VIP and noradrenaline act synergistically to increase cyclic AMP in cerebral cortex

There is growing evidence that two, or possibly more, neurotransmitters can coexist within the same neurone. In particular, the presence of a peptide and a biogenic amine has been demonstrated in the same terminals of central and peripheral neurones. These findings have led to the hypothesis that neurotransmitters, coexisting within the same neurones, can interact at pre- or postsynaptic sites in a functionally coordinated manner. However, interactions between neurotransmitters contained in distinct neuronal systems terminating within the same region of the central nervous system (CNS) can be envisaged. We have examined this last possibility in the cerebral cortex, an area of the CNS where the two neurotransmitters vasoactive intestinal polypeptide and noradrenaline are contained in separate neuronal systems and where they both stimulate the formation of cyclic AMP. We report here that vasoactive intestinal polypeptide and noradrenaline act synergistically to stimulate the formation of cyclic AMP and that this synergistic interaction is antagonized by the specific alpha-adrenergic antagonist phentolamine.     

A neuronal clone derived from a Rous sarcoma virus-transformed quail embryo neuroretina established culture

Neuroretina (NR) is an evagination of the central nervous system (CNS) which is composed of photoreceptors, glial (Müller) cells and horizontal, bipolar, amacrine and ganglion neuronal cells. We describe here the usefulness of Rous sarcoma virus (RSV) in the establishment of a neuronal clone from quail embryo neuroretina. When primary cultures of chick and quail embryo neuroretina cells are transformed by RSV, neuronal markers such as ribbon synapses, choline acetyltransferase (CAT) and glutamic acid decarboxylase (GAD) specific activity are present. These RSV-transformed primary cultures can be established into permanent cell lines from which neuronal clones have been isolated. One of them, clone QNR/D, can generate tetrodotoxin(TTX)-inhibitable action potentials on electrical stimulation, has a high GAD activity and binds monoclonal antibodies raised against chick embryo neuroretina. The presence of these neuronal markers suggests that the QNR/D clone is derived from cells of the amacrine or ganglionic lineage. This is the first time that a neuronal cell clone of defined origin has been obtained from the CNS. The neuronal markers of the QNR/D clone are expressed at both the permissive and the non-permissive temperatures for transformation.     

Neurones express high levels of a structurally modified, activated form of pp60c-src

Neural tissues contain high levels of the cellular homologue of the transforming protein of Rous sarcoma virus (RSV), but neither the specific cell types expressing high levels of c-src, nor the function of the cellular src (c-src) protein has been determined. Using primary culture methods, we have found that pure neurones and astrocytes derived from the rat central nervous system (CNS) contain 15- to 20-times higher levels of the c-src protein than fibroblasts. However, the specific activity of the c-src protein from the neuronal cultures is 6- to 12-times higher than that from the astrocyte cultures. In addition, the c-src protein expressed in neuronal cultures contains a structural alteration within the amino-terminal region of the molecule that causes a shift in the mobility of the c-src protein on the SDS-polyacrylamide gels. These results indicate that a structurally distinct form of the cellular src protein that possesses an activated tyrosylkinase activity is expressed at very high levels in post-mitotic CNS neurones.     

Platelet-derived growth factor promotes division and motility and inhibits premature differentiation of the oligodendrocyte/type-2 astrocyte progenitor cell

The mitogens which modulate cell-cell interactions during development of the central nervous system are unknown. One of the few interactions sufficiently well understood to allow identification of such molecules involves the two glial lineages which make up the rat optic nerve. One population of glial cells in this tissue, the type-1 astrocytes, secrete a soluble factor(s) which promotes division of a second population of bipotential oligodendrocyte/type-2 astrocyte (O-2A) progenitor cells; these progenitors give rise to oligodendrocytes, which myelinate large axons in the CNS, and type-2 astrocytes, which enwrap bare axons at nodes of Ranvier. Type-1 astrocytes also promote progenitor motility, and inhibit the premature differentiation of progenitors into oligodendrocytes which occur when these cells are grown in the absence of type-1 astrocytes. We have now found that platelet-derived growth factor mimics the effects of type-1 astrocytes on O-2A progenitor cells, and antibodies to PDGF block the effects of type-1 astrocytes.     

Membrane conductance oscillations in astrocytes induced by phorbol ester

Glial cells in the central nervous systems (CNS) have complex functions which are difficult to decipher because of the intimate intertwining of glial cells with neurons. We have therefore developed an essentially neuron-free preparation of CNS astrocytes in the kainic acid lesioned hippocampal slice. With this preparation we have examined the effect of activating protein kinase C in astrocytes with a phorbol ester, TPA (12-O-tetradecanoyl-phorbol-13-acetate). In most cells, TPA induced rhythmic oscillations (0.1-3.0 Hz) of membrane potential which were typically 5-10 mV in amplitude and were associated with increases of up to eightfold in input resistance during the depolarizing phase. These large changes in membrane conductance are the first reported observations of endogenously generated conductance changes in astrocytes of the mammalian CNS and they could influence excitability of surrounding neurons, possibly by altering extracellular ion concentrations.     

Location of neuronal tangles in somatostatin neurones in Alzheimer's disease

Senile dementia of the Alzheimer type is a chronic, progressive neuropsychiatric condition characterized clinically by global intellectual impairment and neuropathologically by the presence of numerous argyrophilic plaques and tangles. Neurochemical investigations have established loss of the cholinergic and aminergic projections to the cerebral cortex and a loss of the content of somatostatin, with preservation of cholecystokinin and vasoactive intestinal polypeptide, neuropeptides also located in cells intrinsic to the cortex. We describe here the relationship between cortical somatostatin immunoreactivity and the plaques and tangles of diseased tissue by immunocytochemical and silver impregnation techniques on paraffin-embedded tissue. In sections of Alzheimer's tissue, cortical somatostatin-immunoreactive perikarya exhibited morphological changes consistent with neuronal degeneration. Silver-stained material immunostained subsequently showed that many neurones containing tangles were also somatostatin positive. No such colocalization was observed using antisera to other neuropeptides. Our findings indicate that a subclass of somatostatin-positive neurones are affected selectively in Alzheimer's disease and that these neurones also contain neuronal tangles. Thus, destruction of somatostatin-containing neurones is an early and perhaps critical event in the disease process.     

Differentiation of a bipotential glial progenitor cell in a single cell microculture

Although it is known that most cells of the vertebrate central nervous system (CNS) are derived from the neuroepithelial cells of the neural tube, the factors determining whether an individual neuroepithelial cell develops into a particular type of neurone or glial cell remain unknown. A promising model for studying this problem is the bipotential glial progenitor cell in the developing rat optic nerve; this cell differentiates into a particular type of astrocyte (a type-2 astrocyte) if cultured in 10% fetal calf serum (FCS) and into an oligodendrocyte if cultured in serum-free medium. As the oligodendrocyte-type-2 astrocyte (0-2A) progenitor cell can differentiate along either glial pathway in neurone-free cultures, living axons clearly are not required for its differentiation, at least in vitro. However, the studies on 0-2A progenitor cells were carried out in bulk cultures of optic nerve, and so it was possible that other cell-cell interactions were required for differentiation in culture. We show here that 0-2A progenitor cells can differentiate into type-2 astrocytes or oligodendrocytes when grown as isolated cells in microculture, indicating that differentiation along either glial pathway in vitro does not require signals from other CNS cells, apart from the signals provided by components of the culture medium. We also show that single 0-2A progenitor cells can differentiate along either pathway without dividing, supporting our previous studies using 3H-thymidine and suggesting that DNA replication is not required for these cells to choose between the two differentiation programmes.     

Guidance of optic nerve fibres by N-cadherin adhesion molecules

The dendritic branches (neurites) of developing neurons migrate along specific pathways to reach their targets. It has been suggested that this migration is guided by factors present on the surface of other neurons or glial cells. The molecular nature of such factors, however, remains to be elucidated. N-cadherin is a cell-surface glycoprotein which belongs to the cadherin family of cell-cell adhesion molecules. This adhesion molecule is expressed in various neuronal cells as well as in glial cells of the central and peripheral nervous systems in vertebrate embryos and recent immunological studies suggested that N-cadherin may play a role in guiding the migration of neurites on myotubes or astrocytes. To further examine this possibility, we used a molecular-genetic approach; that is, we examined the outgrowth of chicken embryonic optic axons on monolayer cultures of Neuro 2a or L cells transfected with the complementary DNA encoding chicken N-cadherin. The data indicate that N-cadherin is used as a guide molecule for the migration of optic axons on cell surfaces.     

Noradrenaline and beta-adrenoceptor agonists increase activity of voltage-dependent calcium channels in hippocampal neurons

The predominance of unconventional transmitter release sites at noradrenaline-containing synapses and the diffuse projections of noradrenaline-containing fibres originating in locus coeruleus have led to speculation that noradrenaline may act as a neuromodulator in the central nervous system. Evidence suggests that it has a modulatory function in the plasticity of the developing nervous system, in controlling behavioural states of an organism, and in learning and memory. Recently, Hopkins and Johnston demonstrated that noradrenaline enhances the magnitude, duration and probability of induction of long-term potentiation (LTP) at mossy fibre synapses in the hippocampal formation, and LTP is widely believed to be a cellular substrate for aspects of memory. To investigate the membrane effects of noradrenaline on central neurons, we used a newly developed preparation in which patch-clamp techniques can be applied to exposed adult cortical neurons. We report here that noradrenaline produces an enhancement in the activity of voltage-dependent calcium channels in granule cells of the hippocampal dentate gyrus. This action appears to be mediated by beta-adrenoceptors and can be mimicked by cyclic AMP.     

Substance P raises neuronal membrane excitability by reducing inward rectification

Much interest has recently centred on the properties of peptides that modulate the excitability of nerve cells. Such compounds include the undecapeptide substance P, which is particularly well established as an excitatory neurotransmitter, and we examine here its effects on magnocellular cholinergic neurones taken from the medial and ventral aspects of the globus pallidus of newborn rats and grown in dissociated culture. These neurones have previously been shown to respond to substance P3 and are analogous to the nucleus basalis of Meynert in man, which gives a diffuse projection to the cerebral cortex and whose degeneration is the likely cause of Alzheimer's disease. Substance P depolarizes these cultured neurones by reducing an inwardly rectifying potassium conductances; this conductance has been found in several neuronal types and has similar properties to those of certain other cells. As discussed below, modulation of inward (or anomalous) rectification by substance P implies a self-reinforcing element to the depolarization caused by the peptide.     

Kappa- and delta-opioid receptor agonists differentially inhibit striatal dopamine and acetylcholine release

At least three different families of endogenous opioid peptides, the enkephalins, endorphins and dynorphins, are present in the mammalian central nervous system (CNS). Immunocytochemical studies have demonstrated their localization in neurones, which supports the view that these peptides may have a role as neurotransmitter or neuromodulators. However, the target cells and cellular processes acted upon by the opioid peptides are still largely unknown. One possible function of neuropeptides, including the opioid peptides, may be presynaptic modulation of neurotransmission in certain neuronal pathways, for example, by inhibition or promotion of neurotransmitter release from the nerve terminals. Here we report that dynorphin and some benzomorphans potently and selectively inhibit the release of (radiolabelled) dopamine from slices of rat corpus striatum, by activating kappa-opioid receptors. In contrast, [Leu5]enkephalin and [D-Ala2, D-Leu5]enkephalin selectively inhibit acetylcholine release by activating delta-opioid receptors.     

A VIP-containing system concentrated in the lumbosacral region of human spinal cord

Neuropeptides were first localized in the human spinal cord by immunocytochemistry and substance P has been shown, by the same method, to be reduced ipsilaterally in the dorsal horn after limb amputation and bilaterally in the Riley-Day syndrome. Several neuropeptides increasingly fulfil the criteria to establish them as neurotransmitters or neuromodulators, and they may also have trophic actions in the spinal cord. Using radioimmunoassay and immunocytochemistry, we present here for the first time a quantitative regional distribution and localization of vasoactive intestinal polypeptide (VIP), substance P, somatostatin, bombesin and cholecystokinin (CCK-8) in normal postmortem human spinal cord. A comparison of the distribution of these peptides reveals an exceptional pattern for VIP, with relatively much higher levels in the lumbosacral region. Immunocytochemical analysis shows a distinctive distribution of VIP-containing fibres and terminals at the lumbosacral segments. This VIP-containing system may have an important role in the spinal control of urogenital function in man.     

Evidence for migration of oligodendrocyte--type-2 astrocyte progenitor cells into the developing rat optic nerve

Formation of myelinated tracts in central nervous system (CNS) regions such as the optic nerve seems to depend on two glial cell types, both of which derive from a common progenitor cell. This oligodendrocyte--type-2 astrocyte (O-2A) progenitor cell gives rise to oligodendrocytes, which produce internodal myelin sheaths, and to type-2 astrocytes, which extend fine processes in the region of the nodal axolemma. The optic nerve also contains a third glial cell, the type-1 astrocyte, which derives from a separate precursor. These three glial cells develop in a fixed sequence over a two-week period: type-1 astrocytes appear at embryonic day 16 (E16), oligodendrocytes at the day of birth (E21 or postnatal day P0), and type-2 astrocytes between P8 and P10. Type-1 astrocytes secrete a potent mitogen which causes expansion of the O-2A progenitor cell population in vitro. Here, we report that dividing O-2A progenitor cells are highly motile and seem to migrate from the brain into the optic nerve, beginning at its chiasmal end. Our results indicate that long-distance migration along the neural axis is characteristic only of progenitors of the O-2A lineage and may serve to distribute these cells to regions of the CNS that will become myelinated. These results also suggest that the intrinsic neuroepithelial cells of the optic stalk may be even more restricted than previously thought, giving rise only to type-1 astrocytes.     

Noradrenaline and cyclic AMP--independent growth stimulation in newt limb blastemata

Adult newts regenerate functional limbs after amputation. This process normally depends on the trophic influence of nerves on the regenerating limbs, particularly in the early stages before differentiation of the regeneration blastema, when it stimulates growth by maintaining high rates of macromolecular synthesis. The sequence of biochemical events involved is unknown, but it has been suggested that intracellular cyclic AMP may be a second messenger within the blastema. Many studies have indicated that the neural agent(s) involved might be protein. The recent finding that blastemata contain high levels of catecholamines, however, has implicated noradrenaline (NA) as the neurotrophic agent, and suggested that it works via stimulation of beta-adrenergic receptors on the blastemal cells, thereby raising the intracellular concentrations of cyclic AMP. To test this hypothesis we studied the ability of NA alone and in combination with alpha-and beta-adrenergic antagonists to increase cyclic AMP levels and to mimic the effects of nerves by maintaining high rates of protein synthesis and high mitotic indices (MI) in denervated blastemata in organ culture. We find that although NA raises cyclic AMP levels through a beta-adrenergic effect, it does not maintain high rates of protein synthesis or high MI in cultured blastemata. It is unlikely therefore, that this hypothesis applies.     

A glial progenitor cell that develops in vitro into an astrocyte or an oligodendrocyte depending on culture medium

We have identified a cell type in 7-day-old rat optic nerve that differentiates into a fibrous astrocyte if cultured in the presence of fetal calf serum and into an oligodendrocyte if cultured in the absence of serum. In certain culture conditions some of these cells acquire a mixed phenotype, displaying properties of both astrocytes and oligodendrocytes. These observations suggest that fibrous astrocytes and oligodendrocytes develop from a common progenitor cell and provide a striking example of developmental plasticity and environmental influence in the differentiation of CNS glial cells.     

Nerve growth factor induces adrenergic neuronal differentiation in F9 teratocarcinoma cells

Teratocarcinoma cells have been used as a model to study differentiation and development in vertebrates. Treatment with retinoic acid (RA) and dibutyryl cyclic AMP can in some embryonal carcinoma (EC) cell lines lead to neural differentiation, as judged by neurofilament expression and by the induction of enzymes involved in cholinergic transmission. Short-term culture of F9 line cells with RA and dibutyryl cyclic AMP results in a biochemically demonstrable rise in acetylcholinesterase (AChE) activity. We now report that long-term culture of F9 cells with RA and dibutyryl cyclic AMP induces neurofilament expression, demonstrated by immunofluorescence with specific antibodies. Furthermore, if nerve growth factor (NGF) is also added, the developing neurone-like cells exhibit immunoreactivity to tyrosine hydroxylase, a rate-limiting enzyme of catecholamine synthesis specific for adrenergic neurones. Immunoreactivity for Leu-enkephalin-like peptides is also induced. These results suggest that F9 cells can differentiate into cells with adrenergic characteristics.     

Pericytes are required for blood-brain barrier integrity during embryogenesis

Vascular endothelial cells in the central nervous system (CNS) form a barrier that restricts the movement of molecules and ions between the blood and the brain. This blood-brain barrier (BBB) is crucial to ensure proper neuronal function and protect the CNS from injury and disease. Transplantation studies have demonstrated that the BBB is not intrinsic to the endothelial cells, but is induced by interactions with the neural cells. Owing to the close spatial relationship between astrocytes and endothelial cells, it has been hypothesized that astrocytes induce this critical barrier postnatally, but the timing of BBB formation has been controversial. Here we demonstrate that the barrier is formed during embryogenesis as endothelial cells invade the CNS and pericytes are recruited to the nascent vessels, over a week before astrocyte generation. Analysing mice with null and hypomorphic alleles of Pdgfrb, which have defects in pericyte generation, we demonstrate that pericytes are necessary for the formation of the BBB, and that absolute pericyte coverage determines relative vascular permeability. We demonstrate that pericytes regulate functional aspects of the BBB, including the formation of tight junctions and vesicle trafficking in CNS endothelial cells. Pericytes do not induce BBB-specific gene expression in CNS endothelial cells, but inhibit the expression of molecules that increase vascular permeability and CNS immune cell infiltration. These data indicate that pericyte-endothelial cell interactions are critical to regulate the BBB during development, and disruption of these interactions may lead to BBB dysfunction and neuroinflammation during CNS injury and disease.     

The oligodendrocyte-type-2 astrocyte cell lineage is specialized for myelination

Astrocytes are one of the most numerous cell types in the vertebrate central nervous system (CNS) and yet their functions are largely unknown. In the rat optic nerve there are two distinct types of astrocyte: type-1 astrocytes develop from one type of precursor cell, and type-2 astrocytes develop from bipotential, oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells, that initially give rise to oligodendrocytes (which make myelin in the CNS), and then to type-2 astrocytes. Type-1 astrocytes form the glial limiting membrane at the periphery of the optic nerve and are probably responsible for glial scar formation following nerve transection. The functions of type-2 astrocytes, which, like oligodendrocytes, are found mainly in tracts of myelinated axons throughout the CNS, are unknown. In this report we provide evidence that processes from type-2 astrocytes contribute to the structure of nodes of Ranvier, suggesting that the O-2A cell lineage is specialized for constructing myelin sheaths and nodes in the mammalian CNS.