Differential localisation of the metabotropic glutamate receptor mGluR1a and the ionotropic glutamate receptor GluR2/3 in neurons of the human cerebral cortex

Specimens of human cerebral cortex were obtained during neurosurgical operations and studied by immunocytochemistry and electron microscopy, using antibodies to the metabotropic glutamate receptor subunit mGluR1a and the ionotropic glutamate receptor GluR2/3. A small number of non-pyramidal neuronal cell bodies were labelled for mGluR1a. Double immunolabelling with mGluR1a and GluR2/3 showed that most pyramidal cell bodies were labelled for GluR2/3 but not for mGluR1a. Despite the non-colocalisation of these two receptor subtypes in cell bodies, however, many dendrites and dendritic spines were double-labelled for mGluR1a and GluR2/3 at electron microscopy. As there is evidence that most neurons positive for GluR2/3 are pyramidal cells, this suggests that mGluR1a is present in dendrites of pyramidal neurons, despite absent or low levels of immunoreactivity in their cell bodies. Received: 5 May 1997 / Accepted: 24 July 1997     

An electron microscopic study of plasmatic arterionecrosis in the human cerebral arteries

Summary An electron microscopic study of the intracerebral arteries from 9 hypertensive cases was performed in order to elucidate the morphogenesis of the plasmatic arterionecrosis which was considered to be the direct cause of hypertensive intracerebral hemorrhage. In the preceding stage of the arterial lesions, marked necrosis of medial smooth muscle cells and increase of basement membrane-like substance in the intima and media were observed. The lumina of these arteries were slightly dilated. The dilatation and hemodynamic factors were supposed to cause endothelial injury resulting in blood plasma insudation into the intima through the opened spaces between endothelial cells. The insudated blood plasma dispersed and dissolved the basement membrane-like substance, collagen and elastic fibers in the arterial wall, leading to the development of the plasmatic arterionecrosis.This study was supported by a Grant-in-Aid for Scientific Research of the Japanese Ministry of Education (No. 857052).     

Distribution of GABA and neuropeptides in the human cerebral cortex

Summary Antibodies were used to identify neurons in human frontal and temporal cortex that were immunopositive to -aminobutyric acid (GABA) and the neuropeptides vasoactive intestinal polypeptide (VIP), substance P (SP) and somatostatin (SOM). Specimens were taken at surgical biopsy and fixed immediately after removal. The results described for both light and electron microscopy were obtained when relatively high concentrations of glutaraldehyde (2.5–3%) were present in the fixative. Specimens were examined from three adults and an infant aged 5 months. GABAergic neurons were present in all cortical layers, with fewest in layers I, deep III and V, and were mainly small, and round or oval. No labelled pyramidal neurons were detected. GABAergic puncta were common in the neuropil, probably representing axonal profiles. VIP-neurons were also found in all layers, including layer I, and were approximately twice as numerous as GABA-cells. SP-positive cells were found throughout the layers, but were sparse in layers I and VI. They were about three times commoner than GABAergic neurons. SOM-reactivity was demonstrated in about the same number of cells as that for SP. Again, this involved all layers, but layer I least. Peptidergic neurons were larger, on the average, than GABAergic cells, and were frequently pyramidal in character. In the infant, the distribution, size and frequency of immunoreactive neurons were similar to those in the adult. However, GABAergic puncta were commoner.This paper represents part of a study for the degree of Ph.D. in the National University of Singapore by WYO while at the Department of Anatomy, National University of Singapore.     

An electron microscopic study of neurosecretion in the cerebral ganglion of the earthworm

Non-synaptic, exocytotic release of neurosecretory granules in cerebral ganglion neurons was observed electron microscopically in 3 species of the oligochaete annelids Aporrectodea caliginosa, Octolasion cyaneum and Lumbricus terrestris. In addition to the features indicating exocytotic release of neurosecretory granules into perineuronal space, possible features of neurosecretion into blood vessels were seen within the cerebral ganglion. Axon terminals in synaptic contact with perikaryal profiles of cerebral ganglion neurons were also found.     

An electron microscopic study of dendritic degeneration in the cerebral cortex resulting from laminar lesions

Summary The morphological characteristics of dendrites in layers of the cerebral cortex above laminar lesions induced by ionizing particle irradiation have been studied in the striate field of rat at various survival times. Within two weeks following irradiation an increasing number of dendrites display unusual alterations inferred to be signs of degeneration.Degenerating dendrites can be characterized by a dense cytoplasmic matrix, disruption of mitochondria, presence of dense bodies, irregular outline and a marked alteration of the plasmalemma in its dimensions and staining properties. Some degenerating dendrites possess a large accumulation of dense subsynaptic material and are contacted by synapses with enlarged and altered synaptic clefts. A few dendrites contain extensive membranous whorls. Engulfment by reactive astrocyte processes is a common feature and often includes the presynaptic axonal knob, but only the degenerating dendrite has been observed within glial cytoplasm. The inference that the majority of degenerating dendrites in this material are apical dendrites of pyramidal cells suggests that either shaft synapses are common for these cells, protuberances may retract during degeneration, or spines are lost due to loss of afferent terminals.Supported by research grants from the United States Public Health Service, NB-4578 and NB-6594.     

Distribution of collagen types I and III and basal lamina in human gastric carcinoma: An immunohistochemical and electron microscopic study

Summary Collagen types I and III were examined immunohistochemically in 32 cases of gastric carcinoma classified as poorly differentiated adenocarcinoma with scirrhous stroma, well differentiated adenocarcinoma with intermediate stroma, or poorly differentiated adenocarcinoma with medullary stroma. In the stroma of scirrhous carcinoma, types I and III collagens were distributed abundantly in fibrillar or granular patterns with little difference in the intensity of staining. In well differentiated adenocarcinoma, type I collagen was diffusely distributed in the stroma with type III collagen distributed sparsely. In poorly differentiated adenocarcinoma with medullary stroma, the two types of collagen were only found around capillaries, constituting the tumor interstitium. Electron microscopic examination of scirrhous carcinoma showed tumor cells partially covered with fibroblasts, and discontinuous basal lamina, collagen fibers and microfibrils present between tumor cells and fibroblasts. In well differentiated carcinoma, tumor cells were surrounded by fibroblasts, and well developed basal lamina was observed beneath the tumor cells. In poorly differentiated carcinoma with medullary stroma, the stroma consisted of capillaries and very few fibroblasts with discontinuous basal lamina occasionally being present between tumor cells and fibroblasts.     

Glutamate Receptors GluR1 and GluR4 in the Hamster Superior Colliculus: Distribution and Co-localization with Calcium-Binding Proteins and GABA

We investigated the distributions of AMPA glutamate receptor subtypes GluR1 and GluR4 in the hamster superior colliculus (SC) with antibody immunocytochemistry and the effect of enucleation on these distributions. We compared these labelings to those of GluR2/3 in our previous report (Park et al., 2004, Neurosci Res., 49:139–155) and calcium-binding proteins calbindin D28K, calretinin, parvalbumin, and GABA. Anti-GluR1-immunoreactive (IR) cells were scattered throughout the SC. By contrast, anti-GluR4-IR cells formed distinct clusters within the lower lateral stratum griseum intermediale (SGI) and lateral stratum album intermediale (SAI). The GluR1- and GluR4-IR neurons varied in size and morphology. The average diameter of the GluR1-IR cells was 13.00 µm, while the GluR4-IR cells was 20.00 µm. The large majority of IR neurons were round or oval cells, but they also included stellate, vertical fusiform and horizontal cells. Monocular enucleation appeared to have no effect on the GluR1 and GluR4 immunoreactivity. Some GluR1-IR cells expressed calbindin D28K (9.50%), calretinin (6.59%), parvalbumin (2.53%), and GABA (20.54%). By contrast, no GluR4-IR cells expressed calcium-binding proteins or GABA. Although the function of the AMPA receptor subunits in SC is not yet clear, the distinct segregation of the GluR subunits, its differential colocalization with calcium-binding proteins and GABA, and differential responses to enucleation suggest the functional diversity of the receptor subunits in visuo-motor integration in the SC.     

Electron microscopic study of paired helical filaments and cerebral amyloid using a novel en bloc silver staining method

Summary A one step en bloc silver staining method which was originally established to study nucleolar organizer regions has been applied for the demonstration of both paired helical filaments (PHF) and extracellular cerebral amyloids in semi-thin sections and at the electron microscopic level. The three forms of PHF can be visualized: (1) neurofibrillary tangles are shown in all stages from first appearance in form of intracellular patches of PHF to severely degenerated shadow-like ghost tangles; (2) neuropil threads are distinctly stained in great numbers; and (3) PHF are easily detected as neuritic components in amyloid plaques. All forms of fibrillar extracellular amyloid structures, i.e. diffuse, classical and burnt out plaques, are well demonstrated; congophilic angiopathy reveals amyloid preferentially in arteries and arterioles of the leptomeninges and cortex ranging from small circumscribed patches to large circumferential amounts with occasional plaque-like condensations or broad loose accumulations of amyloid; perivascular cuffs and laminar subpial deposits of amyloid are stained as well. At the electron microscopic level all lesions are clearly visible in non uranyl/lead-stained specimens, characterized by varying numbers of silver grains on a pale background. The detailed demonstration of structures in archival material, which had been stored in paraffin and re-embedded for electron microscopy, is due to the demonstration of argyrophilic structures by the protective colloidal developer of gelatin and formic acid and to the proteolytic resistance of insoluble PHF and extracellular amyloids in plaques and congophilic angiopathy.Parts of this paper were presented at the Annual Meeting of the German Society for Neuropathology and Neuroanatomy, Düsseldorf, FRG, 1991     

Intrahypothalamic connections: An electron microscopic study in the rat

Summary Terminal degeneration within the hypothalamus was studied by electron microscopy 1 or 2 days (1) after carefully placed microlesions in the arcuate, anterior periventricular, ventromedial, premammillary and posterior hypothalamic nuclei and (2) after microlesions placed in the hypothalamus deafferented 3 weeks earlier.In the median eminence terminal degeneration was found after each of these lesions. Projections from the ventromedial nucleus reach the arcuate, suprachiasmatic, and anterior periventricular nuclei.Projections from the arcuate nucleus terminate in the medial preoptic, anterior periventricular, and ventromedial nuclei.After lesioning the premammillary nuclei degeneration was found in the supraoptic, arcuate, anterior hypothalamic and ventromedial nuclei.     

A light and electron microscopic study of betaine/GABA transporter distribution in the monkey cerebral neocortex and hippocampus

The present study aimed to elucidate the distribution of betaine/gamma-aminobutyric acid (GABA) transporter-1 (BGT-1) in the normal monkey cerebral neocortex and hippocampus by immunoperoxidase and Immunogold labelling. BGT-1 was observed in pyramidal neurons in the cerebral neocortex and the CA fields of the hippocampus. Large numbers of small diameter dendrites or dendritic spines were observed in the neuropil. These made asymmetrical synaptic contacts with unlabelled axon terminals containing small round vesicles, characteristic of glutamatergic terminals. BGT-1 label was observed in an extra-perisynaptic region, away from the post-synaptic density. Immunoreactivity was not observed in portions of dendrites that formed symmetrical synapses, axon terminals, or glial cells. The distribution of BGT-1 on dendritic spines, rather than at GABAergic axon terminals, suggests that the transporter is unlikely to play a major role in terminating the action of GABA at a synapse. Instead, the osmolyte betaine is more likely to be the physiological substrate of BGT-1 in the brain, and the presence of the transporter in pyramidal neurons suggests that these neurons utilize betaine to maintain osmolarity.     

A light- and electron-microscopic study of GluR4-positive cells in cerebral cortex,subcortical white matter and corpus callosum of neonatal,immature and adult rats

The distribution of the [³H]alpha-amino-3-hydroxy-5-methylisoxzalepropionic acid (AMPA) receptor subunit GluR4 was studied in frontal, parietal and temporal cerebral cortex, subcortical white matter and corpus callosum of neonatal, immature and mature rats. In 1-to 2-day-old rats, a few oligodendrocyte progenitors and amoeboid microglia in the supraventricular part of the corpus callosum were immunolabelled for GluR4. At 7 to 10 days, the number of amoeboid microglia and oligodendrocyte progenitors in white matter increased; many neurons in cortex, including pyramidal neurons, were also moderately labelled for GluR4. The pattern of GluR4 immunostaining in 14-day-old rats was different from that in 7-to 10-day-old rats, but similar to the adult, in that there was no immunoreactivity in microglia and oligodendrocyte progenitors in subcortical white matter. A proportion of non-pyramidal neurons in cortex were moderately labelled, while some pyramidal neurons were lightly labelled. A population of small glial cells with features of oligodendrocyte progenitors were densely labelled in cortex.     

A transmission electron microscopic study of microglia/macrophages in the hippocampal cortex and neocortex following chronic exposure to valproate

In chronic administration of sodium valproate to rats, significant disorders of structural integrity of the hippocampal gyrus and the neocortex of the temporal lobe, observed in the last two stages of the experiment (after 9 and 12 months), coexisted with increased number of microglial cells and, especially after 12 months, with intense phagocytic activity within these cells. At the ultrastructural level, phagocyte microglial cells were hypertrophied with several broadened processes. Their cytoplasm contained rich lysosomal apparatus, numerous lipofuscin-like structures, lipid droplets and multilaminated bodies. The nuclei of these cells were characteristic oval or round and sometimes triangle in shape with dense and highly clumped heterochromatin, distinctly accumulated under nuclear envelope, and sparse euchromatin. Microglia/macrophages were frequently present in a close vicinity of changed neuronal somata and also close to the altered elements of the neuropil pyramidal layer of the cortex. Microglial response may, together with abnormalities in neurones, astroglia and blood-brain barrier, play a significant role in the development of experimental valproate encephalopathy.     

Correlation of electron microscopic and secretory response of human parathyroid adenomas with different calcium concentrations in organ culture

Summary Twelve parathyroid chief cell adenomas from patients with primary hyperparathyroidism were incubated in a tissue culture system in the presence of different calcium concentrations and for various time periods. The endocrine response of the tissue was examined electron microscopically and radioimmunologically.After incubation in a medium of low calcium concentration the parathyroid adenomas showed ultrastructural signs of stimulation with proliferation of the hormone-synthesizing organelles. The development of the ultrastructural response could first be observed after four hours and increased up to several days. Radioimmunologically, an increase of the hormone secretion could be demonstrated.Converse results were obtained after incubation of the tumor tissue under suppressive culture conditions.To check for de-novo synthesis of the hormone released the tissue was incubated in a ⁷⁵Se-methionine-containing medium. This resulted in radioactivity of the secreted parathyroid hormone, indicating de novo synthesis in our culture system.The biological potency of the released hormone was demonstrated by comparison of the PTH out of the medium with the international human MRC standard using two different radioassays.The technical assistance of Mrs. E. Lehmann and B. Sehringer is gratefully acknowledged. For diagnosis and surgery we are endebted to Prof. Dr. F. Kuhlencordt and Dr. G. Koch (both University of Hamburg), Dr. M. Bressel and Dr. H. Hüsselmann (both General Hospital Hamburg-Harburg). This work was supported by a grant of the Deutsche Forschungsgemeinschaft, SFB 34, Endokrinologie.     

Enhanced activity of GABA receptors inhibits glutamate release induced by focal cerebral ischemia in rat striatum

Cerebral ischemia causes an excess release of glutamate, which can injure neurons. The striatum is one of the important regions vulnerable to hypoxia and ischemia. Using push–pull perfusion technique, we investigated the regulatory role of γ-aminobutyric acid (GABA) and its receptors in modifying the amount of glutamate in rat striatum with ischemia. Perfusion with exogenous GABA (1 mM) inhibited cerebral ischemia-induced glutamate release by as much as 47%. We further characterized relative roles of subtype receptors of GABA on glutamate release by using pharmacological tools. While baclofen (500 μM), a GABA_B receptor agonist, suppressed ischemia-induced glutamate release by 52%, GABA_B receptor antagonist saclofen (500 μM) failed to produce a significant increase of glutamate release. The GABA_A receptor agonist muscimol (500 μM) also reduced by 38% the release of glutamate induced by cerebral ischemia but the GABA_A receptor antagonist bicuculline (500 μM) had very little effect. The present study demonstrates that the excessive release of glutamate or the overly activated glutamate receptor, triggered by cerebral ischemia, can be down-regulated by exogenous GABA or by increased activity of GABA receptors, especially the presynaptic GABA_B receptors, which might be one of the important mechanisms to protect against striatum neuronal damage from over stimulation by excessive glutamate during ischemia.     

Light and electron microscopic findings in five cases of cryoglobulinemic glomerulonephritis

Summary Renal tissue from five patients with cryoglobulinemia was studied by light and electron microscopy and immunofluorescence. None of the histologic features observed at the light microscopic level seems to be specific for cryoglobulinemia. Electron microscopic investigations have shown very large electron dense deposits in almost every examined lobule in all cases. The deposits displayed two main patterns; a homogeneous texture in two cases and tubular or annular structures in three cases. The patients with typically structured deposits had IgG-IgM cryoglobulinemia (2 cases) or monoclonal IgM cryoglobulinemia (1 case). The presence of IgM in cryoglobulinemia may be the cause of the peculiar structure of the deposits.     

Kappa opioid receptors are expressed by interneurons in the CA1 area of the rat hippocampus: a correlated light and electron microscopic immunocytochemical study

A local GABA-system is known to have a mediatory function between several afferents and the principal cells of the hippocampus. This study examines the distribution and fine structure of kappa opioid receptor-immunoreactive elements in the CA1 subfield and reveals some new aspects concerning the structural basis of opioid-GABA interaction in the rat hippocampal formation. Kappa receptors were visualized immunocytochemically with a previously produced and characterized monoclonal antibody, the mAb KA8 (Maderspach, K., Németh, K., Simon, J., Benyhe, S., Szûcs, M., Wollemann, M., 1991. A monoclonal antibody recognizing kappa-, but not mu- and delta-opioid receptors. J. Neurochem. 56, 1897–1904). The antibody selectively recognizes the kappa opioid receptor with preference to the kappa₂ subtype. Neuronal cell bodies, proximal dendrites and occasionally glial processes surrounding neuronal perikarya were labelled in the CA1 area. The immunopositive cells were present mainly in the stratum oriens, followed by the stratum pyramidale in a rostrocaudally increasing number. Their shape was fusiform, or multipolar. Occasionally kappa receptor-immunoreactive boutons surrounding weakly immunopositive somata were also observed. Electron microscopy of immunopositive neurons showed that the DAB labelling was intensive in the perinuclear cytoplasm. The widths and electron densities of the postsynaptic densities of some axosomatic synapses were remarkably increased. Similar increase of postsynaptic densities were observable at some axodendritic and axospinous synapses. On the basis of their location and fine structural properties the labelled cells are suggested to be GABAergic inhibitory interneurons, probably belonging to the somatostatinergic sub-population. The axons of these inhibitory interneurons are known to arborize in the stratum lacunosum-moleculare where the entorhinal afferents terminate. A modulatory effect of opioids on the entorhinal input, mediated by somatostatinergic interneurons is suggested     

Light and electron microscopic examination of fine-needle aspirates in the preoperative diagnosis of cartilaginous tumors

Twenty-eight patients with chondrogenic tumours--2 chondroblastomas, 4 chondromas, 18 chondrosarcomas, 1 clear-cell chondrosarcoma, and 3 mesenchymal chondrosarcomas--underwent fine-needle aspiration biopsy (FNAB) in the preoperative investigation. The cytologic features in smears were compared with the histopathologic findings in the surgical specimens; in 14 cases they were also compared with the light and electron microscopic findings in resin-embedded fine-needle aspirates. The smears of the vast majority of the classical chondrosarcomas presented features that made possible the FNAB diagnosis of a chondrogenic tumor to be made. In the case of the low-grade chondrosarcomas in particular, which were poorly or moderately cellular in smears and showed chondroblastic cells often in lacunary structures of hyaline matrix, consideration of the clinical presentation, size, location, and roentgenographic appearance was essential for the diagnosis of chondrosarcoma. On the other hand, the high-grade chondrosarcomas presented cytologic features that clearly indicated their malignancy and they usually had a myxoid matrix. The possible differential diagnoses that may arise from the FNAB diagnosis of cartilaginous tumors are discussed. The resin-embedding technique for the light and electron microscopic examination of FNABs, along with the histochemical analysis for the demonstration of sulphated glucosaminoglycans and the immunocytochemistry applied to smears, was found to be of value in the definite diagnosis, especially in the distinction of chondrogenic tumors from chordoma and metastatic mucous-producing carcinoma.     

Cytology of embryonal rhabdomyosarcoma: a cytologic, light microscopic, electron microscopic, and immunohistochemical study of seven cases

A correlated cytologic and histologic study of seven cases of embryonal rhabdomyosarcoma is presented. The diagnosis of rhabdomyosarcoma was established by light and electron microscopy and immunohistochemistry of the operative specimens. The cytologic appearance of the smears corresponded well with the histopathologic findings. Cytologically, two main cell types were distinguished: a predominant primitive, small round cell with scant cytoplasm and a large cell with an abundant cytoplasm, sometimes tadpole- or ribbon-shaped. The tumor cells were often enclosed in a background of mucosubstances. The lack of cytologic features proving rhabdomyoblastic differentiation, such as cross-striation, necessitates the use of additional methods in the cytologic diagnosis of embryonal rhabdomyosarcoma. The value of the embedding technique for ultrastructural analysis and immunohistochemistry in the demonstration of desmin in aspirates is emphasized in the diagnosis of embryonal rhabdomyosarcoma.     

An electron microscopic study of the developing caudate nucleus in euthyroid and hypothyroid states

Summary Thyroid hormone exerts a powerful influence on CNS growth and maturation. Hypothyroidism early in life has long been known to cause disturbances in innate behavior, motor performance, severe and frequently irreversible mental retardation. In this deficiency, depressed caudate neurogenesis, cell migration and neuropil development during the rapid period of CNS growth may contribute to the clinical picture of perceptual handicaps often seen in cretins. Light microscopic and Golgi studies of the developing caudate nucleus in thyroid deficiency have been carried out to help attain insights into the mechanisms whereby the extrapyramidal system regulates motor function. The ultrastructural study of caudate nuclear cytogenesis and synaptogenesis in normal and hypothyroid states provides more detailed information for further analysis of the problem.Hypothyroidism was induced from birth by adding propylthiouracil to the food and drinking water of lactating dams. Linear development of the caudate nucleus of both normal and hypothyroid rats at ages 8, 14, 20, 30 and 42 days was studied by electron microscopy. Thyroid glands were examined by light microscopy to assess the normal and deficient states.Immature cells, primitive processes and synapses were the characteristic features of the 8-day-old normal caudate nucleus. Distinctively wide cisternae of the rough endoplasmic reticulum, loosely packed Golgi apparatus and chromatin clumps throughout the nuclei of the neurons were significant early morphologic variations. The dramatic cytoarchitectural maturation in the 14- and 20-day normal caudate neuropil points to the rapidity of developmental rate. After the growth spurt of the first three weeks a maturational plateau occurs which is characterized by well-formed neuronal cytoplasmic organelles, myelinated and non-myelinated axons, axon terminals, dendrites and their spines, and synapses.Thyroid deficiency causes a marked maturational delay of approximately 7 days in caudate neuronal proliferation, the elaboration of neuronal networks and the attainment of mature synaptic contents and membranes. This delay is evidenced by comparison of the structural similarities between 8-day-old normal and 14-day-old deficient rats; and additional comparisons between the 14-day-old normal and 20-day-old hypothyroid rats. A rapid catch up process in fine structural morphogenesis takes place in the period between days 14 and 30 in the deficient animals. Repression of thyroid function does not entirely prevent development of the caudate nucleus but allows a fairly extensive, though critically incomplete degree of maturation. This imperfection is manifested by a decrease in the number of synaptic contacts that persists even after the rapid catch up phenomenon of caudate synaptogenesis.Partially supported by USPHS Grant No. HD-05615