inner hair cells the ribbons adjacent to the nerve fibres are dominant (over 90%) and most of them (88%) are synaptically engaged. In the outer hair cells the presynaptic ribbons dominate the population (up to 60%) during the first postnatal week when the cells acquire afferent synaptic connections. This stage is followed by a marked reduction in the number of all ribbons. In the intact animal the rapid decrease results in a relative increase of misplaced and free ribbons. These changes are presumably due to the loss of some of the afferents. In the denervated hair cells the distribution of ribbons indicated the presence of conspicuous scatter. In the areas of incomplete denervation, however, the ribbons are apposed to the preserved fibres. Despite denervation, most of the ribbons develop the entire presynaptic complex in apposition to non-neuronal structures.The different populations of synaptic ribbons appear to reflect different stages in synapse formation. Possibly, the synaptic body originates in the interior of the hair cell and subsequently migrates to the cell membrane. In any case, a nerve fibre appears critical in influencing the location of the synaptic ribbon. At the apposition of the ribbon to the hair cell membrane, presynaptic densities are formed and the ribbon appears to become anchored. Typically, the nerve fibre membrane apposed to the presynaptic complex responds with the formation of postsynaptic densities.     

Electron microscopic study of GABA-immunoreactive neuronal processes in the superficial gray layer of the rat superior colliculus: their relationships with degenerating retinal nerve endings

Summary GABA-immunoreactive neuronal elements were detected in the stratum griseum superficiale or superficial gray layer of the rat superior colliculus in an electron microscopic study, using postembedding immunocytochemistry with protein A-gold as a marker. In addition to neuronal somata, two types of GABA-immunoreactive neuronal processes were observed. Numerous profiles of axon terminals (1 m in diameter) with clear round or pleomorphic synaptic vesicles and mitochondria were found to establish mostly symmetrical synaptic contacts with GABA-immunonegative dendrites of various diameters. Some axosomatic synapses could also be observed. The gold particle density in this axon terminal compartment was between seven and 13 times the background level. The stratum griseum superficiale also included GABA-immunoreactive dendrites, some of which contained clear synaptic vesicles. These dendritic profiles always formed the presynaptic component of dendrodendritic synaptic contacts. The density of the gold particles in the dendritic compartment, taken as a whole, was between three and 13 times the background level. Furthermore, the relationship between the GABA-immunoreactive neuronal elements and degenerating retinal nerve endings identified in the left stratum griseum superficiale following enucleation of the right eye was investigated after a 7-day survival period. The profiles of degenerating retinal nerve endings (0.7 m in diameter) were found to be devoid of any specific labelling. Most of the retinal boutons established axodendritic synapses of the asymmetrical type with an immunonegative dendrite, which was also contacted in some cases by a GABA-immunopositive axon terminal. Other retinal endings were presynaptic to GABA-immunopositive dendritic profiles with synaptic vesicles, some of which were found to contact in turn an unlabelled dendrite, thereby completing serial synaptic relationships. More rarely, retinal endings formed the presynaptic component of possible axoaxonic synapses with GABA-positive terminals presumed to be axonic in nature. It can be concluded that the retinal input to the superficial gray layer often converges with a GABAergic axonal input on a dendritic target, the neurotransmitter specificity of which is unknown. In other cases, retinal terminals synaptically contact GABA-immunolabelled conventional and presynaptic dendrites and probably also some axon terminals; this might provide an anatomical substrate for the control of GABA release from these GABAergic processes. These results indicate that transmitter GABA plays an important role in retinocollicular transmission.     

Subicular dendritic arborization in Alzheimer's disease correlates with neurofibrillary tangle density

Intracellular accumulation of PHFtau in Alzheimer's disease (AD) disrupts the neuronal cytoskeleton and other neuronal machinery and contributes to axonal and dendritic degeneration, and neuronal death. Furthermore, amyloid-beta (Abeta) has been reported to be toxic to neurons and neurites. While loss of presynaptic elements is an established feature of AD, the nature and extent of dendritic degeneration has been infrequently studied. We investigated MAP2-immunoreactive dendrites using a novel method of high-throughput quantification and also measured cortical thickness and the densities of NeuN-immunoreactive neurons, PHFtau neurofibrillary tangles (NFTs), and Abeta plaque burden in the subiculum in AD and elderly controls. Corrected for atrophy, the "dendritic arborization index" was significantly reduced by up to 66% in all three layers of the subiculum. Laminar thickness was reduced by an average 33% and there was a marked reduction in neuron density of approximately 50%. As expected, NFTs and Abeta plaques were significantly increased in AD. Dendritic arborization indices negatively correlated with NFT densities while no significant correlations were found with Abeta plaque densities. The pattern of dendritic loss in the subiculum and the correlations with NFT densities respectively suggest that deafferentation and intrinsic neurofibrillary degeneration both may contribute to dendritic loss in AD.     

Cholinergic innervation and thalamic input in rat nucleus accumbens

Cholinergic interneurons are the only known source of acetylcholine in the rat nucleus accumbens (nAcb); yet there is little anatomical data about their mode of innervation and the origin of their excitatory drive. We characterized the cholinergic and thalamic innervations of nAcb with choline acetyltransferase (ChAT) immunocytochemistry and anterograde transport of Phaseolus vulgaris-leucoagglutinin (PHA-L) from the midline/intralaminar/paraventricular thalamic nuclei. The use of a monoclonal ChAT antiserum against whole rat ChAT protein allowed for an optimal visualization of the small dendritic branches and fine varicose axons of cholinergic interneurons. PHA-L-labeled thalamic afferents were heterogeneously distributed throughout the core and shell regions of nAcb, overlapping regionally with cholinergic somata and dendrites. At the ultrastructural level, several hundred single-section profiles of PHA-L and ChAT-labeled axon terminals were analyzed for morphology, synaptic frequency, and the nature of their synaptic targets. The cholinergic profiles were small and apposed to various neuronal elements, but rarely exhibited a synaptic membrane specialization (5% in single ultrathin sections). Stereological extrapolation indicated that less than 15% of these cholinergic varicosities were synaptic. The PHA-L-labeled profiles were comparatively large and often synaptic (37% in single ultrathin sections), making asymmetrical contacts primarily with dendritic spines (>90%). Stereological extrapolation indicated that all PHA-L-labeled terminals were synaptic. In double-labeled material, some PHA-L-labeled terminals were directly apposed to ChAT-labeled somata or dendrites, but synapses were never seen between the two types of elements. These observations demonstrate that the cholinergic innervation of rat nAcb is largely asynaptic. They confirm that the afferents from midline/intralaminar/paraventricular thalamic nuclei to rat nAcb synapse mostly on dendritic spines, presumably of medium spiny neurons, and suggest that the excitatory drive of nAcb cholinergic interneurons from thalamus is indirect, either via substance P release from recurrent collaterals of medium spiny neurons and/or by extrasynaptic diffusion of glutamate.