Lithium ion inhibits nerve growth factor-induced neurite outgrowth and phosphorylation of nerve growth factor-modulated microtubule-associated proteins.

LiCl (2.5-20 mM) reversibly suppressed nerve growth factor (NGF)-induced neurite outgrowth by cultured rat PC 12 pheochromocytoma cells. Similar concentrations of LiCl also reversibly blocked NGF-dependent regeneration of neurites by PC12 cells that had been primed by long-term pre-exposure to NGF and by cultured newborn mouse sympathetic neurons. In contrast, transcription-dependent responses of PC12 cells to NGF such as priming and induction of the NGF-inducible large external glycoprotein, occurred despite the presence of Li+. SDS PAGE analysis of total cellular phosphoproteins (labeled by 2-h exposure to 32P-orthophosphate) from neurite-bearing primed PC12 cells revealed that Li+ reversibly inhibited the phosphorylation of a band of Mr 64,000 that was barely detectable in NGF-untreated PC12 cells. However, Li+ did not appear to affect the labeling of other phosphoproteins in either NGF-primed or untreated PC12 cultures, nor did it affect the rapid increase in phosphorylation of several proteins that occurs when NGF is first added to unprimed cultures. Several criteria indicated that the NGF-inducible phosphoprotein of Mr 64,000 is a microtubule-associated protein (MAP). Of the NGF-inducible phosphorylated MAPs that have been detected in PC12 cells (Mr 64,000, 72,000, 80,000, and 320,000), several (Mr 64,000, 72,000, and 80,000) were found to be substantially less phosphorylated in the presence of Li+. Neither a phorbol ester tumor promotor nor permeant cAMP analogs reversed the inhibitory effects of Li+ on neurite outgrowth or on phosphorylation of the component of Mr 64,000. Microtubules are a major and required constituent of neurites, and MAPs may regulate the assembly and stability of neuritic microtubules. The observation that Li+ selectively inhibits NGF-induced neurite outgrowth and MAP phosphorylation suggests a possible causal relationship between these two events.

Differential inhibition of nerve growth factor and epidermal growth factor effects on the PC12 pheochromocytoma line.

Tests have been made of the action of the methyltransferase inhibitors 5'-S-methyl adenosine, 5'-S-(2-methyl-propyl)-adenosine, and 3-deaza-adenosine +/- L-homocysteine thiolactone, on nerve growth factor (NGF)-dependent events in the rat pheochromocytoma line PC12. Each of these agents inhibited NGF-dependent neurite outgrowth at concentrations of the order of millimolar. Slow initiation of neurite outgrowth over several days and more rapid regeneration of neurites (congruent to 1 d) were blocked, as was the priming mechanism necessary for genesis of neurites. The inhibitions were reversible in that PC12 cells maintained for several days in the presence of inhibitors grew neurites normally after washout of these agents. Other NGF-dependent responses of the PC12 line (i.e., induction of ornithine decarboxylase activity [over 4 h], enhancement of tyrosine hydroxylase phosphorylation [over 1 h], and rapid changes in cell surface morphology [30 s onward]) were inhibited by each of the agents. In contrast, corresponding epidermal growth factor-dependent responses in ornithine decarboxylase activity, phosphorylation, and cell surface morphology were not blocked, but instead either unaffected or enhanced, by the methylation inhibitors. These inhibitors did not act by blockade of binding of NGF to high- or low-affinity cell surface receptors, though they partially inhibited internalization of [125I]NGF. The inhibition of rapidly-induced NGF-dependent events and the differential inhibition of responses to NGF and epidermal growth factor imply that the methyltransferase inhibitors specifically block one of the first steps in the mechanistic pathway for NGF.

Spin label and lanthanide binding sites on glyceraldehyde-3-phosphate dehydrogenase.

The electron spin resonance spectrum of rabbit muscle D-glyceraldehyde-3-phosphate dehydrogenase spin-labelled with 4-(2-iodoacetamido)-2,2,6,6-tetramethylpiperidinooxyl has two components. One component is due to a spin label highly immobilized on the enzyme surface and the other to a nitroxyl group able to tumble more rapidly. The spin-labelled enzyme is inactive. Selective modification of the active site cysteine residue (149) and determinations of total sulphydryl content implicate this residue as the site of the immobile spin-label. The mobile spin label is attached to another sulphydryl group. Crystallographic studies on the human muscle enzyme (Watson, H.C., Duee, E. and Mercer, W.D. (1972) Nat. New Biol., 240, 130) have located a binding site for samarium ion in the active centre. Addition of the paramagnetic gadolinium ion to spin-labelled enzyme reduces the intensity of both the spin label signals (by 72% for the mobile and by 11% for the immobile component). This indicates that the metal ion site (Kd equals 0.7 mM) is close to both types of spin label. Measurements of the effect of gadolinium-protein binding on the relaxation rate of solvent water protons enable the enzyme-bound spin label-metal ion distances to be tentatively estimated as 15 angstrom.

Cultured epithelioid astrocytes migrate after transplantation into the adult rat brain.

A highly purified population of dividing epithelioid astrocytes has been prepared from postnatal rat corpus callosum. These cells were labelled in culture by incorporation of either [3H]thymidine or fluorescent microspheres and transplanted in a fibrin clot into the hippocampi of adult syngeneic rats. Transplanted cells divided in vivo and progressively migrated into the host brain from the site of implantation up to distances of about 1 mm. After a 1-week survival, transplant cells stained strongly for glial fibrillary acidic protein and had the thick sinuous processes characteristic of stellate astrocytes. Artefactual transfer of radiolabel to host cells was ruled out by control experiments in which either the proportion of transplant cells that were radiolabelled was varied or radiolabelled transplant cells were killed prior to implantation. Astrocyte migration over the first days after implantation was determined to occur at a rate of approximately 100 microns per day. Transplant cells moved into both grey and white matter areas of the host brain and over the migratory period were commonly observed to be associated with blood vessels. Some transplant cells were directly juxtaposed against neuronal perikarya and dendrites. Many labelled astrocytes were located in areas that were apparently completely free of damage caused by implantation. These results define a class of mature astrocytic cells that have the ability to migrate through the adult brain. The existence of pathways for cell movement in the adult CNS has implications for the mechanisms of tissue remodelling after injury and transplantation, for regenerative repair of the CNS, and for the dynamics of cell-cell contacts in the normal adult mammalian brain.

Intracerebral transplantation of cultured neurons after reaggregation in a plasma clot.

In order to be able to transplant neural cells which have been either manipulated in vitro or maintained in culture for the purpose of cell-type enrichment, we have developed a novel plasma clot method which permits reaggregation of previously dissociated cells such that they can be implanted as highly localized transplants rather than as dispersion-prone cell suspension grafts. To establish the method, enzymatically dissociated cells prepared from hippocampal primordia of late embryonic rats were immediately reaggregated into plasma clots and transplanted to the hippocampal formation of adult recipients. By using fluorescein-labelled bovine plasma to form the plasma clot grafts of reaggregated cells, the fate of the plasma clot protein matrix was followed at different post-operative survival times. Initially, 4-5 days post-operative, the plasma clot maintained the grafted cells in a loose sponge-like sack at the site of implantation. After 2-3 weeks, the transplanted cells were more compact and fused with the host neuropil, and the plasma clot matrix had largely been degraded. At 1 month or longer survival, there was no distinguishable boundary between transplant and host, and there was little or no evidence of any remaining plasma clot matrix or proteins. The plasma clot method was subsequently applied to the transplantation of cultures enriched in pyramidal cells. Enrichment for pyramidal cells was achieved by eliminating mitotic cells (dentate granule cells and glia) by brief (200 rad) irradiation of hippocampal primordia followed by dissociation and maintenance in monolayer culture for 4-6 days. Fibres from host dentate granule cells grew into the pyramidal cell-enriched transplants and established mossy fibre terminals on the donor cells. In transplants between embryonic and adult rats, donor cells were identified at long survival times by prelabelling donor cells in utero or in vitro with [3H]thymidine prior to transplantation. In transplants between embryonic and adult mice, donor tissue from A Thy-1.1 strain mice was transplanted to congenic A strain (Thy-1.2) mice such that the donor cells bearing the Thy-1.1 cell surface glycoprotein could be later identified by immunocytochemical staining with antibodies specific for the Thy-1.1 antigen. Reaggregation and transplantation of dissociated cells in a plasma clot thus provides a novel method whereby prior manipulation of neural tissue (separation of neurons and glia, enrichment for specific types of neurons, or glia etc.) can be used to great advantage in studying host-transplant connectivity and in assessing those factors which are critical in sustaining the survival of grafted neural tissue.

Microtransplantation of neural cells into adult rat brain.

Epithelioid ("type 1") astrocytes and hippocampal neurons have been stereotaxically grafted into adult rat brain by a new "microtransplantation" method and the results of grafting assessed at various survival times by conventional histological methods using light and electron microscopes. The extent of bleeding that accompanied grafting was assessed by peroxidase staining of extravascular red cells. Grafts were small, compact and reproducible in terms of their volume and location. Implantation was achieved with very little trauma to host tissue. There was no apparent disruption of host tissue adjacent to the graft and very little damage to host blood vessels from earliest survival times. Donor astrocytes were in direct contact with cells of the host immediately after grafting. This situation was compared with conventional implantation of astrocytes in which much damage, necrosis and bleeding occurred and for which cells of graft and host were initially isolated from one another. Microtransplanted astrocytes were observed to migrate. Similar, comparatively non-traumatic microtransplantation of hippocampal neurons was also carried out. Long-term survival of grafted neurons was demonstrated.

Selective innervation of embryonic hippocampal transplants by adult host dentate granule cell axons.

Fragments containing different cytoarchitectonic fields were dissected out of late embryonic rat hippocampal primordia and transplanted into the hippocampus or septum of adult syngeneic hosts. Field CA3 transplants contained clusters of large, angular (pyramidal) cell bodies surrounded by a radiating corona of dendrites. These cells stained selectively with our monoclonal antibody Py, and a proportion were labelled by [3H]thymidine administered on the 15th day of embryonic life. Field CA1 transplants contained smaller, angular, Py-negative cells, which formed elongated laminae rather than globular clusters. The ability of the host dentate granule cells to project to the transplants was examined by (1) the Timm stain for mossy fibres, (2) electron microscopy of Golgi-impregnated CA3 pyramidal neurons in the transplants, and (3) quantitative electron microscopic assessment of the proportions of large mossy fibre terminals in the synaptic population of the transplants. The Timm stain showed that CA3 transplants received a projection from host dentate granule cells when the transplants were placed in direct contact with the axons in the host mossy fibre pathway. As in the normal host field CA3, the ingrowing mossy fibres terminated selectively on the juxtacellular regions of the dendritic tree and ignored the major part of the dendrites in the radiating corona. The electron micrographs showed that within this territory the host mossy fibres formed synaptic terminals with all the complex features typical of normal mossy fibres, and were presynaptic to complex spines arising from the juxtacellular region of Golgi-impregnated donor CA3 pyramidal cells. The quantitative electron microscopic study demonstrated that the mossy fibre-innervated juxtacellular regions of the field CA3 transplants had up to 20% of the normal density of mossy fibre synapses found in the stratum lucidum of field CA3 in situ. CA3 transplants which were placed in the septum, remote from the host mossy fibres, had either trivial numbers of mossy fibre synapses or none. This confirmed that the abundant mossy fibre terminals in the intrahippocampal CA3 transplants were of host origin, and not due to donor dentate granule cells inadvertently included in the grafts. The selectivity of the host dentate projection for field CA3 transplants was demonstrated by the observation that CA1 transplants in the same locations received only slight mossy fibre projections in the Timm stain, and in electron micrographs their synaptic population had only insignificant numbers of large mossy fibre terminals.(ABSTRACT TRUNCATED AT 400 WORDS)

Monoclonal antibodies reveal molecular differences between terminal fields in the rat dentate gyrus.

We have derived a number of monoclonal antibodies which detect molecular differences correlating with the afferent inputs to the molecular layer of the adult rat hippocampal dentate gyrus. One group, dubbed OM-1 to OM-4, strongly stain the outer zone of the molecular layer, which receives its major innervation from the ipsilateral entorhinal cortex. A second group, IM-1 and IM-2, show a complementary pattern and preferentially stain the inner molecular layer, which receives inputs from the ipsilateral and contralateral hippocampus. These antigens are not, however, restricted to these layers, being found outside the hippocampus in several other areas of neuropil in the adult brain. In the developing brain the IM-1 antigen appears ubiquitously from the earliest age studied, embryonic day 12. Within the dentate gyrus, its restriction to the inner terminal field of the molecular layer only occurs during the second postnatal week. In contrast, OM staining appears only sparsely and late in the prenatal brain, appearing in developing cortical white matter between embryonic days 18 and 20. The outer dentate molecular layer becomes OM-positive from birth onwards, corresponding to the time of arrival of entorhinal axons during the first postnatal week. These two groups of monoclonal antibodies recognize a number of different glycoproteins. Ultrastructural immunohistochemistry shows they are cell surface molecules, and as such may be involved in the recognition events required for the establishment of specific patterns of neuronal connectivity.

Use of colloidal gold complexes of wheat germ agglutinin as a label for neural cells.

We have made stable complexes between wheat germ agglutinin and either 5 or 10 nm particles of colloidal gold. These complexes were phagocytosed by neuronal and glial cells in embryonic rat hippocampal cultures and the incorporated gold gave intense, low-background staining in the light microscope either directly, for the most heavily labelled cells, or after intensification by physical development of silver. Cells were labelled in a punctate fashion over perikarya and processes. In the electron microscope, particles of gold were observed in lysosomal vesicles, frequently in an aggregated form. Gold complex incorporated into cells in culture was retained by those cells over periods up to 20 days. Embryonic hippocampal cells were labelled in suspension culture by incorporation of wheat germ agglutinin-gold complexes and transplanted into the brains of syngeneic adult host rats. Grafted neurons and glia were observed in the electron microscope to retain high levels of gold label over periods up to 30 days. Receipt of synaptic connections by transplanted neurones was observed. Complexes of wheat germ agglutinin with 10 nm gold particles were injected unilaterally into field CA3 of the hippocampus of adult rats. Specific retrograde transport of gold was observed in the light and electron microscopes to pyramidal and hilar neurones of the contralateral hippocampus and to neurones of the medial septal nucleus. Colloidal gold-wheat germ agglutinin complexes appear to be useful cellular markers that can be visualized at both light and electron microscope levels.

Visualization of migration of transplanted astrocytes using polystyrene microspheres.

Fluorescent polystyrene microspheres have been used to mark rat astrocytes for transplantation. Highly fluorescent cells were generated by incorporation of microspheres over periods of the order of 10 h. Microspheres ranging in average diameter from 50 to 200 nm and derivatized with either rhodamine or fluorescein have been used to label cells. There were no apparent cytotoxic effects of microsphere incorporation. Loaded astrocytes retained their complement of microspheres over periods of at least several weeks. The microspheres could be visualized in the transmission electron microscope. They were most likely phagocytosed by a coated-pit mechanism and accumulated in various types of lysosomal vesicles. Labelled astrocytes from neonatal PVG rats were implanted stereotaxically (as a plasma clot) into hippocampi of recipient syngeneic adult rats. Donor cells migrated from transplant to host over about 7 days. Transplant cells tended to be close to blood vessels over this period. Their location in paravascular spaces was determined from observation of microsphere-bearing cells in electron microscopy. Microsphere-loading of astrocytes is a simple method for marking cells for extended periods of time for transplantation which allows direct identification of marked cells with both fluorescence and electron microscopes.

Neuroprotection by both NMDA and non-NMDA receptor antagonists in in vitro ischemia.

We have investigated the relative contributions of oxygen and glucose deprivation to ischaemic neurodegeneration in organotypic hippocampal slice cultures. Cultures prepared from 10-day-old rats were maintained in vitro for 14 days and then deprived of either oxygen (hypoxia), glucose (hypoglycaemia), or both oxygen and glucose (ischaemia). Hypoxia alone induced degeneration selectively in CA1 pyramidal cells and this was greatly potentiated if glucose was removed from the medium. We have also characterised the effects of both pre- and post-treatment using glutamate receptor antagonists and the sodium channel blocker tetrodotoxin (TTX). Neuronal death following either hypoxia or ischaemia was prevented by pre-incubation with CNQX, MK-801 or tetrodotoxin. MK-801 or CNQX also prevented death induced by either hypoxia or ischaemia if added immediately post-insult, however, post-insult addition of TTX prevented hypoxic but not ischaemic damage. Organotypic hippocampal slice cultures are sensitive to both NMDA and non-NMDA glutamate receptor blockade and thus represent a useful in vitro system for the study of ischaemic neurodegeneration paralleling results reported using in vivo models of ischaemia.

Selective growth of hippocampal neurites on cryostat sections of rat brain.

Dissociated rat hippocampal neurons were cultured on horizontal cryostat sections from neonatal and adult rat brain and their growth patterns visualized by brightfield and interference contrast microscopy. Cells adhered to the sections as individuals or in small clusters and grew extensive neurites. Neurites grew over all areas of neonatal sections without apparent selectivity. For adult sections, however, neurites grew almost exclusively on areas of grey matter: there was no neurite growth on areas of white matter, irrespective of the location of that white matter within the brain. The transition from the neonatal to the adult pattern of growth occurred for sections from animals aged between 14 and 21 days.

Short-latency local actions of nerve growth factor at the growth cone.

Cultures of neurite-bearing pheochromocytoma (PC12) cells and of sympathetic neurons have been examined by time-lapse video microscopy. In the presence of nerve growth factor (NGF), the neurites of such cultures elongated and their growth cones changed geometry, via microspike and lamellipodial motion, on a time scale of minutes. Withdrawal of NGF caused process extension to cease and a progressive reduction in growth-cone area as a result of retraction of lamellipodia and microspikes. By approximately equal to 4 hr after NGF withdrawal, most neurite tips were smooth sided, devoid of conical expansions at their termini, and virtually immobile. Addition of NGF to cultures from which it had been withdrawn induced motion of microspikes and projections from the upper surface of growth cones within 2 min, while lamellipodial spreading and neurite reextension were induced after approximately equal to 20 min. For PC12 cells, these responses to replacement of NGF could not be mimicked by addition of dibutyryl cAMP (less than or equal to 2 mM) or the Ca2+ ionophore A23187 (less than or equal to 5 microM) to NGF-deprived cultures nor inhibited by the presence of EGTA (less than or equal to 2 mM) or calcium antagonists in the culture medium. Since neurite fragments formed by transection of processes of PC12 cells deprived of NGF responded to its replacement in a manner similar to intact neurites, it is concluded that the effects are focal to the neurite and growth cone and independent of the cell body. This influence of NGF on growth-cone shape and motility represents short-term local activation of this structure and has significance for control of neurite extension.

Rapid regulation of neuronal growth cone shape and surface morphology by nerve growth factor.

Scanning electron microscopy was used to study regulation of growth cone shape and surface morphology by nerve growth factor (NGF). The growth cones of cultured rat sympathetic neurons and neuronally-differentiated PC12 cells were observed under conditions of continuous NGF exposure, NGF withdrawal, and NGF readdition. Growth cones of cells cultured in the continuous presence of NGF were mostly spread in shape and about 60% possessed surface ruffles. Ruffles appeared to be largely restricted to growth cones in that few were observed on cell bodies and neurites. Withdrawal of NGF for 4-5 hr caused most of the growth cones to take on a non-spread or contracted appearance and to lose their ruffles. Readdition of NGF promoted rapid changes in growth cone properties. Within 30 sec, ruffling was again evident on the growth cones and remained prominent there throughout the course of treatment (up to 5 hr). This was in contrast to cell bodies on which, as previously reported, ruffling also occurred following NGF readdition, but only transiently (for less than 15 min). Respreading of growth cones also occurred under these conditions. This was evident within 1 min of NGF readdition and reached the levels observed in continuously-treated cultures within 1-2 hr. Neurites were also examined. Ruffles were only rarely present in the continuous presence of NGF and were absent after NGF withdrawal. NGF readdition elicited ruffling along neurites within 30 sec; the prevalence of such ruffles diminished to that seen in continuously-treated cultures within about an hour.(ABSTRACT TRUNCATED AT 250 WORDS)