Neurodegeneration associated with genetic defects in phospholipase A(2).

OBJECTIVE: Mutations in the gene encoding phospholipase A(2) group VI (PLA2G6) are associated with two childhood neurologic disorders: infantile neuroaxonal dystrophy (INAD) and idiopathic neurodegeneration with brain iron accumulation (NBIA). INAD is a severe progressive psychomotor disorder in which axonal spheroids are found in brain, spinal cord, and peripheral nerves. High globus pallidus iron is an inconsistent feature of INAD; however, it is a diagnostic criterion of NBIA, which describes a clinically and genetically heterogeneous group of disorders that share this hallmark feature. We sought to delineate the clinical, radiographic, pathologic, and genetic features of disease resulting from defective phospholipase A(2). METHODS: We identified 56 patients clinically diagnosed with INAD and 23 with idiopathic NBIA and screened their DNA for PLA2G6 mutations. RESULTS: Eighty percent of patients with INAD had mutations in PLA2G6, whereas mutations were found in only 20% of those with idiopathic NBIA. All patients with two null mutations had a more severe phenotype. On MRI, nearly all mutation-positive patients had cerebellar atrophy, and half showed brain iron accumulation. We observed Lewy bodies and neurofibrillary tangles in association with PLA2G6 mutations. CONCLUSION: Defects in phospholipase A(2) lead to a range of phenotypes. PLA2G6 mutations are associated with nearly all cases of classic infantile neuroaxonal dystrophy but a minority of cases of idiopathic neurodegeneration with brain iron accumulation, and genotype correlates with phenotype. Cerebellar atrophy predicts which patients are likely to be mutation-positive. The neuropathologic changes that are caused by defective phospholipase A(2) suggest a shared pathogenesis with both Parkinson and Alzheimer diseases.

Alzheimer disease-like clinical phenotype in a family with FTDP-17 caused by a MAPT R406W mutation.

We report clinical, molecular, neuroimaging and neuropathological features of a Danish family with autosomal dominant inherited dementia, a clinical phenotype resembling Alzheimer's disease and a pathogenic mutation (R406W) in the microtubule associated protein tau (MAPT) gene. Pre-symptomatic and affected family members underwent multidisciplinary (clinical, molecular, neuroimaging and neuropathological) examinations. Treatment with memantine in a family member with early symptoms, based on the clinical phenotype and the lack of specific treatment, appears to stabilize the disease course and increase the glucose metabolism in cortical and subcortical areas, as determined by serial [F(18)]FDG-PET scanning before and after initiation of treatment. Neuropathological examination of a second affected and mutation-positive family member showed moderate atrophy of the temporal lobes including the hippocampi. Microscopy revealed abundant numbers of tau-positive neurofibrillary tangles in all cortical areas and in some brainstem nuclei corresponding to a diagnosis of frontotemporal lobe degeneration on the basis of a MAPT mutation. The clinical and genetic heterogeneity of autosomal dominant inherited dementia must be taken into account in the genetic counselling and genetic testing of families with autosomal dominantly inherited dementia in general.

Evidence implicating BRD1 with brain development and susceptibility to both schizophrenia and bipolar affective disorder.

Linkage studies suggest that chromosome 22q12-13 may contain one or more shared susceptibility genes for schizophrenia (SZ) and bipolar affective disorder (BPD). In a Faeroese sample, we previously reported association between microsatellite markers located at 22q13.31-qtel and both disorders. The present study reports an association analysis across five genes (including 14 single nucleotide and two microsatellite polymorphisms) in this interval using a case-control sample of 162 BPD, 103 SZ patients and 200 controls. The bromodomain-containing 1 gene (BRD1), which encodes a putative regulator of transcription showed association with both disorders with minimal P-values of 0.0046 and 0.00001 for single marker and overall haplotype analysis, respectively. A specific BRD1 2-marker 'risk' haplotype showed a frequency of approximately 10% in the combined case group versus approximately 1% in controls (P-value 2.8 x 10(-7)). Expression analysis of BRD1 mRNA revealed widespread expression in mammalian brain tissue, which was substantiated by immunohistochemical detection of BRD1 protein in the nucleus, perikaryal cytosol and proximal dendrites of the neurons in the adult rat, rabbit and human CNS. Quantitative mRNA analysis in developing fetal pig brain revealed spatiotemporal differences with high expression at early embryonic stages, with intense nuclear and cytosolar immunohistochemical staining of the neuroepithelial layer and early neuroblasts, whilst more mature neurons at later embryonic stages had less nuclear staining. The results implicate BRD1 with SZ and BPD susceptibility and provide evidence that suggests a role for BRD1 in neurodevelopment.

Creutzfeldt-Jakob disease segregating in a three generation Danish family.

A three generation family is presented in which rapidly progressive, early-onset Creutzfeldt-Jakob disease without typical EEG changes segregates as an autosomal dominant disease. An aspartic acid to asparagine mutation at codon 178 of the prion gene, PRNP, co-segregates with the disease. As expected, the disease allele also carries the valine codon of the polymorphic valine/methionine codon 129 of the gene. In family members homozygous for this valine codon the disease was more rapidly progressive than in a heterozygous family member, who had a variant clinical phenotype. Definite neuropathological diagnosis required prion staining with specific antibodies.

Hippocampus of the domestic pig: a stereological study of subdivisional volumes and neuron numbers.

With the aim of establishing a quantitative structural basis for comparative and experimental studies, the volumes of the hippocampal subdivisions and the total number of neurons in each subdivision were estimated in domestic pig brains using modern stereological techniques. In addition to a detailed description of the stereological methods used in the analysis, comprehensive descriptions of the architectonic boundaries of the subdivisions are included. The absolute and relative volumes of the subdivisions were compared to those of a number of other species and the relationship between the number of neurons and the volume of the subdivisions was compared to that in homologous subdivisions of laboratory rats and humans. The methodology used to estimate the volumes and the total number of neurons in the individual subdivisions were evaluated with regard to the sensitivity that they can provide in experimental studies.

Cholecystokinin-, enkephalin-, and substance P-like immunoreactivity in the dentate area, hippocampus, and subiculum of the domestic pig.

The distribution of cholecystokinin-like, enkephalin-like, and substance P-like immunoreactivities is described in the dentate area, hippocampus, and subiculum of the domestic pig (Sus scrofa domesticus) as a baseline for future experimental studies. The distributions in the pig are compared with previous observations in other species. Cholecystokinin-like immunoreactive nerve cell bodies were intensely stained and present in large numbers in all subfields studied. Cholecystokinin-like immunoreactive terminals appeared as stained puncta, whereas fibers were only rarely encountered. The puncta were mainly seen in the dentate molecular layer and dentate granule cell layer, the pyramidal cell layer of the hippocampal regio inferior, stratum moleculare of the hippocampal regio superior, and in the subiculum. Enkephalin-like immunoreactive nerve cell bodies were faintly stained and generally present in very small numbers, except for some pyramidal cells in the subicular cell layer. Enkephalin-like immunoreactive fibers were few in number, whereas stained puncta appeared with variable densities. Puncta of particularly high densities were found in the dentate molecular layer, whereas they appeared of moderate density in the dentate hilus, stratum moleculare of the hippocampal regio superior, and in the subiculum. Substance P-like immunoreactive nerve cell bodies were few and very faintly stained. They primarily occurred in the dentate hilus, stratum oriens of the hippocampus, and in the subicular cell layer. Stained fibers were few in number, whereas stained puncta were present in abundant numbers corresponding to the mossy fiber projection in the dentate hilus and the layer of mossy fibers of the hippocampal regio inferior, and in moderate numbers in stratum moleculare of the hippocampal regio superior and in the subiculum. For all three neuropeptides there were consistent and very characteristic variations in the distribution of immunoreactivity along the septotemporal axis of the hippocampus. When viewed in a comparative perspective the distribution of enkephalin-like and substance P-like terminals in the domestic pig displayed striking differences from the basic pattern observed in other species. This contrasted with the distribution of cholecystokinin-like neurons and terminals, which resembled more closely these species.

Somatostatin- and neuropeptide Y-like immunoreactivity in the dentate area, hippocampus, and subiculum of the domestic pig.

With the principal aim of providing baseline observations for future experimental studies, the distribution of somatostatin-like and neuropeptide Y-like immunoreactivities is described in the dentate area, hippocampus, and subiculum of the domestic pig (Sus scrofa domesticus) and compared with the distribution described in other mammals. Intensely stained somatostatin-like immunoreactive nerve cell bodies were present throughout the region, with highest densities in the dentate hilus, stratum radiatum and stratum oriens of the hippocampal regio inferior, stratum oriens of the hippocampal regio superior, and in the subicular cell layer. Somatostatin-like immunoreactive terminals were represented by both stained fibers and stained puncta. Scattered somatostatin-like immunoreactive nerve fibers were seen in most areas, but regular fiber plexuses were present in the dentate molecular layer and dentate hilus, stratum moleculare of the hippocampus, and in the subicular plexiform layer. Somatostatin-like immunoreactive puncta were seen in the dentate molecular layer, stratum moleculare of the hippocampus, and in the subicular plexiform layer. Neuropeptide Y-like immunoreactive nerve cell bodies were less numerous than somatostatin-like immunoreactive ones. They were mainly seen in the dentate granule cell layer and dentate hilus, stratum radiatum and stratum oriens of the hippocampus, and in the subicular cell layer. Intensely stained neuropeptide Y-like immunoreactive fibers were numerous, and present in all areas examined. They formed fiber plexuses in the dentate molecular layer and dentate hilus, stratum moleculare of the hippocampal regio superior, and in the subicular plexiform layer. Neuropeptide Y-like immunoreactive puncta were present in the dentate molecular layer, stratum moleculare of the hippocampus, and in the subicular plexiform layer. Consistent and very characteristic variation in the distribution of somatostatin-like and neuropeptide Y-like immunoreactivity was found along the septotemporal axis of the hippocampus. The distribution of somatostatin-like and neuropeptide Y-like neurons and terminals in the domestic pig displayed striking similarities with the basic pattern of organization of these neuropeptides in other species, although more subtle species-specific characteristics were also observed in the pig.

Tyrosine hydroxylase and acetylcholinesterase in the domestic pig mesencephalon: an immunocytochemical and histochemical study.

The mesencephalon of the young domestic pig was studied by tyrosine hydroxylase (TH) immunocytochemistry and acetylcholinesterase (AChE) histochemistry with focus on the substantia nigra (SN), the ventral tegmental area (VTA), and related areas. The purpose was to obtain information on the organization of the mesencephalic, TH immunoreactive (TH-i), and dopaminergic areas of the pig, in order to provide the necessary background for the possible use of the pig as an alternative large animal experimental model for research on Parkinson's disease, including the use of encapsulated pig dopaminergic neurons for intracerebral xenotransplantation. Significant findings in the pig, compared to observations in other species, included the presence of prominent bundles of TH-i dendrites passing in a dorsoventral direction from pars compacta into pars reticulata at middle and caudal levels of the SN, and the presence of a distinct TH-i substantia nigra pars lateralis (SNL). Caudally in the pig mesencephalon, the retrorubral field (RRF) was found to be very extensive. The view of the RRF, SN, and VTA as parts of the same integrated system was indicated by the crisscrossing of TH-i dendrites at the transitions between these areas. Estimation of the number of TH-i neurons in the SN and the VTA showed that these nuclei were of equal size in the pig. Further, it was found that TH-i nerve cells were present in the midline between the VTA in the interfascicular and rostral linear groups. TH-i nerve cells were also present in the otherwise serotoninergic dorsal raphe nuclei, just as other TH-i cells formed a perirubral cell group. AChE-positive neurons were present in both SN and VTA, and appeared to have the same size and morphology as the TH-i neurons in these areas. Within both nuclei, there were local differences in the AChE staining density, but perhaps more significantly were some marked differences in the structure of the AChE-positive neuropil of the two areas. We anticipate that the present description of the cellular organization of the TH-i dopaminergic areas in the domestic pig ventral mesencephalon will be useful for the development of a nonprimate, large animal, experimental model of Parkinson's disease.

Histochemical demonstration of zinc in the hippocampal region of the domestic pig: III. The dentate area.

The distribution of zinc was described in the dentate area, a part of the hippocampal region, of the domestic pig. A modification of Timm's sulphide silver procedure, the Neo-Timm method, was used for the histochemical demonstration of zinc. The staining of the dentate area exhibited a well-defined stratified pattern, the predominant part of the staining being restricted to the neuropil, although weakly stained nerve cell bodies were observed in the hilus fasciae dentatae. In the molecular layer, three distinct sublaminae were seen at most septotemporal levels. The outer and inner sublaminae displayed medium staining intensity, whereas the intermediate sublamina appeared extremely pale. The granular cell layer was well stained in its superficial two thirds, because of dense masses of staining occupying the interstices between the unstained granular cells. In the hilus fasciae dentatae, extreme differences in staining intensity were seen between the layers, ranging from very intense staining of the outer hilar cell layer to generally weak staining of the inner plexiform layer. The distribution of zinc in the pig was compared with that in the guinea pig and rat, described previously. The staining pattern of the molecular layer showed striking species differences, whereas the granular cell layer appeared very near identical. The stratified staining pattern seen in the hilus of the pig is very similar to the distribution observed in the guinea pig, but differs from the essentially homogeneous staining of the rat hilus.(ABSTRACT TRUNCATED AT 250 WORDS)

Computer-assisted three-dimensional reconstruction of the hippocampal region based on serial sections.

A large series of rabbit hippocampal Neo-Timm stained sections were manually aligned, digitized, and by a modified median filtration noise reduced and reconstructed into a three-dimensional object. From the presented simulated grey tone cuts of this object, the reader may assemble a rabbit hippocampal model, that spatially illustrates its anatomy.

Histochemical demonstration of zinc in the hippocampal region of the domestic pig: II. Subiculum and hippocampus.

The distribution of zinc has been described in two areas of the hippocampal region of the domestic pig, viz., the subiculum and the hippocampus. Zinc was demonstrated histochemically according to the Neo-Timm method, a modification of the sulphide-silver procedure. In each of the examined areas the staining displayed a distinctly stratified pattern which has been compared in detail to fields and layers defined on the basis of cyto- and fibroarchitecture, resulting in a combined chemo- and cytoarchitectonic map. Most of the staining was confined to the neuropil, but a considerable number of stained nerve cell bodies were seen in both the subiculum and the hippocampus. In the subiculum, the plexiform layer was divided into a superficial, weakly stained subzone and a deep, better stained subzone. The cell layer was generally well stained, but displayed a complex staining pattern with differences in staining intensity of both the cell bodies and neuropil. In regio superior of the hippocampus, the stratum moleculare appeared weakly stained, with the exception of a tapering process of more darkly stained tissue projecting from the plexiform layer of the subiculum into the deepest part of the layer. Stratum radiatum and the superficial subzone of stratum oriens showed a weak staining intensity, contrasting to the relatively darkly stained pyramidal cell layer and the intensely stained deep subzone of stratum oriens. In regio inferior, the stratum moleculare was divided into a moderately stained superficial part and an unstained deep part. Stratum radiatum and stratum oriens both appeared weakly stained. The layer of mossy fibers was very intensely stained and appeared almost homogeneously black in its main suprapyramidal part, whereas the infrapyramidal part was looser in character. The pyramidal cell layer was darker than in regio superior. The distribution of zinc in the pig was compared with that in the guinea pig and rat, described previously. The staining pattern is fundamentally similar in all three species, though notable species-specific traits do exist.

Densitometric analysis of the local bleaching of the Neo-Timm staining pattern following intrahippocampal injection of diethyldithiocarbamate.

Treatment with certain metal chelating agents causes a time-dependent bleaching of the Neo-Timm staining pattern of zinc visualized in synaptic vesicles. In the present study, the extent and time course of the reversible chelation of hippocampal vesicular zinc was investigated following intrahippocampal injection of the chelating agent diethyldithiocarbamate. The carbamate (1.0 microliters 45 mg ml-1, 200 mM) was injected unilaterally into the hippocampal region of adult rats, which were allowed to survive 15 min-6 h before sacrifice. Control animals either received injections of distilled water or were untreated. Computerized optical densitometry was performed on cryostat sections of brains stained with the Neo-Timm method. Injection of diethyldithiocarbamate into the hippocampal region resulted in a localized bleaching of the Neo-Timm staining pattern. The extent of the bleaching varied with time being most pronounced at 15 min survival and gradually decreasing with time. After 6 h survival, a faint bleaching of the injected hippocampal region was barely seen. Computerized optical densitometry confirmed and extended the observations providing a semi-quantitative measure of zinc in synaptic vesicles.

Vasoactive substances in subchondral bone of the dog knee.

The purpose of the present study was to investigate regulatory mechanisms for subchondral bone blood flow. A model including elevation of joint cavity pressure in the immature dog knee was applied. The role of prostaglandins in bone blood flow regulation was indirectly examined by indomethacin blockade. In six puppies, both venous tamponade of the joint cavity [50% of the mean arterial blood pressure (MAP)] and arterial tamponade (150% of MAP) resulted in a significant increase in the intraosseous pressure of the distal femoral epiphyses (p less than 0.05). During venous tamponade no changes were registered in pO2, pCO2, pH, potassium, and lactate in blood withdrawn from the distal femoral epiphyses. Arterial tamponade resulted in hypoxia, a decrease in pH, and increased lactate. Inhibition of the prostaglandin synthesis did not alter this response pattern. Thus, the present study suggests the presence of a regulatory mechanism for subchondral bone blood flow since venous tamponade did not significantly alter intraosseous gas tensions, pH, lactate, or potassium in spite of elevated venous outlet resistance. The study does not allow any conclusion as to the exact nature of the regulatory mechanism, but local metabolic regulation is likely to be involved as indicated by accumulation of vasoactive substances at higher tamponade levels. Prostaglandins are probably of minor importance in this regulation.

Immunocytochemical demonstration of the calcium-binding proteins calbindin-D 28k and parvalbumin in the subiculum, hippocampus and dentate area of the domestic pig.

The distribution of the calcium-binding proteins calbindin-D 28k (CaBP) and parvalbumin (PV) in the hippocampal region of the domestic pig was demonstrated by immunocytochemistry. Scattered CaBP-immunoreactive cell bodies were present in the subiculum, stratum oriens, pyramidal cell layer and stratum radiatum of the hippocampal regio superior and inferior, and the outer plexiform layer and outer hilar cell layer of the dentate hilus. Other cell bodies and bundles of stained fibers were present in stratum moleculare of regio superior and inferior, and in the outer third of the molecular layer of the fascia dentata. Terminal-like CaBP-immunoreactivity was seen in the subiculum and around cell bodies in the pyramidal cell layer of regio superior and inferior and the dentate granular cell layer. Scattered PV-immunoreactive cell bodies were present in stratum oriens and the pyramidal cell layer of regio superior and inferior, and in the outer plexiform layer and outer hilar cell layer of the dentate hilus. Terminal-like PV-immunoreactivity surrounded the cell bodies in the pyramidal cell layer of regio superior and inferior and in the dentate granular cell layer. The distribution of CaBP and PV in the pig hippocampus is compared to that of other more commonly used experimental animals. Whereas the distribution of PV-immunoreactivity in the pig hippocampus appears identical to that of the rat hippocampus, the distribution of CaBP-immunoreactivity in the pig hippocampus differs markedly from that of the rat hippocampus, the most prominent feature being a lack of CaBP-immunoreactivity in the granule cells, mossy fibers and pyramidal cells in the pig. The functional implications of calcium-binding proteins in the brain are discussed.

Histochemical demonstration of zinc in the hippocampal region of the domestic pig: I. Entorhinal area, parasubiculum, and presubiculum.

A detailed description is given of the distribution of zinc in three areas of the domestic pig hippocampal region, viz., the entorhinal area, the parasubiculum, and the presubiculum. Zinc was demonstrated histochemically with use of the Neo-Timm method, a recent modification of the sulphide silver procedure. Each of the studied areas showed a distinctly stratified staining pattern, which has been correlated in detail to fields and layers defined on the basis of cyto- and fibroarchitecture, providing a combined chemo- and cytoarchitectonic map. The staining was primarily confined to the neuropil, although stained nerve cell bodies were encountered in all three parts of the hippocampal region. Two main subfields were identified in the entorhinal area that have been designated pars medialis and pars lateralis, in accordance with their topographical positions, but both the cytoarchitecture and Neo-Timm staining pattern are indicative of further subdivision. In pars medialis, the deep half of layer I, the interstices between the stellate cell bodies in layer II, and layer III were medium to heavily stained, whereas layer IV stained weakly. Layers V-VI were slightly darker than layer IV and were inseparable on the basis of the Neo-Timm staining. The staining of pars lateralis differed in many respects from that of pars medialis, the most conspicuous feature being a much lighter layer III. In the parasubiculum, the deep half of layer I together with layers II-III had the appearance of an intensely stained triangle wedged in between the entorhinal area and the presubiculum. The latter showed moderate staining of the inner half of layer I and posterior part of layer II, while layer IV was stained intensely. Layers III and V-VI exhibited only weak staining. The distribution of zinc in the pig was compared with that in the guinea pig and rat, described previously. Although many histochemical features are shared by the staining patterns of the three species, striking differences exist in the pig, the most notable being the virtually reverse staining of the entorhinal layer IV. The possible functional implications of zinc in synaptic vesicles are considered.

Computerized analysis of the acetylcholinesterase distribution in the rabbit hippocampal region.

The distribution of acetylcholinesterase (AChE) was analysed in the hippocampal region of the rabbit, employing computerized optical densitometry (COD). AChE was demonstrated histochemically according to a modification of the copper thiocholine method, and computerized analysis was performed on a selected section with the use of a graphic operating processor, resulting in images with pixel values ranging between 0 and 255, measuring 512 X 512 pixels. Examples of the densitometric analysis include presentation of pixel values in single points, in profiles through selected areas and in slicing procedures. The results of the densitometric analysis basically agreed with the subjective visual impression of different staining intensities in the sections and corresponding photomicrographs. However, the densitometric analysis provided an objective and more exact expression of the relative AChE content of the different subfields and layers of the hippocampal region. In particular, zones with the same enzyme content as well as zones differing only minimally in activity can be recognized easily and unequivocally. In view of this, the promising future uses of COD are considered briefly.

Effect of indomethacin on regulation of juxta-articular bone blood-flow during joint tamponade. An experimental study in puppies.

Prostaglandins are vasoactive substances which are assumed to play a major role in bone metabolism and bone repair. The purpose of the present study was to investigate the effect of indomethacin on the control of epiphyseal bone blood-flow. By means of simultaneous intra-osseous pressure (IOP) and regional blood flow (RBF) measurements in the distal femoral epiphysis (DFE), aspects of vascular control mechanisms in the distal femoral epiphysis were investigated during knee joint tamponade (50% of mean arterial pressure) before and after administration of indomethacin 7.5 mg/kg. Six dogs aged 3-4 months were investigated in fentanyl anaesthesia. Knee joint tamponade resulted in a significant increase in IOP and calculated venous resistance in the DFE, while no significant changes in regional blood-flow or arterial resistance were encountered. Administration of indomethacin did not affect this reaction. The results suggests that indomethacin 7.5 mg/kg does not influence the regulation of epiphyseal blood-flow during elevation of joint pressure indicating that prostaglandins play only a minor or no role in this regulation.

99mTc-DPD uptake in juvenile hemarthrosis. Scintimetry and autoradiography of the knee in dogs.

The pathogenesis of subchondral bone lesions and growth plate affection in hemophilic arthropathy was studied in puppies by means of repeated regional 99mTc-diphosphonate scintimetry and contact autoradiography. Unilateral hemarthrosis of the knee was induced by biweekly intraarticular injections of autologous blood for 12 weeks. Hemarthrosis caused an early (2 to 4 weeks) decrease in uptake of 99mTc-diphosphonate in the juxtaarticular growth plates (ratio 0.7) and a delayed (8 to 10 weeks) increase in epiphyseal uptake (ratio 1.5). In a recovery phase after hemarthrosis, growth plate uptake returned to normal, while the epiphyseal uptake remained elevated for 8 to 10 weeks. By contact autoradiography, the growth plate uptake was localized to the calcification layer at the metaphyseal aspect of the growth plates, while the epiphyseal uptake mainly was seen in the thin subchondral and subsynovial bone layer and around osteophytes. The changes in uptake of 99mTc-diphosphonate following hemarthrosis for 3 months were reversible and could be ascribed to the presence of synovial inflammation.

Neo-Timm and selenium stainable glial cells of the rat telencephalon.

The Neo-Timm and selenium methods predominantly stain the neuropil of the rat brain and have been found to visualize zinc in synaptic vesicles. A fraction of glial cells and neuronal somata is also stained, especially when the Neo-Timm method is used. In the present study the localization and appearance of stained glial cells in the rat telencephalon are described using the two methods and the effect of metal chelating agents on the stained glial cells is examined. Neo-Timm stained glial cells were observed in both white and grey matter, with a preponderance in the major fiber tracts of the telencephalon, and were seen to contain rather large silver grains in their cytoplasm. Chelation with diethyldithiocarbamate (DEDTC) or dithizone prevented this staining. Brains from rats treated intravitally with selenium contained only occasionally stained glial cells. However, when present they showed the same characteristics as the Neo-Timm stained glial cells, including the reaction to chelation. Although both the Neo-Timm and selenium methods primarily visualize zinc in the neuropil of the rat brain, the possibility that copper could contribute to the glial cell staining cannot be ruled out. This possibility is further discussed.

Electron microscopic analysis of glial cells in the rat telencephalon stained with the Neo-Timm and selenium methods.

Two histochemical methods for visualization of zinc in synaptic vesicles, the Neo-Timm and selenium methods, have been shown to additionally stain glial cells and neuronal somata. In a previous light microscopic study the majority of stained glial cells were seen in the major fiber tracts of the rat telencephalon. The aim of the present study was to further characterize the stained glial cells with respect to glial cell type and ultrastructural localization of the silver grains responsible for the staining. Electron microscopic analysis of brains treated according to either method revealed that the vast majority of stained glial cells belonged to the dark oligodendroglial cell type. However, a smaller number of stained astrocytes was also seen, especially in the grey matter. The silver grains responsible for the staining were located in electron-dense rounded cytoplasmic organelles, suggestive of lysosomes.