Postembedding Staining of Brain Tissue for Ultrastructural Visualization of Amyloid in Alzheimer's Disease

A postembedding staining method is presented for ultrastructural visualization of amyloid deposits in brain sections from patients with Alzheimer's disease. Methenamine silver stain is applied to thin sections of tissue embedded in the acrylic resin LR Gold. Senile plaques are easily labeled by silver granules and the ultrastructural detail is well preserved. When staining time is prolonged, silver precipitate also is deposited on neuronal paired helical filaments. This method overcomes the drawbacks of previously reported applications of the stain on Vibra-tome and Epon sections. Thin sections from the same tissue block can be immunostained with antibodies to various plaque components, thus allowing comparative studies at the electron microscope level.     

Evidence for local corticotropin releasing factor (CRF)-immunoreactive neuronal circuits in the paraventricular nucleus of the rat hypothalamus. An electron microscopic immunohistochemical analysis

The interrelationships of corticotropin-releasing factor (CRF) immunoreactive neuronal cell bodies and processes have been examined in the paraventricular nucleus (PVN) of adrenalectomized-dexamethasone treated rats. Antisera generated against ovine CRF (oCRF) were used in the peroxidase-anti-peroxidase-complex (PAP)-immunocytochemical method at both the light and electron microscopic levels. In this experimental model, a great number of CRF-immunoreactive neurons were detected in the parvocellular subdivisions of the PVN and a few scattered labelled parvocellular neurons were also observed within the magnocellular subunits. Characteristic features of immunolabeled perikarya included hypertrophied rough endoplasmic reticulum with dilated endoplasmic cisternae, well developed Golgi complexes and increased numbers of neurosecretory granules. These features are interpreted to indicate accelerated hormone synthesis as a result of adrenalectomy. Afferent fibers communicated with dendrites and somata of CRF-immunoreactive neurons via both symmetrical and asymmetrical synapses. Some neurons exhibited somatic appendages and these structures were also observed to receive synaptic terminals. Within both the PVN and its adjacent neuropil, CRF-immunoreactive axons demonstrated varicosites which contained accumulations of densecore vesicles. CRF-containing axons were observed to branch into axon collaterals. These axons or axon collaterals established axo-somatic synapses on CRF-producing neurons in the parvocellular regions of the PVN, while in the magnocellular area of the nucleus they were found in juxtaposition with unlabeled magnocellular neuronal cell bodies or in synaptic contact with their dendrites. The presence of CRF-immunoreactive material in presynaptic structures suggests that the neurohormone may participate in mechanisms of synaptic transfer. These ultrastructural data indicate that the function of the paraventricular CRF-synthesizing neurons is adrenal steroid hormone dependent. They also provide morphological evidence for the existence of a neuronal ultrashort feed-back mechanism within the PVN for the regulation of CRF production and possibly that of other peptide hormones contained within this complex.     

Evidence for local corticotropin releasing factor (CRF)-immunoreactive neuronal circuits in the paraventricular nucleus of the rat hypothalamus

Summary The interrelationships of corticotropin-releasing factor (CRF) immunoreactive neuronal cell bodies and processes have been examined in the paraventricular nucleus (PVN) of adrenalectomized-dexamethesone treated rats. Antisera generated against ovine CRF (oCRF) were used in the peroxidase-anti-peroxidase-complex (PAP)-immunocytochemical method at both the light and electron microscopic levels. In this experimental model, a great number of CRF-immunoreactive neurons were detected in the parvocellular subdivisions of the PVN and a few scattered labelled parvocellular neurons were also observed within the magnocellular subunits. Characteristic features of immunolabeled perikarya included hypertrophied rough endoplasmic reticulum with dilated endoplasmic cisternae, well developed Golgi complexes and increased numbers of neurosecretory granules. These features are interpreted to indicate accelerated hormone synthesis as a result of adrenalectomy. Afferent fibers communicated with dendrites and somata of CRF-immunoreactive neurons via both symmetrical and asymmetrical synapses. Some neurons exhibited somatic appendages and these structures were also observed to receive synaptic terminals. Within both the PVN and its adjacent neuropil, CRF-immunoreactive axons demonstrated varicosites which contained accumulations of densecore vesicles. CRF-containing axons were observed to branch into axon collaterals. These axons or axon collaterals established axo-somatic synapses on CRF-producing neurons in the parvocellular regions of the PVN, while in the magnocellular area of the nucleus they were found in juxtaposition with unlabeled magnocellular neuronal cell bodies or in synaptic contact with their dendrites. The presence of CRF-immunoreactive material in presynaptic structures suggests that the neurohormone may participate in mechanisms of synaptic transfer.These ultrastructural data indicate that the function of the paraventricular CRF-synthesizing neurons is adrenal steroid hormone dependent. They also provide morphological evidence for the existence of a neuronal ultrashort feedback mechanism within the PVN for the regulation of CRF production and possibly that of other peptide hormones contained within this complex.Supported by NIH grant NS 19266 to WKP     

1-naphthol-pyronin B as a novel substrate for silver intensification: application to light and electron microscopic immunocytochemistry of neuroendocrine systems

We describe a modification of silver intensification of immunoperoxidase end-product using 1-naphthol (1N) and 1N enhanced by pyronin B after suppressing nonspecific tissue argyrophilia with a solution of penicillamine and merthiolate buffered near neutral pH. This approach facilitates the preservation of a second antigen sequentially labeled in the same tissue section for light microscopic double immunolabeling experiments and also allows retention of ultrastructural detail. Using this protocol, we obtained rapid and uniform silver intensification of somatostatin (SRIF)-immunoreactive (IR) neuronal perikarya and processes in the rat hypothalamic paraventricular nucleus (PVN). Ultrastructurally, 1N- and 1N-pyronin B-silver intensified reaction product was clearly recognized by the presence of a coarse intracellular precipitate of high electron density. Light microscopic double-immunolabeling studies demonstrated the association between SRIF- and thyrotropin-releasing hormone (TRH)-IR neuronal systems in the PVN. We propose that silver intensification of 1N and 1N-pyronin B is a useful alternative to standard methods of silver intensification of immunoperoxidase reaction product at both light and ultrastructural levels and may be particularly amenable for double-immunolabeling studies.     

Tachykinin-related neuropeptide in the crayfish olfactory midbrain

Immunoreactivity indicative of tachykinin-related peptide (TRP) was detected in the olfactory midbrain of the crayfish Pacifastacus leniusculus when using an antiserum to the insect neuropeptide locustatachykinin I (LomTK-I). A monoclonal antibody to the mammalian tachykinin substance P was shown in double-labeling experiments to label structures in the crayfish brain identical to those labeled with the LomTK antiserum. Within the midbrain LomTK-like immunoreactive (LomTK-LI) material was observed in a limited population of neuronal somata and their varicose processes. A single pair of large interneurons gave rise to numerous varicose LomTK-LI processes innervating a cluster of cell bodies (cluster 10) as well as the olfactory neuropils. The latter neuropil was also innervated by a population of LomTK-LI globuli cells with cell bodies in cluster 9. Radioimmunoassay (RIA), utilizing the LomTK antiserum, and reverse-phase high-performance liquid chromatography (HPLC) were used to partially characterize the immunoreactive material in extract of the portion of the midbrain that houses the olfactory (OL) and accessory (AL) lobes and cell clusters 9 and 10 on the one hand, and in extract of the remaining parts of the brain on the other. Approximately the same amounts of LomTK-LI material were observed for the two extracts. RIA showed that the immunoreactive material of both extracts diluted roughly in parallel to synthetic LomTK-I and HPLC analysis of the extracts revealed immunoreactive material in both tissues which eluted with retention times in the range of synthetic LomTK-I and LomTK-II. These results suggest that TRPs similar to LomTKs are present in the olfactory midbrain of Pacifastacus. The distribution of immunolabeled neuronal structures suggests that in the crayfish, peptide(s) closely related to insect TRPs may act as a neuroactive substance released from nerve fibers in olfactory neuropil areas and at certain neuronal cell bodies.     

Localization of immunoreactive tyrosine hydroxylase in the goldfish retina with pre-embedding immunolabeling with one-nanometer colloidal gold particles and gold toning.

The goal of this study was to develop an alternative to silver intensification for visualizing small colloidal gold particles by light and electron microscopy. The isolated goldfish retina was labeled with rabbit antiserum to tyrosine hydroxylase and 1-nm colloidal gold-conjugated goat anti-rabbit IgG. The gold particles were enlarged by toning with gold chloride, followed by reduction in oxalic acid. Dopaminergic interplexiform cells were clearly visible by light microscopy and, in lightly-fixed material treated with detergent, they were labeled in their entirety. Labeling was qualitatively similar, although less extensive, in material fixed and processed for electron microscopy. The labeled processes were apparent in ultra-thin sections viewed at low magnification, but the gold-toned particles were not so large that they obscured subcellular structures. The procedure apparently had no deleterious effects on the tissue, since the ultrastructural preservation was comparable to that seen with other pre-embedding immunolabeling methods. The technique was simple, reliable and, since the gold solutions were so dilute, relatively inexpensive.     

A combined light and electron microscopic immunocytochemical method for the simultaneous localization of multiple tissue antigens. Tyrosine hydroxylase immunoreactive innervation of corticotropin releasing factor synthesizing neurons in the paraventricular nucleus of the rat

In order to study the morphological interrelationships between immunocytochemically identified neuronal systems, a double labelling procedure - suitable for correlative light and electron microscopic observations - is introduced. The technique is based on the consecutive use of the silver-gold (SG) intensified and non-intensified forms of the oxidized 3,3'-diaminobenzidine (DAB) chromogen in the framework of the peroxidase-antiperoxidase complex (PAP) indirect immunocytochemical procedure. The first tissue antigen is detected by the SG intensified DAB chromogen, which has a black color and high electron density. The structures containing the second antigen are visualized by the non-intensified DAB-endproduct, which is less electron-dense than the silver-gold amplified form and is brown. The metallic shield that forms around the labeled antibody sequences associated with the first antigen prevents non-specific binding of immunoglobulins used for the detection of the second tissue antigen. The application of this method for the simultaneous detection of tyrosine hydroxylase (TH)- and corticotropin releasing factor (CRF)-immunoreactive structures revealed that black colored TH-immunopositive fibers contacted brown colored CRF-synthesizing neurons in the hypothalamic paraventricular nucleus. The juxtaposition of TH- and CRF-containing elements was apparent in both thick vibratome (40 micron) and semithin (1 micron) sections. At the ultrastructural level, TH-positive terminals - labeled by silver-gold grains - were observed to establish asymmetric synapses with both CRF- and TH-immunoreactive neurons. The former finding indicates a direct, TH-immunopositive, catecholaminergic influence upon the hypothalamic CRF system, while the latter demonstrates the existence of intrinsic connections between TH-positive elements.     

Immunocytochemical localization of neuropeptide Y-like immunoreactivity in adrenergic and non-adrenergic neurons of the rat gastrointestinal tract

The immunocytochemical location of neuropeptide Y (NPY)-like immunoreactivity (LI) within the neuronal structures of the rat gastrointestinal (GI) tract was investigated with the indirect immunofluorescence method. NPY immunoreactive neurons were found throughout all regions of the GI tract with the largest number in the duodenum. NPY immunoreactive perikarya were mainly located in the submucosal ganglia. NPY labeled processes were extensively seen in the submucosal and myenteric plexuses, smooth muscles, muscularis mucosa, mucosa and surrounding blood vessels. Following 6-hydroxydopamine (6-OHDA) treatment, NPY immunoreactive nerve fibers around blood vessels disappeared completely and the reactive fibers in other regions were reduced in number. NPY immunoreactive nerve cell bodies in the ganglionic plexuses, however, were not affected by 6-OHDA treatment. Serial sections of the coeliac ganglion showed that NPY-LI was present in cell bodies which also displayed tyrosine hydroxylase (TH) immunoreactivity. Our results suggest that NPY is abundantly contained in both adrenergic and non-adrenergic neurons of the gut and may play an important role in the regulation of the GI tract.     

Ultrastructural localization of immunolabeled substance P and methionine-enkephalin-octapeptide in the surface layer of the dorsal horn of rat spinal cord

Summary A preembedding dual immunolabeling technique and electron microscopy were utilized to demonstrate the localization of immunoreactive substance P and methionine-enkephalin-octapeptide (Enk-8) in ultrathin sections of the surface layer (laminae I and II) of rat spinal dorsal horn. The immunoreaction of Enk-8 was visualized as goldtoned silver particles and that of substance P as diaminobenzidine reaction products. Axonal terminals with immunoreactive substance P, and also unlabeled axonal terminals, formed synaptic junctions with the perikarya and dendritic processes of Enk-8-containing neurons. Dendritic profiles immunolabeled for substance P were synaptically linked with unlabeled axons but not with Enk-8-positive ones. Furthermore, it was found that Enk-8 axons and substance P axons terminated synaptically in juxtaposition to one another on the same immunonegative dendrites. Among the Enk-8-containing neurons axonal profiles also appeared to be synaptically associated with immunoreactive Enk-8 dendritic processes.     

Expression of nerve growth factor and its receptors in the somatosensory-motor cortex of Macaca nemestrina

The present study was designed to examine the nerve growth factor (NGF) system (ligand and receptor-expressing neurons) in the somatosensory (areas 1, 3a, and 3b) and motor (area 4) cortices of the mature macaque. Light and electron microscope immunohistochemistry was used to assess the distribution and identity of NGF-, p75-, and trk-expressing elements. In each cortical area examined, NGF-positive neuronal somata were distributed through all laminae; most immunolabeled neurons were in layers II, III, and V. Based upon light microscope criteria (e.g., the morphology of proximal dendrites), both pyramidal and stellate neurons expressed NGF. Of the identifiable NGF- immunoreactive cells, 92% were pyramidal neurons and the remainder was stellate neurons. The electron microscope study showed that most (88%) NGF-positive somata formed symmetric synapses, whereas the others formed both symmetric and asymmetric synapses. As the somata of pyramidal neurons form only symmetric synapses and those of inhibitory stellate neurons form both symmetric and asymmetric somatic synapses, the ultrastructural data support the light microscopic analyses. In contrast, neurotrophin receptors, p75 and trk, were expressed chiefly by the cell bodies of layer V pyramidal neurons and the supragranular neuropil. At the ultrastructural level, receptor-positive profiles were post-synaptic elements (e.g., dendritic shafts and spines) and the concentration of immunoreactivity was greatest in the vicinity of post-synaptic densities. Thus, NGF regulatory systems parallel excitatory and inhibitory neurotransmitter systems. Cortex contains the morphological framework by which pyramidal and/or inhibitory stellate neurons can affect the activity of post-synaptic pyramidal neurons via anterograde and autocrine/paracrine NGF systems.     

Electron microscopic localization of chromogranin A in osmium-fixed neuroendocrine cells with a protein A-gold technique

An antibody (LK2H10) to chromogranin A has been recommended for use in ultrastructural identification of neuroendocrine secretory granules. Previous studies have demonstrated immunoreactive chromogranin A in specimens prepared for electron microscopy by glutaraldehyde fixation only. In this study, the effect of specimen post-fixation by osmium tetroxide on post-embedding localization of chromogranin A was evaluated. Human tissues from benign endocrine glands, neuroendocrine tumors, and non-neuroendocrine tumors were post-fixed in osmium, embedded in epoxy resin, and the sample thin sections immunolabeled using a protein A-gold technique. Chromogranin A-positive neurosecretory granules were detected in pancreatic islets, adrenal medulla, stomach, ileum, anterior pituitary, and parathyroid. Mid-gut carcinoids, bronchial carcinoids, pheochromocytomas, paragangliomas, carotid body tumors, and thyroid medullary carcinomas contained immunoreactive granules. Cytoplasmic granules in non-neuroendocrine tumors did not react for chromogranin A. Tissues post-fixed in osmium tetroxide had optimally preserved ultrastructural features, and use of this fixative is compatible with postembedding localization of chromogranin A in neurosecretory granules.     

Fluorescence spectral properties of cyanine dye labeled DNA near metallic silver particles

Recent studies have demonstrated that silver metallic particles can increase the quantum yield and decrease the lifetimes of nearby fluorophores. These studies are extended to double stranded DNA oligomers labeled with N,N'-(dipropyl)-tetramethylindocarbocyanine (Cy3) or N,N-(dipropyl)-tetramethylindodicarbocyanine (Cy5). The proximity to silver particles increases the apparent quantum yields and decreases the lifetimes of the double helical DNA 23-mer labeled individually with Cy3 or Cy5. The decreased lifetimes are accompanied by apparently increased photostability of the labeled oligomers near silver particles. Because of spatial averaging across the sample these results are likely to significantly underestimate the effects of silver particles on labeled DNA localized at an optimal distance from the metallic surface. These results suggest that DNA arrays fabricated on substrates with silver particles can display increased sensitivity and photostability in the analysis of gene expression.     

Electron microscopic autoradiographic localization of prolactin mRNA in rat pituitary

Recent immunoelectron microscopic studies have shown that immunoreactive prolactin (PRL) in rat pituitary can be detected not only in typical PRL cells, characterized by large secretory granules, but also in another type of cell, which contains small secretory granules. To determine whether or not these two cell types are involved in PRL biosynthesis, we developed a procedure to investigate PRL gene expression by using in situ hybridization at the ultrastructural level. Rat pituitary was fixed and vibratome sections were incubated with a PRL [35S]-cDNA probe and subsequently flat-embedded in Araldite. Semi-thin and ultra-thin sections were processed for autoradiography. The results indicate that only the two PRL cell types were labeled. When immunolabeling for PRL was applied to ultra-thin sections, only immunopositive cells were seen to contain silver grains. In these cells the silver grains were associated with the rough endoplasmic reticulum and nucleus. When a growth hormone (GH) [35S]-cDNA probe was used as a control, only GH-secreting cells were labeled. This study confirms that the two PRL cell types are involved in biosynthesis of PRL. Moreover, this simple in situ hybridization technique provides a new approach to accurately localize mRNA in complex tissue and to investigate the subcellular distribution of mRNA under differing experimental conditions.     

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

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

Silver amplification of mercury sulfide and selenide: a histochemical method for light and electron microscopic localization of mercury in tissue

A method for light and electron microscopic demonstration of mercury sulfides and mercury selenides in mammalian tissue is presented. Silver ions adhering to the surface of submicroscopic traces of mercury sulfides or selenides in the tissue are reduced to metallic silver by hydroquinone. Physical development thereupon renders deposits of mercury sulfides or mercury selenide visible as spheres of solid silver. Examples of localization of mercury in the central nervous system and various organs from animals exposed to mercury chloride or methyl mercury chloride with or without additional sodium selenide treatment are presented. Selenium treatment results in a considerable increase in the amount of mercury that can be made visible by silver amplification. After mercury chloride treatment, most of the mercury is localized in lysosomes and is only rarely seen in secretory granules. After simultaneous selenium treatment, mercury is also found in nuclei of proximal tubule cells in the kidney and in macrophages. The "sulfide-osmium" method for ultrastructural localization of mercury suggested by Silberberg, Lawrence, and Leider (Arch Environ Health 19:7, 1969) and the light microscopic method using a photographic emulsion suggested by Umeda, Saito, and Saito (Jpn J Exp Med 39:17, 1969) have been experimentally analyzed and commented on.     

Preembedding colloidal gold immunostaining of hypothalamic neurons: light and electron microscopic localization of beta-endorphin-immunoreactive perikarya

A preembedding immunogold staining (IGS) procedure was developed to identify beta-endorphin/adrenocorticotropic hormone immunoreactive neurons at the light and electron microscopic levels. Colchicine-treated rats were perfused with Nakane's periodate-lysine-paraformaldehyde fixative. Vibratome sections were incubated in primary antisera followed by goat anti-rabbit immunoglobulin G coupled to 16 nm colloidal gold, and, in some cases, rabbit immunoglobulin G coupled to gold. The appearance to pink to light red perikarya, corresponding to colloidal gold deposition at antigenic sites, was monitored under the light microscope. Positive cell bodies in the arcuate region sometimes extended lateral to the nucleus. Only proximal portions of neuronal processes were stained. At the ultrastructural level, colloidal gold labeled the periphery of 90-110 nm dense neurosecretory granules in the perikaryal cytoplasm and a few proximal axons. Clusters of gold particles, appearing free in the neuroplasm, actually labeled secretory granules in adjacent thin sections. Granules associated with the Golgi apparatus were not stained. Colloidal gold labeling of mature beta-endorphin granules, but not progranules, in rat hypothalamic neurons was confirmed using the peroxidase-antiperoxidase technique. The results correlate well with data on the intracellular processing of pro-opiomelanocortin in pituitary cells and prepropressophysin in the paraventricular nucleus. These data demonstrate the first application of the preembedding colloidal gold staining method for the identification of intracellular antigens within the central nervous system. The IGS method provides a definitive marker for single or double labeling of nervous tissue at both the light and electron microscopic levels.     

A combined light and electron microscopic immunocytochemical method for the simultaneous localization of multiple tissue antigens

Summary In order to study the morphological interrelationships between immunocytochemically identified neuronal systems, a double labelling procedure — suitable for correlative light and electron microscopic observations — is introduced. The technique is based on the consecutive use of the silver-gold (SG) intensified and non-intensified forms of the oxidized 3,3-diaminobenzidine (DAB) chromogen in the framework of the peroxidase-antiperoxidase complex (PAP) indirect immunocytochemical procedure. The first tissue antigen is detected by the SG intensified DAB chromogen, which has a black color and high electron density. The structures containing the second antigen are visualized by the non-intensified DAB-endproduct, which is less electron dense than the silver-gold amplified form and is brown. The metallic shield that forms around the labeled antibody sequences associated with the first antigen prevents non-specific binding of immunoglobulins used for the detection of the second tissue antigen.The application of this method for the simultaneous detection of tyrosine hydroxylase (TH)- and corticotropin releasing factor (CRF)-immunoreactive structures revealed that black colored TH-immunopositive fibers contacted brown colored CRF-synthesizing neurons in the hypothalamic paraventricular nucleus. The juxtaposition of TH-and CRF-containing elements was apparent in both thick vibratome (40 m) and semithin (1 m) sections. At the ultrastructural level, TH-positive terminals — labeled by silvergold grains — were observed to establish asymmetric synapses with both CRF- and TH-immunoreactive neurons. The former finding indicates a direct, TH-immunopositive, catecholaminergic influence upon the hypothalamic CRF system, while the latter demonstrates the existence of intrinsic connections between TH-positive elements.Supported by NIH Grant NS19266     

Subcellular localization of cadmium in the root cells of<Emphasis Type="Italic">Allium cepa</Emphasis> by electron energy loss spectroscopy and cytochemistry

The ultrastructural investigation of the root cells ofAllium cepa L. exposed to 1 mM and 10 mM cadmium (Cd) for 48 and 72 h was carried out. The results indicated that Cd induced several obvious ultrastructural changes such as increased vacuolation, condensed cytoplasm with increased density of the matrix, reduction of mitochondrial cristae, severe plasmolysis and highly condensed nuclear chromatin. Electron dense granules appeared between the cell wall and plasmalemma. In vacuoles, electron dense granules encircled by the membrane were aggregated and formed into larger precipitates, which increase in number and volume as a consequence of excessive Cd exposure. Data from electron energy loss spectroscopy (EELS) confirmed that these granules contained Cd and showed that significantly higher level of Cd in vacuoles existed in the vacuolar precipitates of meristematic or cortical parenchyma cells of the differentiating and mature roots treated with 1 mM and 10 mM Cd. High levels of Cd were also observed in the crowded electron dense granules of nucleoli. However, no Cd was found in cell walls or in cells of the vascular cylinder. A positive Gomori-Swift reaction showed that small metallic silver grains were abundantly localized in the vesicles, which were distributed in the cytoplasm along the cell wall.     

Distribution of calbindinlike immunoreactive structures in the optic tectum of normal and eye-enucleated cyprinid fish

Summary Using the ABC immunohistochemical method, we investigated the distribution of calbindinlike immunoreactive structures in the optic tectum of normal fish, Tinca tinca, and from normal and unilaterally eye-enucleated fish, Cyprinus carpio. In nonoperated individuals of both species the optic tectum contained numerous immunoreactive neurons with strongly positive somata located in the stratum periventriculare and a thick immunolabeled dendritic shaft ascending radially toward the stratum fibrosum et griseum superficiale. The retinorecipient layers contained many fibrous immunoreactive structures. Some varicose fibers, isolated or in small bundles, were localized to the stratum album centrale, especially in the dorsal tectal half. Unilateral eye removal produced the disappearance of the immunoreactive fibrous structures located in the retinorecipient layers of the tectum contralateral to the enucleation. The present work shows that calbindinlike immunoreactive substances are localized in specific neural circuits of the fish optic tectum and suggests that the calbindin-like immunoreactive fibers in the retinorecipient strata are of retinal origin.