Role of growth factors and estrogen as modulators of growth, differentiation, and expression of gonadotropin subunit genes in primary cultured sheep pituitary cells.

Although the pituitary is known to produce several growth factors, their effects on pituitary cell growth and differentiation are still unclear, particularly in normal tissue. Using primary cultures of aged ewe pituitaries cultured in serum-free conditions, we studied the effects of basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), transforming growth factor-beta (TGF beta 1), insulin, and 17 beta-estradiol (E2) on the growth, differentiation, and expression of gonadotropin subunit genes. After 72-h incubation of the monolayer (day 5) with optimal concentrations of each factor, [3H]thymidine incorporation was increased significantly (P < 0.01) over the control values by 33 +/- 8% (mean +/- SEM; n = 3; 10 nM E2), 36 +/- 10% (1 ng/ml TGF beta 1), 83 +/- 12% (10 ng/ml bFGF), and 118 +/- 12% (1 nM EGF). Insulin showed a two-phase dose-response curve, increasing [3H]thymidine uptake by 34 +/- 9% at 10 ng/ml and by 63 +/- 13% at 10 micrograms/ml. Cell counting using a Coulter counter confirmed these results. Characterization of cell types by immunocytochemistry (avidin-biotin-peroxidase complex technique) revealed that the cell cultures were predominantly gonadotrophs. However, the cultures contained cells that did not stain with any specific ovine or human antiserum against LH beta, FSH, TSH beta, PRL, GH, ACTH, or glial fibrillary acidic protein, but were of epithelial cell lineage, as shown by positive keratin staining. Treatment with EGF and bFGF increased the proportion of these undifferentiated pituitary cells and induced changes in their morphology to large cuboidal cells containing large nuclei. After treatment with E2, insulin, and TGF beta 1, pituitary cells remained differentiated, although with E2, staining for gonadotrophs was much reduced. Northern blot analysis revealed that E2 treatment for 0-48 h progressively reduced the mRNA for FSH beta (16 +/- 4.5% of control values) and LH beta (12.4 +/- 2.5%), but had little effect on the common alpha-subunit (88.4 +/- 4.6%). TGF beta 1, however, stimulated the expression of FSH beta subunit gene by 142 +/- 4.6% (P < 0.01) of the control value, but had no significant effect on LH beta and common alpha-subunit genes. Insulin, EGF, and bFGF showed no significant effect on the expression of these three subunit genes. The data define the direct effects of growth factors and E2 on the growth and differentiation of normal sheep pituitary cells and gonadotrophs in particular, which may be of relevance to the pathophysiology of the pituitary and in the multistage process of pituitary tumorigenesis.

The role of magnetic resonance imaging in the diagnosis of Alzheimer disease in adults with Down syndrome.

OBJECTIVE: To correlate findings from magnetic resonance imaging (MRI) with neuropathological analysis and clinical assessment of Alzheimer disease in patients with Down syndrome. DESIGN AND METHODS: Case study of 1 elderly man with trisomy 21 and Alzheimer disease who had been followed up prospectively over a 5-year period. The patient was a resident in a supervised community unit and died with end-stage dementia. The MRI changes were correlated with the results of clinical psychopathological analysis and neuropathological brain tissue findings. To our knowledge, this is the first case in which clinical, MRI, and neuropathological data were available in a case involving an elderly patient with Down syndrome and Alzheimer disease. RESULTS: The MRI findings correlated with the clinical deterioration and neuropathological features of Alzheimer disease. Marked changes in temporal and hippocampal regions were found. CONCLUSION: Magnetic resonance imaging is potentially a valuable tool in the diagnosis of Alzheimer disease in adults with Down syndrome, particularly in individuals for whom standard intellectual assessments are not possible.

Primary intracranial Hodgkin's disease. A case report and discussion.

We report here a case in which a frontotemporal meningeal tumor was found to be an isolated, apparently primary, deposit of nodular sclerosing Hodgkin's disease in an otherwise well man. The presentation of Hodgkin's disease as a primary, solitary intracranial lesion is rare; only 13 cases have been found in the world literature. The histological appearance of the tumor in the present case fulfills all the requirements for nodular sclerosing Hodgkin's disease according to current criteria of classification; the morphologic diagnosis is supported by appropriate immunohistochemical staining of the neoplastic cells for Leu M1 and LN2 markers, with absence of staining for the common leucocyte antigen. No recurrent local or disseminated disease has been manifest in a 14-month follow-up of the patient.

Distribution and characterization of the [3H]granisetron-labelled 5-HT3 receptor in the human forebrain.

The present study has demonstrated the distribution of [3H]granisetron-labelled 5-HT3 receptors in the human forebrain with relatively high levels of this receptor in homogenates of hippocampus, caudate nucleus, putamen, nucleus accumbens and amygdala. Lower levels of 5-HT3 receptors were found in other brain regions and the cervical vagus nerve. Pharmacological characterization of the labelled 5-HT3 receptor in human putamen homogenates identified a relatively low affinity for d-tubocurarine compared to the 5-HT3 receptor in NG108-15 neuroblastoma-glioma cell homogenates. In contrast, the affinities of 19 other 5-HT3 receptor ligands were not significantly different for the [3H]granisetron-labelled receptor in these two preparations. Such findings indicate that the human putamen 5-HT3 receptor displays a unique pharmacology which may have significance given the reported clinical potential of compounds active at this receptor when assessed in animal models of disease.

Ulex europeus agglutinin I binds exclusively to primary olfactory neurons in the rat nervous system.

Lectin binding histochemistry was used to investigate the distribution of binding sites for the fucose-selective lectin Ulex europeus agglutinin I in the rat nervous system. It was found that this lectin bound exclusively to a surface membrane component, probably a glycoprotein, on primary olfactory and vomeronasal sensory neurons, and to no other structure in the nervous system. A similar pattern of Ulex europeus agglutinin I binding was found in adult, immature and embryonic rats. Binding was also demonstrated on olfactory axons regenerating through a peripheral nerve graft. Three other lectins, including another fucose-selective lectin, and the N-acetyl galactosamine-binding soybean lectin failed to show similar exclusive binding. The presence of a specific surface glycoconjugate solely on olfactory neurons suggests that the molecule may play some role in development or maintenance of organization of this neuronal pathway.

Regeneration of olfactory sensory axons into transplanted segments of peripheral nerve.

Isolated segments of cervical sympathetic trunk were transplanted onto the olfactory mucosa and bulb in adult rats, and olfactory nerve fascicles were sectioned in the region of the transplant. Olfactory axons, presumably arising from newly-formed sensory neurons, were observed to grow into the transplanted segments of peripheral nerve and were ensheathed by Schwann cells of the transplant. The axons were ensheathed as large bundles containing many axons in direct contact, an arrangement characteristic of the normal olfactory nerves, and not of the normal sympathetic trunk. However, single Schwann cell 'units' were each surrounded by basal lamina, an arrangement typical of the sympathetic trunk, not of olfactory nerves. The results indicate that olfactory axons, like other peripheral axons, are capable of directing some aspects of the manner in which they are ensheathed by Schwann cells.

Schwann cells of the olfactory nerves contain glial fibrillary acidic protein and resemble astrocytes.

Two antisera to glial fibrillary acidic protein from human brain and an antiserum to a 49 k dalton glial filament protein from human brain detected a cross-reacting antigen in the Schwann cells of the olfactory and vomeronasal nerves. The antigen was demonstrated at light- and electron-microscope levels. It was found throughout the cytoplasm and in association with cytoplasmic filaments of olfactory nerve Schwann cells in intact tissue and in Schwann cells grown in vitro. This observation, together with observations on the ultrastructure of olfactory nerve Schwann cells, relates them to central astroglia and to glial cells of the myenteric plexus, rather than to Schwann cells of other peripheral nerves. The unusual properties of olfactory nerve Schwann cells are of interest in relation to the regenerative abilities of the olfactory nerves.

Differentiation of neurons containing olfactory marker protein in adult rat olfactory epithelium transplanted to the anterior chamber of the eye.

Olfactory marker protein is a cytoplasmic component unique to fully-differentiated olfactory sensory neurons. It has been proposed that expression of this protein occurs only if the neurons make synaptic contact with the central nervous system. In the present experiments, adult olfactory epithelium was transplanted as an autograft to the anterior chamber of the eye. This procedure destroys the mature sensory neurons, which are subsequently replaced by division and differentiation of stem cells. The newly-formed sensory neurons differentiate sufficiently to produce olfactory marker protein, without apparently contacting central nervous tissue. We conclude that contact with the central nervous system is not necessary for expression of olfactory marker protein.

Fixation of Thy-1 in nervous tissue for immunohistochemistry: a quantitative assessment of the effect of different fixation conditions upon retention of antigenicity and the cross-linking of Thy-1.

It has proved difficult to obtain good immunohistochemical localization of cell surface antigens in nerve for a number of reasons, prominent among which are problems of fixing this class of molecule without destroying their antigenicity. In the course of developing a fixation procedure suitable for one such antigen. Thy-1, we have quantitatively assessed the effect of different fixation parameters upon the retention of Thy-1 antigenicity and upon the extent of cross-linking of the antigen in the tissue. The former was measured using radioimmunoassays adapted for membrane antigens in fixed tissue, the latter by measuring the proportion of antigen rendered insoluble to the detergent, sodium deoxycholate, and by examining the size of the antigen on sodium dodecyl sulfate--polyacrylamide gels. These approaches demonstrated that minor modifications of the standard vascular perfusion fixation of brain, using both glutaraldehyde and paraformaldehyde, were sufficient to fix the Thy-1 molecule, and at the same time substantially spare its antigenicity. In this study we measured Thy-1 using both a conventional rabbit antiserum and a mouse monoclonal antibody to the Thy-1.1 antigenic determinant. The multiple antigenic determinants recognized by the rabbit antibodies were cumulatively more resistant to fixation than the single antigenic determinant recognized by the monoclonal antibody.

Evidence that the atypical 5-HT3 receptor ligand, [3H]-BRL46470, labels additional 5-HT3 binding sites compared to [3H]-granisetron.

1. The radioligand binding characteristics of the 3H-derivative of the novel 5-HT3 receptor antagonist BRL46470 were investigated and directly compared to the well characterized 5-HT3 receptor radioligand [3H]-granisetron, in tissue homogenates prepared from rat cerebral cortex/hippocampus, rat ileum, NG108-15 cells, HEK-5-HT3As cells and human putamen. 2. In rat cerebral cortex/hippocampus, rat ileum, NG108-15 cell and HEK-5-HT3As cell homogenates, [3H]-BRL46470 bound with high affinity (Kd (nM): 1.57 +/- 0.18, 2.49 +/- 0.30, 1.84 +/- 0.27, 3.46 +/- 0.36, respectively; mean +/- s.e. mean, n = 3-4) to an apparently homogeneous saturable population of sites (Bmax (fmol mg-1 protein): 102 +/- 16, 44 +/- 4, 968 +/- 32 and 2055 +/- 105, respectively; mean +/- s.e. mean, n = 3-4) but failed to display specific binding in human putamen homogenates. 3. In the same homogenates of rat cerebral cortex/hippocampus, rat ileum, NG108-15 cells, HEK-5-HT3As cells and human putamen as used for the [3H]-BRL46470 studies, [3H]-granisetron also bound with high affinity (Kd (nM): 1.55 +/- 0.61, 2.31 +/- 0.44, 1.89 +/- 0.36, 2.03 +/- 0.42 and 6.46 +/- 2.58 respectively; mean +/- s.e. mean, n = 3-4) to an apparently homogeneous saturable population of sites (Bmax (fmol mg-1 protein): 39 +/- 4, 20 +/- 2, 521 +/- 47, 870 +/- 69 and 18 +/- 2, respectively; mean +/- s.e. mean, n = 3-4). 4. Competition studies with a range of structurally different 5-HT3 receptor ligands indicated that in both rat cerebral cortex/hippocampus and rat ileum homogenates, [3H]-BRL46470 binding exhibited a pharmacological profile consistent with the labelling the 5-HT3 receptor with compounds competing with Hill coefficients close to unity.5 In HEK-5-HT3As cell homogenates, [3H]-BRL46470 and [3H]-granisetron associated rapidly((3.84+/-0.4)106 M-1S-1 and (5.85+/-0.2)106 M-1S-1, respectively, mean+/-s.e.mean, n=3-4) in an apparently monophasic manner. Following the establishment of equilibrium, both [3H]-BRL46470 and [3H]-granisetron at a saturating concentration ([3H]-BRL46470 approximately 16 nM; [3H]-granisetron approximately 18 nM) and at a sub-Kd concentration (approximately 1 nm for both radioligands)dissociated biphasically in HEK-5-HT3As cell homogenates (saturating concentration; [3H]-BRL464704.05 x 10-3+/-2.53 x I0-3 s-1 and 5.83 x 10-5+0.91 x I0-5 s-1; [3H]-granisetron 3.20 x 10-3+ 1.70 x IO-3 s-1 and18.58 x 10-5 +/- 4.19 x I0-5 s-1: sub-Kd concentration; [3H]-BRL46470 2.47 x 10-3+/- 1.18 x 10-3 s-1 and 9.30x 10-5+/-2.59x 10-5 S-1; [3H]-granisetron 65.91 x 10-3+/-22.14x I0-3 s-1 and 49.96x 10-5+/-12.26x 10-5s- 1 mean+/- s.e.mean, n = 4-8) when induced by a 300 fold dilution in ice-cold Tris/Krebs.6 In conclusion, the present study provides evidence that [3H]-BRL46470 specifically labels the 5-HT3receptor in rat cerebral cortex/hippocampus, rat ileum, NG108-15 cell and HEK-5-HT3As cell homogenates, but fails to label the 5-HT3 receptor expressed in human putamen. Whilst the pharmacological profile of the site labelled by [3H]-BRL46470 is directly comparable to that labelled by [3H]-granisetron, [3H]-BRL46470 consistently labelled approximately twice the density of sites compared to [3H]-granisetron in the same tissue homogenates prepared from rat cortex/hippocampus, ratileum, NG108-15 cells and HEK-5-HT3As cells.

Identification of angiotensin II receptor subtypes in human brain.

The present study assessed the binding characteristics of [125I]angiotensin II to slices of human cerebellum adhered to glass slides using quantitative receptor autoradiography. Specific [125I]angiotensin II binding, defined by the inclusion of unlabelled angiotensin II (1.0 microM), was detected in the molecular layer of the cerebellum (0.09 +/- 0.02 fmol/mg tissue equivalent, mean +/- s.e.m., n = 3). The angiotensin II-2 receptor subtype selective ligand, PD123177, competed for approximately 65% of the specific binding in the molecular layer whilst the remainder of the specific binding was displaced by the angiotensin II-1 receptor subtype selective ligand, DuP753. It is concluded that angiotensin II receptor subtypes exist in human brain tissue and provide potential therapeutic sites of action.

Adjacent laminar terminations of two centrifugal afferent pathways to the accessory olfactory bulb in the mouse.

Anterograde and retrograde axonal tracing methods have been combined with transection of the stria terminalis to investigate the centrifugal afferent connections of the accessory olfactory bulb in the mouse. Injection of tritiated proline into the postero-medial cortical amygdaloid nucleus (C3) gives rise to anterograde autoradiographic labelling of a pathway terminating in the internal granular layer of the accessory olfactory bulb (AOB). Transection of the ipsilateral stria terminalis completely abolishes labelling of this pathway. Injections further rostral, in the bed nucleus of the accessory olfactory tract (bnAOT) and medial amygdaloid nucleus (M), give rise to labelling of a second ipsilateral afferent pathway to the AOB which terminates in the internal plexiform layer (IPL) and is unaffected by strial transection. Injections of wheat germ lectin-HRP conjugate into the AOB confirm that it receives afferents from the ipsilateral bnAOT, M and C3, and from a few cells in the contralateral C3. Transection of the ipsilateral stria terminalis prevents retrograde labelling of any cells in the ipsilateral C3, but does not affect labelling of cells in M or bnAOT (or contralateral C3). The conjugate is also transported anterogradely in this system, labelling the efferent projections of the AOB to bnAOT, M and C3. It is concluded that the AOB receives at least two sets of ipsilateral afferents: one set from C3, via the stria terminalis, terminating in the internal granular layer, and a second set from M and/or bnAOT terminating in the IPL and probably running in the accessory olfactory tract.

Axonal growth by newly-formed vomeronasal neurosensory cells in the normal adult mouse.

Vomeronasal neurosensory cells which are continuously formed in adult mice have been shown to possess axons running in the vomeronasal nerves, since they undergo a reaction of retrograde cell death after vomeronasal nerve transection. Retrograde axonal transport of horseradish peroxidase has been combined with [3H]thymidine labelling of dividing cells to show that the axons of the newly-formed neurosensory cells reach their appropriate target, the accessory olfactory bulb.

Regeneration of vomeronasal nerves into the main olfactory bulb in the mouse.

After surgical section of the vomeronasal nerves the neurosensory cells in the vomeronasal epithelium die. Electron microscopy has been used to demonstrate that their axons, and synaptic terminals in the accessory olfactory bulb degenerate and are removed by phagocytic astroglia. The vacated postsynaptic sites in the accessory bulb persist, and are not re-innervated, either by vomeronasal or olfactory axons, as long as 150 days post-operatively. However, new neurosensory cells which are produced in the vomeronasal epithelium after vomeronasal nerve section do form axons. Light and electron microscope autoradiography of axonally transported material has been used to show that some of these axons grow back into the cranial cavity and form glomeruli in the main olfactory bulb, in regions where it is damaged or de-afferented. The regenerated vomeronasal glomeruli contain synapses between vomeronasal nerve terminals and dendrites of main bulb neurons.

Replacement of receptor neurones after section of the vomeronasal nerves in the adult mouse.

Eight days after vomeronasal nerve section or removal of the accessory olfactory bulb, the majority of receptor cells of the vomeronasal neuroepithelium degenerate and disappear, leaving a regular framework consisting of supporting cells and their radial processes. The cell clusters at the boundaries of the epithelial sheet (which have been shown to be actively dividing in the normal, unoperated adult mouse) are also spared. The epithelium is subsequently repopulated by receptor cells appearing first in the basal part of the receptor cell layer and later occupying the full width of the receptor layer. These cells are anatomically fully differentiated receptor cells with normal sensory dendrites. Their axons form conspicuous intraepithelial neuromatous masses. Administration of [3H]thymidine on days 10-20 postoperatively labels some clusters of supporting cells and virtually all of the receptor cells, indicating that the repopulation of the epithelium is due to new formation of receptor cells.

Inhibition by puromycin of transneuronal transport in the mouse accessory olfactory bulb.

Application of [3H]proline to the vomeronasal organ (VNO) in mice results in the transport of labelled material along the vomeronasal axons to their terminals in the glomerular layer of the accessory olfactory bulb (AOB). In addition labelled material leaves the vomeronasal nerve terminals and is found over the external plexiform layer (EPL), where a previous electron microscopic autoradiographic study showed that it is preferentially accumulated in mitral cells. Grain densities over the glomerular layer and the EPL were counted in light micrographs. After subtracting background, the overall density of grains in the EPL is about 10% of that over the glomerular layer at 6 h after administration of [3H]proline to the VNO (5 mice). In a further 7 mice, puromycin (or saline) was applied directly to the AOB at hourly intervals during the 6 h after [3H]proline administration. Under these circumstances the labelling in the EPL is only 2--4% of that in the glomerular layer (9% for the 2 saline controls). These observations are evidence that a major part of the transsynaptic transfer mechanism is dependent on protein synthesis, and also favour the view that free amino acids are an important component of the material transferred.

Cell division in the vomeronasal organ of the adult mouse.

Using [3H]thymidine labelling we could demonstrate the presence of a population of dividing cells in the vomeronasal neurosensory epithelium of the adult mouse. These cells are localised in the regions of the epithelium adjacent to its boundaries with the ciliated respiratory epithelium. With increasing survival times after thymidine administration, the labelled cells become situated progressively further away from the boundary region. The cluster of cells with labelled nuclei forms a loose column, consisting of labelled receptor cells, but in addition the immediately overlying supporting cell nuclei are also labelled. By 56 days after thymidine administration the cluster of labelled cells is separated from the epithelial boundary by a distance equivalent to about one-fifth of the total width of the epithelial sheet. There is little further change in position at 102 days. It is not clear to what extent this represents a turnover process as opposed to a continuing growth of the epithelium by accretion at the edges.

Electron microscope autoradiographic evidence for specific transneuronal transport in the mouse accessory olfactory bulb.

The distribution of radioactive material was examined autoradiographically 8 h after application of [3H] proline to the vomeronasal organ in mice. Labelled material was transported along the axons of the vomeronasal nerves to their terminals in the glomerular layer of the accessory olfactory bulb (AOB). A lesser but consistent amount of radioactivity was found in the external plexiform layer (EPL) of the AOB. Electron microscopic autoradiography was used to determine which of the components of the EPL contained this labelled material. The method of proportional grain counts showed that the highest concentration of silver grains lay over the mitral cell dendrites, which are the elements immediately postsynaptic to the vomeronasal nerve axons. However, a fairly high proportion of grains also lay over the peripheral processes of granule cells. By application of a method of 'crossfire analysis' (which is explained in detail) it was possible to show that the observed grain distribution is best explained by the assumption that the radioactive material is confined to mitral cells, and the labelling over granule cell processes is due to crossfire from these sources. Im one animal at 5 days after [3H]proline administration label was found to have extended from mitral cells to granule cells, suggesting that the transsynaptically transported radioactive material, which was confined to the mitral cells at 8 h, may have become further redistributed at longer survivals. In a control experiment, [3H]proline was applied directly to the surface of the AOB. This gave rise to a completely different distribution of radioactivity in the EPL: radioactive material was present in all tissue components.

Astrocyte cultures from adult rat brain. Derivation, characterization and neurotrophic properties of pure astroglial cells from corpus callosum.

It has not as yet been routinely possible to derive primary cultures of glial cells from adult rat brain tissue even when adopting strategies that have proven successful with perinatal tissue. We now report that in response to a surgical lesion and a period of postoperative 'priming' in vivo, proliferating cultures of astroglial cells can be derived from the normally quiescent glia of the corpus callosum region of the adult rat brain. In such cultures the predominance of astroglia and the virtual absence of oligodendroglia and neurons has been established by the use of a variety of cell-type specific antisera. Fibroblasts, the only other cell type identified, when not numerous could be successfully eliminated by treatment of the cultures with anti-Thy-1 antibodies and guinea pig complement. Pure astroglial cells from adult brain have been sub-cultured and maintained for up to 4 months in vitro, providing suitable quantities of cells for studies on the trophic interaction between glia and neurons. In long-term culture the adult astrocytes maintain a flattened undifferentiated morphology but readily assume a stellate shape with long branching processes upon the addition of a crude homogenate from bovine pituitary.

Identification and characterisation of angiotensin II receptor subtypes in human brain.

Autoradiographic and homogenate binding studies using the radioligand, [125I]angiotensin II, identified a heterogeneous distribution of specific binding sites (defined by angiotensin II, 1.0 microM) throughout the human forebrain. Highest AT receptor densities were detected in the paraventricular nucleus, median eminence, substantia nigra, putamen and caudate nucleus (2.4, 1.2, 1.0, 0.30 and 0.24 fmol/mg tissue equivalent, respectively). The AT1 receptor antagonist, losartan (1.0 microM) competed for the majority of the specific binding. [125I]Angiotensin II-specific binding (although not consistently above non-specific binding levels) was also detected in various other brain regions (e.g. amygdala, entorhinal cortex, frontal cortex, hippocampus, inferior colliculus, nucleus accumbens, parietal cortex, periaquaductal grey, superior colliculus, striate cortex, temporal cortex, thalamus). In the presence of losartan (1.0 microM), angiotensin II, saralasin, losartan and PD123177 competed for [125I]angiotensin II binding to membranes prepared from the cerebellum or substantia nigra with a rank order of affinity; angiotensin II = saralasin > PD123177 > losartan. In the presence of PD123177 (1.0 microM), the rank order of affinity of losartan and PD123177 was reversed. These studies indicate the presence of both AT1 and AT2 receptor subtypes within various regions of the human forebrain.