Post-embedding Immunogold Method Research Articles

Identification of gamma-aminobutyric acid-immunoreactive axon endings associated with mesencephalic periodontal afferent terminals and morphometry of the two types of terminals in the cat supratrigeminal nucleus.

A previous study has shown that mesencephalic periodontal afferent terminals receive contacts more frequently from axonal endings containing pleomorphic, synaptic vesicles (P-endings) in the supratrigeminal nucleus (Vsup) than in the trigeminal motor nucleus, suggesting that interneurons in Vsup play an important role in modulating the jaw-closing reflex. The present study was attempted to identify neurotransmitters in P-endings associated with mesencephalic periodontal afferents in cat Vsup through the use of intracellular staining of horseradish peroxidase combined with the postembedding immunogold methods. A morphometric analysis was carried out to compare the ultrastructural features of these two types of terminals. Serial sections of 31 labeled boutons and of their associated 38 P-endings were examined. They were processed for postembedding immunogold labeling with antibodies to the neurotransmitter gamma-aminobutyric acid (GABA). The 38 P-endings presynaptic to periodontal afferents showed GABA-like immunoreactivity, but the afferent terminals were free from the labeling. The morphometric analysis indicated that bouton volume, apposed surface area, total active zone size, and mitochondrial volume were smaller in GABA-immunoreactive P-endings than in periodontal afferents, but the pooled data of the two types of terminals showed that each synaptic parameter was highly correlated in a positive, linear manner with bouton volume. These observations provide evidence that P-endings presynaptic to mesencephalic periodontal afferents contain the neurotransmitter GABA and that their axoaxonic synapses are organized in accordance with the ultrastructural "size principle" proposed by Pierce and Mendell (Pierce and Mendell [1993] J. Neurosci. 13:4748-4763) on Ia-motoneuron synapses.

Synaptology of the nigrostriatal projection in relation to the compartmental organization of the neostriatum in the rat

The patch-matrix organization of the striatal complex, which is fundamental to the structural and functional organization of the basal ganglia, is characterized on the basis of both connections and neurochemistry. In order to determine whether differences in the connections and neurochemistry are reflected in differences in synaptic organization, we examined the synaptology of the dopaminergic nigrostriatal projection in the patch-matrix complex of the rat. Three approaches were used. First, deposits of the anterograde tracer, biotinylated dextran amine, were placed in the substantia nigra. Sections of perfuse-fixed neostriatum were then processed to reveal anterogradely-labelled nigrostriatal axons and calbindin-D28k immunoreactivity, a marker for the patch-matrix complex. Secondly, sections of perfuse-fixed neostriatum were immunolabelled to reveal both tyrosine hydroxylase, a marker for dopaminergic structures and calbindin-D28k. Labelled axons in the patches and the matrix were examined at both the light and the electron microscopic levels. Finally, in order to test for the presence of fixed GABA in sub-types of anterogradely-labelled terminals in the neostriatum, ultrathin sections were immunolabelled by the post-embedding immunogold method. Based on morphological analysis, anterogradely-labelled nigrostriatal axons were divided into two types (Type I and Type II). The density of tyrosine hydroxylase labelling in the neostriatum prevented the classification of immunolabelled nigrostriatal axons. The Type I anterogradely-labelled axons and tyrosine hydroxylase-positive axons were found both in the patches and in the matrix. They both formed symmetrical synapses with spines, dendrites and occasionally somata. The morphology, dimensions, type of synaptic specialization and the distribution of postsynaptic targets of axons labelled by both methods were similar in the patches and the matrix. The Type I anterogradely-labelled axons were immunonegative for GABA. The Type II anterogradely-labelled axons were GABA-immunopositive, were found only in the matrix and were only present in those animals in which retrograde labelling was observed in the globus pallidus, they are thus not part of the dopaminergic nigrostriatal projection. It is concluded that although the patch-directed and matrix-directed dopaminergic projections from the ventral mesencephalon arise from different populations of dopaminergic neurons, their innervation of neurons in the patches and matrix is similar. The anatomical substrate, and therefore probably also the mechanism, for dopaminergic modulation of the flow of cortical information through the striatal complex is essentially the same in patches and in the matrix sub-divisions of the striatal complex.

Lysozyme expression in the rat parotid gland: light and electron microscopic immunogold studies.

Lysozyme (muramidase) is capable of direct bacteriolytic action by hydrolyzing glycosidic bonds in bacterial cell walls. Although it is broadly distributed in vertebrate tissues and secretions, the cellular and subcellular localizations of the enzyme are still not well known. The present study examines the distribution of lysozyme expression in the various cell types of LR gold-embedded rat parotid gland, applying a postembedding immunogold-silver staining technique for light microscopy. Simultaneously, a postembedding immunogold method for electron microscopy was used to determine the cellular compartments engaged in the biosynthesis and exocytosis of lysozyme. Silver-amplified immunogold staining for lysozyme demonstrated identical localization in both paraffin and semithin LR-gold sections: in the supranuclear parts of acinar and intercalated duct cells. Staining intensity varied even between adjacent cells. In the electron microscope, immunogold labeling was detected over the cell compartments associated with protein synthesis and exocytosis in acinar and intercalated duct cells. Lysozyme antigenic sites were visible over endoplasmic reticulum and throughout the Golgi apparatus, being intense over the trans-Golgi network, but even stronger in the condensing vacuoles and most prominent over secretory granules in both cell types. The findings provide the first immunocytochemical evidence of the synthesis and secretion of lysozyme in parotid acinar and intercalated duct cells.

Ultrastructural localization of the Na-K-Cl cotransporter in the lateral wall of the rabbit cochlear duct

Localization of the immunoreactivity in the lateral wall of the rabbit cochlear duct was examined using a post-embedding immunogold method with a polyclonal antiserum raised against the rabbit parotid Na-K-Cl cotransporter. In the stria vascularis, the labeling was significant on the basolateral membrane infolding of marginal cells, whereas no labeling was seen on the luminal membrane of these cells. Immunoreactivity was also detected on the cell membranes of various other cells. These include fibrocytes of the spiral ligament and the spiral prominence, and vascular endothelial cells in the stria vascularis and the spiral ligament. In contrast, virtually no gold particles were seen on the membrane of intermediate cells, basal cells of the stria vascularis, the epithelial cells of the spiral prominence, or Reissner's membrane. Our result on the localization of the Na-K-Cl cotransporter in marginal cells is consistent with electrophysiological studies (Wangemann et al. (1995) Hear. Res. 84, 19–29). Our result on fibrocytes is discussed in relation to K+ circulation into endolymph from perilymph (Schulte and Steel (1994) Hear. Res. 78, 65–76). © Elsevier Science B.V. All rights reserved.

Ultrastructural subtypes of glutamate-immunoreactive terminals on rat trigeminal motoneurones and their relationships with GABA-immunoreactive terminals.

Electron-microscopic immunolabelling methods were used to study the relationships between glutamate-immunoreactive and gamma-aminobutyric acid (GABA)-immunoreactive synapses on trigeminal motoneurones labelled by the retrograde transport of horseradish peroxidase. Serial sections were cut through the motor nucleus, alternate sections were incubated with antibodies to glutamate and GABA, and the immunopositive nerve terminal profiles were recognized using a quantitative, post-embedding immunogold method. Boutons exhibiting high levels of glutamate immunoreactivity and GABA-immunoreactive boutons both formed axo-dendritic and axo-somatic synaptic contacts on labelled motoneurones. Boutons strongly immunopositive for glutamate were not immunopositive for GABA, and vice versa. Strongly glutamate immunoreactive boutons received axo-axonic synaptic contacts but did not form such contacts, while GABA-immunoreactive boutons formed axo-axonic synapses but did not receive them. The presynaptic elements at all axo-axonic synapses on to glutamate-immunoreactive boutons sampled were GABA-immunopositive. These data provide ultrastructural evidence in support of the roles of glutamate and GABA as transmitters at synapses on trigeminal motoneurones, and for presynaptic control of transmission at glutamatergic synapses by GABA acting at receptors at axo-axonic synapses. The vast majority (more than 90%) of strongly glutamate immunoreactive boutons contained spherical synaptic vesicles, in contrast to GABA-immunoreactive boutons, which contained pleomorphic vesicles. Most of the glutamate-immunoreactive boutons (67%) formed asymmetrical synaptic active zones, many of which (47% of total) were associated with subsynaptic dense "Taxi" bodies (T-terminals), while a smaller population of boutons (21%) formed symmetrical synapses, and a few (11%) made synapses associated with subsynaptic cisternae (C-terminals). The heterogeneity of active zone ultrastructure of boutons identified as being glutamatergic on the basis of their high levels of immunolabelling is discussed in relation to possible differences in co-transmitters released, origins of the synaptic input or post-synaptic receptor subtypes activated.

Cellular, Subcellular, and Subsynaptic Distribution of AMPA-Type Glutamate Receptor Subunits in the Neostriatum of the Rat

Glutamate released in the basal ganglia is involved in the expression of clinical symptoms of neurodegenerative diseases like Parkinson's or Huntington's. Neostriatal neurons are the targets of glutamatergic inputs derived from the cortex and the thalamus acting via AMPA-type as well as other glutamate receptors. To determine the location of subunits of the AMPA subclass of glutamate receptors (GluR) in the rat neostriatum, we applied multiple immunocytochemical techniques using anti-peptide antibodies against the GluR1, GluR2/3, and GluR4 subunits at both the light and electron microscopic levels. All medium spiny efferent neurons, some of which were identified as striatonigral neurons, displayed immunoreactivity for GluR1 and GluR2/3 subunits. Double immunofluorescence revealed that at least 70-90% of parvalbumin-immunopositive GABAergic interneurons were immunoreactive for each of GluR1, GluR2/3, or GluR4 subunits and that at least 40% of choline acetyltransferase-immunopositive cholinergic interneurons were immunopositive for GluR1 or GluR4 subunits. The majority of nitric oxide synthase-immunopositive neurons had no detectable immunoreactivity for any of the AMPA receptor subunits. Electron microscopic analysis confirmed the presence of immunoreactivity for GluR1 and GluR2/3 in the perikarya of spiny neurons and interneurons and GluR4 in perikarya of interneurons only. GluR1 and GluR2/3 subunits were detected in dendrites and spines. A significant population of extrasynaptic receptors was revealed by pre-embedding immunogold labeling along the plasma membranes of perikarya, dendrites, and spines. Receptors were concentrated in the postsynaptic membrane specialization of asymmetrical synapses, as revealed by the postembedding immunogold method. Quantitative analysis demonstrated that immunoreactivity for the GluR1 and GluR2/3 subunits is higher at the periphery than at the middle of the postsynaptic membrane specialization. Our results demonstrate that AMPA receptor subunits are distributed widely and heterogeneously among striatal neurons and are concentrated on the postsynaptic membrane of asymmetrical synaptic specializations, although extrasynaptic receptors are also present.

Ultrastructural localization of osteopontin immunoreactivity in phagolysosomes and secretory granules of cells in human intestine.

A post-embedding ultrastructural immunogold method was used to detect osteopontin in human intestinal biopsies with special emphasis on secretory and phagocytic organelles. Osteopontin immunoreactivity was localized to phagolysosomes of macrophages, fibroblasts, absorptive epithelial cells of the small intestine and Paneth cells. The mucigen secretory granules and Golgi structures of mucous epithelial cells of the small intestinal epithelium contained osteopontin, but secretory granules of numerous other cells, including Paneth cells, did not. Extracellular and phagocytosed Tropheryma whippelii within macrophage phagolysosomes also bound osteopontin. These localizations are supportive of a role for osteopontin in phagocytic and some secretory cell functions in human intestine.

Perisynaptic location of metabotropic glutamate receptors mGluR1 and mGluR5 on dendrites and dendritic spines in the rat hippocampus.

Ionotropic and metabotropic (mGluR1a) glutamate receptors were reported to be segregated from each other within the postsynaptic membrane at individual synapses. In order to establish whether this pattern of distribution applies to the hippocampal principal cells and to other postsynaptic metabotropic glutamate receptors, the mGluR1a/b/c and mGluR4 subtypes were localized by immunocytochemistry. Principal cells in all hippocampal fields were reactive for mGluR5, the strata oriens and radiatum of the CA1 area being most strongly immunolabelled. Labelling for mGluR1b/c was strongest on some pyramids in the CA3 area, weaker on granule cells and absent on CA1 pyramids. Subpopulations of non-principal cells showed strong mGluR1 or mGluR5 immunoreactivity. Electron microscopic pre-embedding immunoperoxidase and both pre- and postembedding immunogold methods consistently revealed the extrasynaptic location of both mGluRs in the somatic and dendritic membrane of pyramidal and granule cells. The density of immunolabelling was highest on dendritic spines. At synapses, immunoparticles for both mGluR1 and mGluR5 were found always outside the postsynaptic membrane specializations. Receptors were particularly concentrated in a perisynaptic annulus around type 1 synaptic junctions, including the invaginations at 'perforated' synapses. Measurements of immunolabelling on dendritic spines showed decreasing levels of receptor as a function of distance from the edge of the synaptic specialization. We propose that glutamergic synapses with an irregular edge develop in order to increase the circumference of synaptic junctions leading to an increase in the metabotropic to ionotropic glutamate receptor ratio at glutamate release sites. The perisynaptic position of postsynaptic metabotropic glutamate receptors appears to be a general feature of glutamatergic synaptic organization and may apply to other G-protein-coupled receptors.

Structural continuity of filtration slit (slit diaphragm) to plasma membrane of podocyte

Murine monoclonal antibody 5-1-6 was reported to bind to the slit membrane and closely related structures in rat renal glomeruli; it induced heavy, reversible proteinuria and appeared to redistribute onto the plasma membrane of epithelial cells after binding at the original target sites. This phenomenon of antigenic movement has not been analyzed in detail to date. In addition to normal kidneys we also studied localization of the antigen recognized by monoclonal antibody 5-1-6 in protamine sulfate-perfused rat kidneys, in which slit diaphragms are known to be functionally modified. Isolated glomeruli as well as ultrathin kidney cryosections were labeled by the immunogold technique to clarify the relation between this antigen and the slit diaphragm. Sequential localization of injected monoclonal antibody was visualized using a post-embedding immunogold method in rats 2 hours to 12 days after injection of antibody. Ultrastructural immunogold labeling demonstrated that under normal conditions antigenic molecules were expressed mainly in the area beneath the slit diaphragms. Occasionally labeling was found at the base of the foot process, facing the glomerular basement membrane. After protamine sulfate treatment antigenic sites were dislocated due to the lifting and disruption of slit diaphragms, indicating that this antigen is associated with slit diaphragms. Injected antibody was localized at the filtration slits at 2 hours, and by 12 hours it had moved onto the apical plasma cell membrane of foot process. In addition, from 3 days onwards patch or cap-like formation on the plasma cell membrane of podocytes was seen. Possible shedding of antibody from podocyte cell surface membrane was occasionally encountered, but internalization of antibody was a minor event. Elution experiments in isolated glomeruli at day 3 indicated that antigen and antibody were both localized on the podocyte cell surface membrane, suggesting redistribution of immune complexes. In conclusion, filtration slits (slit diaphragms) and the apical membrane of foot process of podocytes demonstrate structural continuity, as revealed by the movement of the antigen recognized by monoclonal antibody 5-1-6 as antigen-antibody complexes.

Ultrastructural immunogold localization of vimentin and S-100 protein in guinea pig pars tuberalis.

Ultrastructural features of vimentin- and S-100 protein-positive cells in guinea pig pars tuberalis were examined by immunoelectron microscopy with the postembedding immunogold method. Interstitial cells that exhibited elongated cytoplasm and partly surrounded the specific secretory cells were filled with intermediate filament bundles on which immunogold particles for vimentin were densely located. Small colloid-containing follicles were occasionally distributed in the cranial region surrounding the median eminence. The follicular cells lining the luminal cavities contained considerable amounts of intermediate filaments immunoreactive for vimentin. Some specific secretory cells displayed various amounts of intermediate filaments immunoreactive for vimentin, which were scattered throughout the cytoplasm or concentrated around the nucleus. The specific secretory cells in which many parallel profiles of rough endoplasmic reticulum (RER) or a large number of mitochondria were dispersed throughout cytoplasm, i.e., being highly metabolically active, were devoid of intermediate filaments. The ventrocaudal region in continuity with the pars distalis was occupied by cells that were packed with vesicular inclusions, considered to be dilated cisternae of RER, and frequently formed colloid-containing follicles. Immunoreactivity for S-100 protein was located exclusively in this novel cell type. Immunogold particles for S-100 protein were distributed in the cytoplasmic matrix and were also associated with the membrane of vesicular inclusions. Gold particles were also detected on the tight junctions, desmosomes, and fibril materials at the apical cell regions of the colloid-containing follicles. The finding of two separate populations of cells containing vimentin and S-100 protein, respectively, supports the idea of functional separation in the pars tuberalis.

Ultrastructural localization of G-protein G S in the lateral wall of the guinea pig cochlear duct

Immunocytochemical localization of a GTP-binding protein G S, in the various cells of the lateral wall of guinea pig cochlear duct was investigated using a post-embedding immunogold method with antibody raised against a synthetic decapeptide (RMHLRQYELL) encoding the C-terminus of the α-subunit of G S. In the stria vascularis, labeling was observed on the basolateral membrane infoldings of marginal cells, on the juxtaposed membrane of intermediate cells, and on the cell membrane of basal cell. In contrast, no significant labeling was observed on the luminal membrane of marginal cells. Immunoreactivity also was detected on the cell membranes of various other cells. These include spiral prominence epithelial cells, fibrocytes of spiral ligament, external sulcus cells, and epithelial and mesothelial cells of Reissner's membrane. Adenylylcyclase has been functionally implicated in some of the cell types with membranes labeled in this study. The significance of these findings is briefly discussed.

Morphology and soma‐dendritic distribution of synaptic endings from the rostral interstitial nucleus of the medial longitudinal fasciculus (riMLF) on motoneurons in the oculomotor and trochlear nuclei in the cat

The morphology and soma-dendritic distribution of anterograde biocytin-labelled rostral interstitial nucleus of the medial longitudinal fasciculus (riMLF) synaptic endings in the oculomotor and trochlear nuclei have been examined by electron microscopy by using both preembedding immunoperoxidase and postembedding immunogold methods. The results indicate that three morphological types of riMLF synaptic endings are distinguishable on the basis of synaptic vesicle morphology (spheroidal, pleiomorphic, or ellipsoidal) and postsynaptic membrane specializations (asymmetrical or symmetrical). All three morphological types of riMLF synaptic endings establish synaptic connections predominantly with dendrites. Synaptic endings that contain ellipsoidal synaptic vesicles have a more proximal soma-dendritic distribution than those that contain either spheroidal or pleiomorphic synaptic vesicles. Furthermore, all three morphological types of synaptic endings are encountered in the same motoneuron subdivisions of the oculomotor and trochlear nuclei in the same experiments. The findings suggest that subregions of the riMLF contain coexistent populations of excitatory and inhibitory premotor neurons that are related to opposite directions of vertical saccadic eye movements but that project to the same motoneuron subgroups on the ipsilateral side. Both the morphology and the mode, pattern, and soma-dendritic distribution of saccade-related riMLF synaptic endings that establish synaptic connections with vertical motoneurons differ from those of excitatory and inhibitory second-order vertical vestibular synaptic endings. These differences in the synaptic organization of riMLF and second-order vestibular inputs to oculomotor and trochlear motoneurons may be related to differences in the information transferred by each source, the riMLF input conveying eye-velocity signals, and the vestibular input conveying eye-position signals. © 1996 Wiley-Liss, Inc.

Morphology and soma-dendritic distribution of synaptic endings from the rostral interstitial nucleus of the medial longitudinal fasciculus (riMLF) on motoneurons in the oculomotor and trochlear nuclei in the cat.

The morphology and soma-dendritic distribution of anterograde biocytin-labelled rostral interstitial nucleus of the medial longitudinal fasciculus (riMLF) synaptic endings in the oculomotor and trochlear nuclei have been examined by electron microscopy by using both preembedding immunoperoxidase and postembedding immunogold methods. The results indicate that three morphological types of riMLF synaptic endings are distinguishable on the basis of synaptic vesicle morphology (spheroidal, pleiomorphic, or ellipsoidal) and postsynaptic membrane specializations (asymmetrical or symmetrical). All three morphological types of riMLF synaptic endings establish synaptic connections predominantly with dendrites. Synaptic endings that contain ellipsoidal synaptic vesicles have a more proximal soma-dendritic distribution than those that contain either spheroidal or pleiomorphic synaptic vesicles. Furthermore, all three morphological types of synaptic endings are encountered in the same motoneuron subdivisions of the oculomotor and trochlear nuclei in the same experiments. The findings suggest that subregions of the riMLF contain coexistent populations of excitatory and inhibitory premotor neurons that are related to opposite directions of vertical saccadic eye movements but that project to the same motoneuron subgroups on the ipsilateral side. Both the morphology and the mode, pattern, and soma-dendritic distribution of saccade-related riMLF synaptic endings that establish synaptic connections with vertical motoneurons differ from those of excitatory and inhibitory second-order vertical vestibular synaptic endings. These differences in the synaptic organization of riMLF and second-order vestibular inputs to oculomotor and trochlear motoneurons may be related to differences in the information transferred by each source, the riMLF input conveying eye-velocity signals, and the vestibular input conveying eye-position signals.

High-resolution immunogold localization of AMPA type glutamate receptor subunits at synaptic and non-synaptic sites in rat hippocampus.

The cellular and subcellular localization of the GluRA, GluRB/C and GluRD subunits of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) type glutamate receptor was determined in the rat hippocampus using polyclonal antipeptide antibodies in immunoperoxidase and immunogold procedures. For the localization of the GluRD subunit a new polyclonal antiserum was developed using the C-terminal sequence of the protein (residues 869-881), conjugated to carrier protein and absorbed to colloidal gold for immunization. The purified antibodies immunoprecipitated about 25% of 3[H]AMPA binding activity from the hippocampus, cerebellum or whole brain, but very little from neocortex. These antibodies did not precipitate a significant amount of 3[H]kainate binding activity. The antibodies also recognize the GluRD subunit, but not the other AMPA receptor subunits, when expressed in transfected COS-7 cells and only when permeabilized with detergent, indicating an intracellular epitope. All subunits were enriched in the neuropil of the dendritic layers of the hippocampus and in the molecular layer of the dentate gyrus. The cellular distribution of the GluRD subunit was studied more extensively. The strata radiatum, oriens and the dentate molecular layer were more strongly immunoreactive than the stratum lacunosum moleculare, the stratum lucidum and the hilus. However, in the stratum lucidum of the CA3 area and in the hilus the weakly reacting dendrites were surrounded by immunopositive rosettes, shown in subsequent electron microscopic studies to correspond to complex dendritic spines. In the stratum radiatum, the weakly reacting apical dendrites contrasted with the surrounding intensely stained neuropil. The cell bodies of pyramidal and granule cells were moderately reactive. Some non-principal cells and their dendrites in the pyramidal cell layer and in the alveus also reacted very strongly for the GluRD subunit. At the subcellular level, silver intensified immunogold particles for the GluRA, GluRB/C and GluRD subunits were present at type 1 synaptic membrane specializations on dendritic spines of pyramidal cells throughout all layers of the CA1 and CA3 areas. The most densely labelled synapses tended to be on the largest spines and many smaller spines remained unlabelled. Immunoparticle density at type 1 synapses on dendritic shafts of some non-principal cells was consistently higher than at labelled synapses of dendritic spines of pyramidal cells. Synapses established between dendritic spines and mossy fibre terminals, were immunoreactive for all studied subunits in stratum lucidum of the CA3 area. The postembedding immunogold method revealed that the AMPA type receptors are concentrated within the main body of the anatomically defined type 1 (asymmetrical) synaptic junction. Often only a part of the membrane specialization showed clustered immunoparticles. There was a sharp decrease in immunoreactive receptor density at the edge of the synaptic specialization. Immunolabelling was consistently demonstrated at extrasynaptic sites on dendrites, dendritic spines and somata. The results demonstrate that the GluRA, B/C and D subunits of the AMPA type glutamate receptor are present in many of the glutamatergic synapses formed by the entorhinal, CA3 pyramidal and mossy fibre terminals. Some interneurons have a higher density of AMPA type receptors in their asymmetrical afferent synapses than pyramidal cells. This may contribute to a lower activation threshold of interneurons as compared to principal cells by the same afferents in the hippocampal formation.

Changes in the pattern of glutamate-like immunoreactivity in rat superior colliculus following retinal and visual cortical lesions.

We have investigated the pattern of glutamate-like immunoreactivity in the superficial layers of the rat superior colliculus by means of postembedding immunocytochemical methods for light and electron microscopy. At the light microscopic level, labelling was faintly to moderately intense in most perikarya of the stratum zonale, stratum griseum superficiale and stratum opticum. Furthermore, strong glutamate-immunoreactive terminal-like elements were accumulated most densely in stratum zonale, stratum griseum superficiale and stratum opticum. At the electron microscopic level, a postembedding immunogold method revealed that the vast majority of those labelled elements corresponded to retinal and visual cortical terminals. These profiles were about twice as heavily labelled as their postsynaptic partners. To determine the contribution of retinal and cortical afferents to the pattern of glutamate-like immunoreactivity, rats were subjected to right retinal ablation, left cortical ablation or combined right retinal and left cortical ablations. After retinal ablation, strongly labelled perikarya were observed in the retinorecipient layers. Furthermore, a prominent loss of glutamate-immunoreactive terminal-like elements occurred in stratum zonale and stratum griseum superficiale. Ipsilateral superior colliculus to cortical ablation exhibited subtle changes characterized by a moderate increase in perikaryal immunostaining in stratum zonale, stratum griseum superficiale and stratum opticum and by an apparent discrete reduction of labelled dots in stratum griseum superficiale and stratum opticum. In cases with combined lesions, strongly immunoreactive cell bodies and dendrites were accompanied by a massive disappearance of labelled terminal-like elements in stratum zonale, stratum griseum superficiale and stratum opticum. The effect of retinal and visual cortical ablations on the pattern of glutamate-like immunoreactivity suggests that these afferents are the major sources for glutamate-immunoreactive terminals in the rat superior colliculus. In addition, these findings provide further evidence for glutamate as neurotransmitter in the visual pathways studied.

Immunocytochemical Localization of the α1 and β2/3 Subunits of the GABAA Receptor in Relation to Specific GABAergic Synapses in the Dentate Gyrus

Dentate granule cells receive spatially segregated GABAergic innervation from at least five types of local circuit neurons, and express mRNA for at least 11 subunits of the GABAA receptor. At most two to four different subunits are required to make a functional pentamer, raising the possibility that cells have on their surface several types of GABAA receptor channel, which may not be uniformly distributed. In order to establish the subcellular location of GABAA receptors on different parts of dentate neurons, the distribution of immunoreactivity for the alpha 1 and beta 2/3 subunits of the receptor was studied using high-resolution immunocytochemistry. Light microscopic immunoperoxidase reactions revealed strong GABAA receptor immunoreactivity in the molecular layer of the dentate gyrus. Pre-embedding immunogold localization of the alpha 1 and beta 2/3 subunits consistently showed extrasynaptic location of the GABAA receptor on the somatic, dendritic and axon initial segment membrane of granule cells, but failed to show receptors in synaptic junctions. Using a postembedding immunogold technique on freeze-substituted, Lowicryl-embedded tissue, synaptic enrichment of immunoreactivity for these subunits was found on both granule and non-principal cells. Only the postembedding immunogold method is suitable for revealing relative differences in receptor density at the subcellular level, giving approximately 20 nm resolution. The immunolabelling for GABAA receptor occupied the whole width of synaptic junctions, with a sharp decrease in labelling at the edge of the synaptic membrane specialization. Both subunits have been localized in the synaptic junctions between basket cell terminals and somata, and between axo-axonic cell terminals and axon initial segments of granule cells, with no qualitative difference in labelling. Receptor-immunopositive synapses were found at all depths of the molecular layer. Some of the boutons forming these dendritic synapses have been shown to contain GABA, providing evidence that some of the GABAergic cells that terminate only on the dendrites of granule cells also act through GABAA receptors. Double immunolabelling experiments demonstrated that a population of GABA-immunopositive neurons expresses a higher density of immunoreactive GABAA receptor on their surface than principal cells. Interneurons were found to receive GABAA receptor-positive synapses on their dendrites in the hilus, molecular and granule cell layers. Receptor-immunopositive synapses were also present throughout the hilus on presumed mossy cells. The results demonstrate that both granule cells and interneurons exhibit a compartmentalized distribution of the GABAA receptor on their surface, the postjunctional membrane to GABAergic terminals having the highest concentration of receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Relative densities of synaptic and extrasynaptic GABAA receptors on cerebellar granule cells as determined by a quantitative immunogold method

Ion channels gated by the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) are thought to be located in synaptic junctions, but they have also been found throughout the somatodendritic membrane of neurons independent of synapses. To test whether synaptic junctions are enriched in GABAA receptors, and to determine the relative densities of synaptic and extrasynaptic receptors, the alpha 1 and beta 2/3 subunits of the GABAA receptor were localized on cerebellar granule cells using a postembedding immunogold method in cats. Immunoparticle density for the alpha 1 and beta 2/3 subunits was approximately 230 and 180 times more concentrated, respectively, in the synaptic junction made by GABAergic Golgi cell terminals with granule cell dendrites than on the extrasynaptic somatic membrane. Quantification of immunoreactivity revealed one synapse population for the beta 2/3, but appeared to show two populations for the alpha 1 subunit immunoreactivity. The concentration of these subunits on somatic membrane was significantly lower than on the extrasynaptic dendritic membrane. Synaptic junctions with glutamatergic mossy fiber terminals were immunonegative. The results demonstrate that granule cells receiving GABAergic synapses at a restricted location on their distal dendrites exhibit a highly compartmentalized distribution of GABAA receptor in their plasma membrane.

Quantitative ultrastructural localization of glutamate dehydrogenase in the rat cerebellar cortex.

Glutamate dehydrogenase is one of the main enzymes involved in the formation and metabolism of the neurotransmitter glutamate. In the present study we investigated the enzyme ultrastructurally in the cerebellar cortex, a region rich in well defined glutamatergic neurons, by pre-embedding immunocytochemical staining (peroxidase-antiperoxidase), as well as by post-embedding immunogold labelling employing a new system for quantitation and for specificity testing under the conditions of the immunocytochemical procedure. A new antiserum against immunologically purified bovine liver glutamate dehydrogenase or antibodies isolated from this by affinity chromatography were used in rats fixed by perfusion with aldehydes. The pre-embedding method displayed peroxidase reaction preferentially in mitochondria of astroglial cells (including the Bergmann glia). Mitochondria of neuronal tissue elements were usually free of peroxidase-reaction product. Extra-mitochondrial staining was not observed. The post-embedding immunogold method was employed to overcome penetration problems and allow semiquantitative analysis of localization and specificity. The highest densities of gold particles were found over the mitochondria in astroglial cell elements (including the Bergmann glia). Mitochondria in cell bodies of Bergmann glia had a lower particle density than those in astrocytic processes. In the latter, analysis of frequency distribution revealed no evidence of a population of mitochondria lacking glutamate dehydrogenase, but suggested the presence of populations with different levels of immunoreactivity. Comparison with the labelling of embedded bovine liver glutamate dehydrogenase indicated that the enzyme constitutes a high proportion (10%) of the total matrix protein of these mitochondria. A weaker but significant labelling was found in oligodendrocytes of the white matter. The labelling of mitochondria in neuronal elements including glutamatergic mossy fibre terminals was of the order of 15% of that in astroglial mitochondria. No difference was detected between glutamatergic neurons (mossy and parallel fibres, granular cells) and non-glutamatergic neurons (Purkinje cells). The particle density over non-mitochondrial areas was very close to background over empty resin. The results, obtained with different methods of tissue and antibody preparation, agree to show that the present form of glutamate dehydrogenase is restricted to mitochondria and preferentially localized in astrocytes.

Peripheral connections of FMRFamide-like immunoreactive neurons in the snail, helix pomatia: an immunogold electron microscopic study.

Using a postembedding immunogold electron microscopic method, the ultrastructure and synaptic connections of FMRFamide-like immunoreactive varicosities were investigated in different peripheral organs of the snail Helix pomatia, including the heart (auricle), intestine, hepatopancreas, upper tentacle and salivary gland. The FMRFamide-like immunoreactive varicosities contained granules and vesicles as described in a previous study of the CNS of this species, and additionally, based on their granule content, two novel types of varicosities were found in the auricle. A selective accumulation of gold particles over the granules could be demonstrated. The FMRFamide-like immunoreactive varicosities formed unspecialized contacts with postsynaptic target cells in all peripheral organs investigated, with the exception of the tentacle retractor muscle. Both the neuro-muscular and the neuro-glandular contacts were characterized by either unspecialized close apposition of the 'pre- and postsynaptic' membranes or the immunoreactive elements faced the target cell(s) across a relatively wide extracellular space. In the tentacle retractor muscle some of the neuromuscular contacts showed appositions of electron dense material along the presynaptic membrane, clustering of agranular synaptic vesicles and intersynaptic cleft material. The present observations support previous electrophysiological findings and suggest a versatile modulatory role of FMRFamide and related substances in the Helix PNS.

Ultrastructural evidence for innervation of the endothelium and interstitial cell in the atrioventricular valves of the Japanese monkey

A rich supply of nerves to the atrioventricular valve has been demonstrated. The role of the valvular nerves is still controversial because the target sites of the nerves have not been confirmed. The innervation of the atrioventricular valves of the Japanese monkey (Macaca fuscata) was examined by acetylcholinesterase staining and electron microscopy. Immunoreactivity for neuropeptide Y (NPY) was also investigated by a post-embedding immunogold method. The valvular nerve elements were clearly concentrated between the endothelium and interstitial cells on the atrial side of cusps. Naked axon terminals were observed to make direct contact (20-nm gaps) with interstitial cells and also to be in close proximity (approximately 200-nm cleft) to the endothelium. NPY immunoreactivity was clearly detected on the large granular vesicles in some terminals that were in close proximity to interstitial cells and/or the endothelium. The present study suggests that the extensive innervation of the atrioventricular valve, which includes NPY-containing nerves, might affect valvular function via interstitial cells and/or the endothelium.