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This study evaluated ¹⁸F-labeled IMPY [6-iodo-2-(4‘-N,N-dimethylamino)phenylimidazo[1,2-a]pyridine] derivatives as agents for imaging β-amyloid plaque with positron emission tomography (PET). The precursor for radiolabeling and reference compounds was synthesized in up to five steps from commercially accessible starting materials. One of the two N-methyl groups of IMPY was substituted with either a 3-fluoropropyl (FPM-IMPY) or a 2-fluoroethyl (FEM-IMPY) group. FPM-IMPY and FEM-IMPY were found to have moderate affinity for Aβ-aggregates with Ki = 27 ± 8 and 40 ± 5 nM, respectively. A “one-pot” method for ¹⁸F-2-fluoroethylation and ¹⁸F-3-fluoropropylation of the precursor was developed. The overall decay-corrected radiochemical yields were 26−51%. In PET experiments with normal mouse, high uptake of activity was obtained in the brain after iv injection of each probe:  6.4% ID/g for [¹⁸F]FEM-IMPY at 1.2 min, and 5.7% ID/g for [¹⁸F]FPM-IMPY at 0.8 min. These values were similar to those of [¹²³I/¹²⁵I]IMPY (7.2% ID/g at 2 min). Polar and nonpolar radioactive metabolites were observed in both plasma and brain homogenates after injection of [¹⁸F]FEM or [¹⁸F]FPM-IMPY. In contrast to the single-exponential washout of [¹²³I/¹²⁵I]IMPY, the washouts of brain activity for the two fluorinated analogues were biphasic, with an initial rapid phase over 20 min and a subsequent much slower phase. Residual brain activity at 2 h, which may represent polar metabolites trapped in the brain, was 4.5% ID/g for [¹⁸F]FEM-IMPY and 2.1% ID/g for [¹⁸F]FPM-IMPY. Substantial skull uptake of [¹⁸F]fluoride was also clearly observed. With a view to slow the metabolism of [¹⁸F]FEM-IMPY, an analogue was prepared with deuteriums substituted for the four ethyl hydrogens. However, D4-[¹⁸F]FEM-IMPY showed the same brain uptake and clearance as the protio analogue. Metabolism of the [¹⁸F]FEM-IMPY was appreciably slower in rhesus monkey than in mouse. Autoradiography of postmortem brain sections of human Alzheimer's disease patients with [¹⁸F]FEM-IMPY showed high displaceable uptake in gray matter and low nonspecific binding in the white matter. This study demonstrates that the IMPY derivatives have favorable in vivo brain pharmacokinetics and a moderate affinity for imaging β-amyloid plaques; however, further improvements are needed to reduce radioactive metabolites, increase binding affinity, and reduce lipophilicity.

A rabbit antiserum against mouse neonatal brain cell surface membranes labeled by immunoperoxidase (PAP) the cells of the central and peripheral nervous systems of adult and neonatal mice and their processes, as well as the differentiating neuroepithelial cells of three OTT-6050 mouse teratoma-derived tumors. Indirect immunofluorescence on living 14-day-old monolayer cultures of neonatal mouse brain demonstrated reaction of the immune serum with external surface membrane antigens of neuroblasts and of primitive and mature glial cells. Radioimmune assays (RIA) showed almost complete loss of antiserum binding to neonatal mouse brain plasma membranes after absorption with adult or neonatal mouse brain membranes, and no loss of binding after absorption by liver, spleen, kidney, and heart membranes.

Summary Neurotensin, an endogenous peptide and putative neurotransmitter, exhibits a wide range of interactions with dopaminergic neurons and displays some actions akin to neuroleptics. Moreover, neurotensin receptors are abundant in specific layers of the entorhinal cortex where cytoarchitectural abnormalites have been reported in schizophrenia. We therefore examined the entorhinal cortex from postmortem specimens of five control patients and six schizophrenic patients for alterations in neurotensin receptor quantitation and distribution using receptor autoradiography.

An expansion of the CAG triplet in the human gene called atrophin-1 or CTG-B37 causes the neuropsychiatric disease known as dentatorubral-pallidoluysian atrophy (DRPLA). We have examined the genomic DNA from brains and peripheral blood leukocytes (PBLs) of schizophrenic and non-schizophrenic individuals and PBLs from a cohort of monozygotic twins discordant or concordant for schizophrenia, for expansions of the CTG-B37 region. All samples, including non-schizophrenic/normal controls, had 7–22 CAG repeats in this region. Thus we found no evidence for an expansion of the DRPLA triplet repeat associated with schizophrenia when compared to controls. Our data suggest that an expansion in the CTG-B37 gene is not linked to or responsible for the disease schizophrenia.

The toxic effects of inorganic lead feedings on the immature brain were studied in the rat pup. Beginning when litters were two weeks old, PbCO3 was fed to nursing mothers and then to pups directly after weaning. Results in lead-fed pups were compared to age-matched controls and to lead-fed young adult males (60 days old). Anaemia and growth failure developed in both pups and adults. In the second week, more than half the pups developed an encephalopathy, with haemorrhage and oedema predominately in the cerebellum and lead-containing densities in the cerebellar molecular layer. The latter were confirmed by X-ray microanalysis. No lead-fed adults showed signs of an encephalopathy.

Using a four-step immunoperoxidase (PAP) method and the monoclonal antibody MAbA9-C6 (MAbA9-C6), which defines an epitope of the retinal S-antigen (S-Ag), we investigated the S-Ag immunoreactivity in human fetal, newborn, infantile and adult pineal glands and in 13 human pineal parenchymal tumors. S-Ag immunoreactivity was demonstrated in a few cells in one of the four fetal and in both infantile glands. Eight of nine adult pineal glands contained isolated MAbA9-C6-positive cells. In two of seven pineocytomas showing neuronal or gangliogliomatous differentiation a few scattered cells displayed S-Ag positivity; two of four pineoblastomas contained small groups of strongly immunoreactive neoplastic cells; two malignant pineocytomas did not demonstrate any S-Ag immunoreactivity. Our results indicate that isolated cells in human pineal gland retain some of the cytochemical characteristics of photoreceptor cells recognized by the MAbA9-C6, and that S-Ag immunoreactivity may be occasionally expressed in pineal parenchymal tumors.

Five human glioblastomas maintained in an organ culture system were studied by autoradiography to determine, after 8 days in vitro, the growth fraction (GF) of the explants, their total cell cycle time (TC) and cell cycle phase durations (TS,TG1,TG2 andTM), and their potential doubling time (Tpot) after pulse-labeling with [3H] TdR for 1 h. These parameters were derived from computer analysis of fraction of labeled mitoses (FLM) curves. The results fell into two groups.

Mouse neonatal brain cell fractions enriched for surface membranes were used as immunogens to produce a heterologous immune serum. Following absorption to remove non-neural anti-mouse activity, this serum demonstrated by microcomplement fixation an anti-brain activity that was completely removed by absorption with neonatal mouse brain or with solid tumors of the mouse transplantable teratoma OTT-6050. Indirect immunofluorescence applied to living monolayer cultures of differentiating teratoma embryoid bodies showed the absorbed serum's reaction with neural cell surfaces only. In material studied with frozen sections, the absorbed serum recognized antigenic sites in all examined areas of both neonatal and adult mouse brain, and only within neuroepithelial cell populations of solid transplants of the teratoma.

While the embryonal central neuroepithelial tumors present complex conceptual and clinical problems, advances in cell type identification by special neurohistological, immunohisto- and immunocytochemical techniques have permitted discrimination of distinct cytomorphogenetic entities. These are based in part on their resemblance to the normal phases of neurocytogenesis. Four of these tumors, medulloepithelioma, desmoplastic infantile ganglioglioma, pineoblastoma and medulloblastoma, are designated as multipotential in light of their capacity to undergo divergent differentiation. Cytomorphogenetic, clinical and experimental data implicate fetal neural cell targets for transformation and raise the possibility that aberrant developmental regulatory mechanisms may contribute to the biologic behavior of these tumors. Growth factors and some neuroregulatory neurotransmitters (such as serotonin) are known to act as modulators of normal neuromorphogenesis. They could play a regulatory role in central neuroepithelial tumors on the hypothesis that the aberrant behavior of the embryonal neoplasms could either be modified by fuctional receptor responses or result from abnormal receptor responses to these substances. Future challenges include 1) the definition of new cytomorphogenetic entities and subgroups of the currently defined forms of embryonal CNS tumors based on the presence of specific growth factors and neuroregulatory neurotransmitters, or their receptors, 2) the characterization of neoplastic receptor responses mediating any modulatory role of the presently known growth factors or neuroregulatory neurotransmitters on the growth and maturation potential of the embryonal central neuroepithelial tumors and 3) the further definition of developmental, stage-specific modulators that might be operative in these tumors.

Cerebrovascular amyloid is the main constituent of the perivascular and neuritic plaques typical of Alzheimer disease, whereas neurofilaments and microtubule-associated tau protein have been considered primary contributors to the formation of the characteristic Alzheimer tangles. Plaques and tangles and their constituents have at times been ascribed a role in pathogenesis of the disease. Normally, neurofilaments become phosphorylated only upon axonal entry. In many neurologic disorders, neurofilament phosphorylation, as detected by any of the available monoclonal antibodies (mAbs) to neurofilament phosphorylated epitopes is shifted from an axonal to a cell-body location. An exception is provided by Alzheimer disease, where tangles (which are neuronal cell-body-derived structures) exhibit only one phosphorylated epitope. However, the very presence of neurofilaments in tangles and plaques has been questioned because of a reported cross-reaction of mAbs to phosphorylated neurofilaments with tau protein. On reinvestigating this cross-reactivity we found that four of five mAbs to phosphorylated neurofilaments and four of five mAbs to nonphosphorylated neurofilaments failed to react with tau protein. A fifth mAb (07-5) to phosphorylated neurofilament cross-reacted with partially denatured tau protein at an affinity 1/1700th of that for denatured neurofilaments; nondenatured tau protein in tissue sections did not cross-react. A fifth mAb (02-40) to nonphosphorylated neurofilament also cross-reacted weakly. In Alzheimer disease normal-appearing axons were revealed with all the mAbs to phosphorylated neurofilaments, but tangles were revealed with only one of them (mAb 07-5). mAb to tau protein did not stain or did so indistinctly. Four of five mAbs to nonphosphorylated neurofilaments failed to reveal axons. Upon dephosphorylation of tissue, staining by mAbs to phosphorylated neurofilaments disappeared, and axons were revealed with the mAb to tau protein and all mAbs to the nonphosphorylated neurofilaments. Tangles became stained with tau mAb and one mAb to the nonphosphorylated neurofilaments (mAb 10-1). Quantitative evaluation of immunocytochemical staining intensities and immunoblot cross-reactivity showed that neurofilaments are, indeed, constituents of tangles--apparently exceeding the concentration of tau protein 17-fold. Contribution of both conformation and primary structure to IgG specificity may explain the lack of any cross-reaction of mAbs to neurofilaments with tau protein in intact tissue and the appearance of cross-reaction in immunoblots where conformation specificity may be largely lost. The present data extend earlier findings of abnormal processing of neurofilaments and tau protein in Alzheimer disease and, together with reported abnormal processing of cerebrovascular amyloid beta-protein, suggest that inhibition of the processing of multiple proteins is basic to the pathogenesis of Alzheimer disease, whereas formation of plaques and tangles could be merely the most striking histologic result.

The cellular distribution and intracellular localization of neuron-specific enolase (NSE) has been studied by electron microscopic immunocytochemistry in the brain of the rat and of the mouse. Although the intensity of staining was less in the mouse, the same structures were positive in both species. In the cerebrum, the neuronal perikarya and dendrites were intensely stained, but staining was almost entirely absent in the presynaptic terminals. The deep neurons of the brain stem were also positive. In the cerebellum, perikarya, axons, and parallel fibers of the granule cell neurons were stained as were the synaptic vesicles and presynaptic membranes of the synapses between the parallel fibers and the Purkinje cell dendrites. Golgi cell dendrites, basket cells and their axons, and mossy fibers were also positive. In contrast, the Purkinje cells including their dendrites, and the climbing fibers that formed synapses with the Purkinje cell dendrites were not stained. The majority of the myelinated axons in both the cerebrum and the cerebellum did not stain, but the fibrillary astrocytic processes between myelinated axons in the white matter did. Oligodendroglia, protoplasmic astrocytes, Bergmann glia, astrocytes investing capillaries, and vascular endothelial cells were negative for reaction product. In the positively staining cells and their processes, the positivity was dispersed throughout the cytoplasm and corresponded most closely to the distribution of ribosomes, the granular endoplasmic reticulum, and microtubules. Nuclei, mitochondria, the cisternae of the Golgi complex, myelin lamellae, and most membranes were not stained.

The distribution of lithium in the rat brain following chronic i.p. injections of lithium chloride has been determined by a radiographic dielectric track registration technique. The study indicated significant differences in lithium distribution in various areas of the brain. Consistently high levels were observed in the neocortex and putamen, and low concentrations in the cerebellum and white matter structures such as the corpus callosum and basis pedunculi. Intermediate levels were found in the thalamus, hypothalamus, septal nuclei, dentate gyrus, hippocampus and substantia grisea centralis.

Laminin immunoreactivity is thought to be masked in formalin-fixed sections since proteolytic treatment is required to unmask it. We analyzed this masking with frozen and formalin-fixed human autopsy brains obtained at various postmortem periods. In unfixed, frozen sections, intense immunoreactivity was invariably detected in vascular walls of entire sections. When such sections were postfixed in formalin, immunoreactivity was not diminished even after prolonged fixation. In vibratome sections of brain fixed in formalin in situ, immunoreactivity varied with postmortem delay: in most cases, immunoreactivity was weak and restricted to superficial cortical layers. However, the extent of immunoreactivity increased with postmortem delay. Two cases fixed after prolonged postmortem periods revealed moderate immunoreactivity throughout the sections. We also investigated rat brains processed without postmortem delay. In unfixed frozen sections, immunoreactivity again was observed throughout the sections, independent of the length of any postfixation. In vibratome sections of fixed rat brain, immunoreactivity was restricted to the cutting margins of the brain blocks and around a trauma-induced cortical lesion, regardless of how long the blocks had been kept in fixative. Our data suggest that postmortem proteolysis accomplishes similar unmasking of laminin antigen as digestion on paraffin sections and that such unmasking can also be effected by proteolysis induced by damaging tissue during cryostat sectioning of fresh tissue.

Four examples of human cerebral medulloepithelioma were studied immunohistochemically with a panel of antibodies and antisera to neuronal and glial proteins. The tumors, in addition to primitive medullary epithelium, contained areas of neuroblastic, ganglionic, astrocytic, ependymoblastic and ependymal differentiation, and, in one tumor, areas resembling polar spongioblastoma. Tumor cells throughout the primitive medullary epithelium displayed focal immunocreactivity for vimentin, glial fibrillary acidic (GFA) protein and for the neuron-associated class III ß-tubulin isotype. Neuroblasts showed immunoreactivity for the class III ß-tubulin isotype, microtubule-associated protein 2 and neuron-specific enolase. Immunoreactivity for neurofilament epitopes and synaptophysin was detected in areas of ganglionic differentiation and coincided with the demonstration of neurofibrils in Bielschowsky's silver impregnations. Vimentin was the only marker detected in ependymoblastic and ependymal rosettes or in areas of polar spongioblastoma, as well as in mesenchymal, cells. The results indicate that, even in very primitive neoplastic neuroepithelium, immunocytochemical evidence of early commitment of some of the cells to a neuronal or glial lineage can be demonstrated. The neuron-associated class III ß-tubulin isotype appears to be one of the earliest markers indicative of neuronal differentiation in normal and neoplastic primitive neuroepithelium.

Two examples of cerebral astroblastoma have been studied by electron microscopy and immunohistochemistry, one of them having been maintained in vitro in an organ-culture matrix system for 8 months and the explants studied by light and electron microscopy at different time intervals. The fine structural characteristics were those of a glial cell type with features intermediary between those of astrocytes and ependymocytes. They recapitulated the structure of the tanycyte, a glial precursor cell which is normally found scattered along the ependymal lining of the embryonal and neonatal mammalian brain, but is distinct from epithelial ependymocytes. The possible origin of some astroblastomas from such a cell would account for a number of characteristics in this enigmatic type of glioma.

The entorhinal cortex (ERC) has been implicated in schizophrenia by a number of studies. There is anatomical observation of neuronal heterotopias in the rostral ERC, which is consistent with a hypothesis of neurodevelopmental abnormalities in this disease. In view of the significant cytoarchitectonic variation of the ERC throughout its rostro-caudal extent, we performed a detailed subareal analysis of the rostral two-thirds of the entorhinal cortex (ERCr) in 14 postmortem schizophrenic brains and 14 matched controls (mean ages of 48 and 47 respectively). This systematic evaluation included both a qualitative microscopic analysis of morphogenetic anomalies that would be consistent with neurodevelopmental pathology and quantitative measurements of total neuronal number, average neuronal density, laminar volume and laminar depth from the cortical surface in cytoarchitectonically matched subareas of schizophrenic and control brains. Parcellation of the entire ERC on the basis of cytoarchitectonic criteria identified five distinct regions, similar to those described in the macaque, except that in the human brain three of the regions were further divisible into two or three subareas, yielding nine distinct cellular compartments. Five rostral areas, prorhinal (Pr), lateral (28L), intermediate rostral and caudal (281r and 281c), and sulcal (28S), comprise the ERCr. Gross and microscopic examination of these subdivisions throughout the ERCr failed to reveal laminar disorganization in any of the schizophrenic brains. The brains also did not differ significantly with respect to total neuronal number, total volume and neuronal density per laminar and subareal subdivision, or laminar thickness per entorhinal subarea. However, neuronal number and density were reduced by 12-18% in Pr and 28L, suggesting that mild quantitative abnormalities may exist in the ERCr and might possibly be revealed in a larger sample of schizophrenic brains. We have failed to confirm previous reports of laminar disorganization in the ERCr in brains of patients with schizophrenia; to the extent that this region is implicated in schizophrenia, the structural changes are likely to consist of more subtle cellular disturbances.

The entorhinal cortex (ERC) has been implicated in the pathophysiology of Alzheimer's disease, schizophrenia and other disorders affecting cognitive functions. While powerful anatomical and histochemical methods (immunohistochemistry, in situ hybridization, etc.) may be applied (although with limitations) to postmortem human brain, each analysis should utilize a cytoarchitectonic approach to provide appropriate comparisons within the subdivisions of the ERC. Accordingly, we describe here the normal cyto- and myeloarchitecture of the human ERC as a prerequisite for the accompanying study of this region in schizophrenia. Our parcellation of this cortex differs from previous treatments in three ways. First, we adopted specific criteria of inclusion to define each subdivision of the region. Although distinctive ERC features are most prominent in the intermediate portion of this region, at least one of these features was considered the minimum necessary criterion to include adjacent tissue in the entorhinal area. Second, we used morphometric measurements (neuronal size and density as well as subdivisional volume and laminar thickness) to support our qualitative evaluation. Third, we have applied to the human ERC the conventional cytoarchitectonic nomenclature of the entorhinal cortex used previously in studies of non-human primates. This allows a more accurate extrapolation of the available numerous experimental anatomical, physiological and psychological data on this region to the human. As in the monkey, the five main subareas were recognized in the human (prorhinal, lateral, intermediate, sulcal and medial) but three required further subdivision (intermediate, sulcal and medial). The morphometric results obtained suggested a progression of the human entorhinal cortex from the peripheral to the central subareas, with the intermediate subarea (281) as the most complete entorhinal subdivision. Compared with non-human primates, the human ERC not only retains the basic periallocortical organization but also demonstrates further evolution. Taken together with available experimental data on the connectivity of this brain region, these results provide an anatomical basis for evaluating the ERC in human behavior.

AMONG neural cell lines the C-6 glioma, a cell line cloned from an N-methylnitrosourea (MNU)-induced experimental glioma in rat, has generated considerable interest as a model system for the study of neuroglia1. The clone was originally selected on the basis of a high production of the protein S-100, a protein believed to be characteristic of glial cells in vivo2. Since its introduction, the line has been further characterised with regard to a number of other biochemical features3. Among these is the recent demonstration of the glial fibrillary acidic (GFA) protein by immunofluorescent staining of C-6 cells maintained in certain in vitro conditions4. The GFA protein, isolated from multiple sclerosis plaques and areas of sub-ependymal gliosis in the central nervous system, has been shown to be a specific marker for fibrillary astrocytes, is believed to be a component of the 80–90 Å diameter glial filaments5–8, and has not been reported in non-glial neural cell lines. It is, therefore, a useful biochemical marker for the differentiation of a type of mature astrocyte. In previous work with the C-6 line, GFA protein was found to be present by immunofluorescence in only an occasional C-6 cell maintained in monolayer or suspension cultures, and to be markedly increased in many C-6 cells when they were maintained for 12–14 d in an organ culture system using Gelfoam matrices4. Such a system is known to favour structural differentiation in vitro9. No increase of filaments was observed, however, in any of the in vitro systems used, suggesting that the synthesis of GFA protein could proceed without the formation of intracytoplasmic glial filaments4. We compared the quantitative levels of GFA protein in C-6 and other cell types in various culture conditions, using a “two-site” immunoradiometric assay recently developed in this laboratory10.

Transformation of tobacco leaf discs with the ‘cytokinin’ ipt gene yielded several transgenic callus tissue lines, respective to the kind of ipt construction present in the A. tumefaciens cointegrates. Those calli containing an active ipt gene were able to grow hormone-autotrophically and showed an increased endogenous cytokinin level in comparison with controls. Analysis of endogenous IAA level did not allow any quantitative correlation with the cytokinin content. However, a minimal level of auxin seems to be necessary to obtain hormone-autotrophic growth. Exogenously supplied NAA significantly reduced the endogenous cytokinin content without modifying growth characteristics. The varying chlorophyll content in the different callus lines elicited the study of the ultrastructure of the plastids. The controls contained small plastids, often filled with starch or accumulated vesicles that did not allow observation of the internal membrane system. The ‘Pssu-ipt’ line, having a higher cytokinin content, showed plastids with an internal membrane system consisting of stroma and grana thylakoids, but this structure was lost during subculture. Swollen thylakoids appeared, the amount of starch was reduced and vesicles were accumulating.

The effect of introducing a supplementary ipt-gene into the genome of Nicotiona tabacum L. cv. Petit Havana SR 1 is studied on the morphological plant development. The ipt-gene, accounting for the biosynthesis of cytokinins, was coupled to the heat-inducible hsp70- promoter from Drosophila melanogaster. Besides the influence of the hormonal changes involved, the effects of the experimental conditions are examined, namely the in vitro growth conditions for selecting transformed plants and the heat treatment to induce ipt-gene expression. The phenotype of the plants is determined by the tissue sensitivity to three factors: (1) heat treatment reduces stem elongation and diameter growth; (2) in vitro pre-cultivation also reduces stem elongation; and (3) expression of the ipt-gene stimulates diameter growth, induces debudding of the axillary shoots and inhibits root development. In addition, axillary bud development indicates that in vitro cultivation affects ipt-gene expression.