Retinal ganglion cells lose response to laminin with maturation

The decisive role played by adhesive interactions between neuronal processes and the culture substrate in determining the form and extent of neurite outgrowth in vitro has greatly influenced ideas about the mechanisms of axonal growth and guidance in the vertebrate nervous system. These studies have also helped to identify adhesive molecules that might be involved in guiding axonal growth in vivo. One candidate molecule is laminin, a major glycoprotein of basal laminae which has been shown to induce a wide variety of embryonic neurones to extend neurites in culture. Moreover, laminin is found in large amounts in injured nerves that can successfully regenerate but is absent from nerves where regeneration fails. However, it is unclear to what extent the mechanisms that regulate axonal regeneration also operate in the embryo when axon outgrowth is initiated. Here we have examined the substrate requirements for neurite outgrowth in vitro by chick embryo retinal ganglion cells, the only cells in the retina to send axons to the brain. We show that while retinal ganglion cells from embryonic day 6 (E6) chicks extend profuse neurites on laminin, those from E11 do not, although they retain the ability to extend neurites on astrocytes via a laminin-independent mechanism. This represents the first evidence that central nervous system neurones may undergo a change in their substrate requirements for neurite outgrowth as they mature.     

Octopamine.

Octopamine is highly concentrated in neurones of several invertebrate species. Unlike in mammals, octopaminergic neurones in invertebrates are spatially separated from catecholaminergic neurons. In identified nerve cells of Aplysia, however, this amine coexists with other putative neurotransmitters. Octopamine is synthesized in nerves from tyrosine and tyramine and metabolised mainly by monoamine oxidase. When lobster nerves are depolarized, octopamine is liberated by a Ca2+-dependent process. A specific adenylate cyclase is stimulated by octopamine in several invertebrates to activate phosphorylase in the cockroach, induce a light-flash in firefly lattern or inhibit rhythm contractions in locust muscle. All of these observations provide compelling evidence that octopamine is a neurotransmitter in invertebrates. In mammals octopamine is localised in nerves in peripheral tissues and brain where it seems to coexist with noradrenaline, the catecholamine being present in much higher concentrations. Octopamine is released from nerves together with noradrenaline and it may under certain conditions modify the actions of the adrenergic neurotransmitter. Octopamine is present in unusually high concentrations in certain neurological and hepatic diseases and may have a pathophysiological role.     

Presynaptic neurones may contribute a unique glycoprotein to the extracellular matrix at the synapse

As the extracellular matrix at the original site of a neuromuscular junction seems to play a major part in the specificity of synaptic regeneration, considerable attention has been paid to unique molecules localized to this region. Here we describe an extracellular matrix glycoprotein of the elasmobranch electric organ that is localized near the nerve endings. By immunological criteria, it is synthesized in the cell bodies, transported down the axons and is related to a glycoprotein in the synaptic vesicles of the neurones that innervate the electric organ. It is apparently specific for these neurones, as it cannot be detected elsewhere in the nervous system of the fish. Therefore, neurones seem to contribute unique extracellular matrix glycoproteins to the synaptic region. Synaptic vesicles could be involved in transporting these glycoproteins to or from the nerve terminal surface.     

Magnocellular axons in passage through the median eminence release vasopressin

Vasopressin (arginine vasopressin, AVP) is present in two types of nerve fibres in the median eminence (ME). First, it is found in nerve terminals that originate in the parvicellular neurones of the hypothalamic paraventricular nucleus (PVN) and abut on the pericapillary space surrounding the fenestrated capillaries of the primary pituitary portal plexus in the external zone (EZ) of the ME. These neurones also synthesize corticotropin-releasing factor (CRF), which acts synergetically with vasopressin to stimulate release of adrenocorticotropin (ACTH) from the pituitary gland (see ref. 7). Second, vasopressinergic axons of the magnocellular neurosecretory system pass through the internal zone (IZ) of the ME to terminate in the neurohaemal contact zone of the neurohypophysis. The involvement of vasopressinergic magnocellular neurones in the control of ACTH secretion is much debated. Of particular interest in this context is the origin of the vasopressin found in pituitary portal blood. Although it has been demonstrated that vasopressin and CRF are present in the same neurosecretory granules of EZ fibres, parallel determinations of vasopressin and CRF in pituitary portal blood have shown alterations of the concentration of vasopressin without a concomitant change in that of CRF. Such a dissociation suggests that either differential release of vasopressin and CRF can occur from a single population of nerve endings, or there are fibres in the pituitary-stalk ME which release vasopressin but not CRF. Here we present evidence for the latter. Our results indicate that stimuli causing depolarization of the axonal membrane in vitro elicit release of vasopressin from nerve fibres in the external and internal zones of the ME.     

Somatostatin selectively inhibits noradrenaline release from hypothalamic neurones

Somatostatin in a hypothalamic peptide hormone which inhibits growth hormone release from the anterior pituitary. However, biochemical and morphological investigations have revealed that somatostatin is located not only in the hypothalamus but also in other brain areas (for example the cerebral cortex) where it occurs and in nerve cell bodies and fibres from which it can be released in a Ca2+-dependent manner. It has therefore been suggested that the neuropeptide may have functions in the central nervous system other than its effect on growth hormone release; one possible action is that of a neuromodulator. Therefore, hypothalamic and cerebral cortical slices of the rat were used to examine whether somatostatin modifies the electrically or CaCl2-evoked release of tritiated monoamines from monoaminergic neurones. it is reported here that somatostatin inhibits 3H-noradrenaline release from the hypothalamus (but not from the cerebral cortex) but does not affect the release of 3H-dopamine and 3H-serotonin.     

Evidence for neurotensin as a non-adrenergic, non-cholinergic neurotransmitter in guinea pig ileum

Neurotensin is a 13-amino acid peptide that is widely distributed in central and peripheral tissues of various mammalian species. In peripheral tissues, the highest concentration of neurotensin-like immunoreactivity is found in the ileum, where it is present in endocrine-like cells and nerve fibres. The longitudinal smooth muscle layer of the guinea pig ileum, where neurotensin has both a direct relaxant and an indirect contractile action, has been used extensively as a biological assay system for neurotensin. We report here that the majority of specific 3H-neurotensin binding sites is present in the guinea pig ileum circular smooth muscle layer, which is known to be innervated by a large proportion of the ileal non-adrenergic inhibitory nerves. Neurotensin produces a dose-dependent, tetrodotoxin-resistant relaxation, whereas the relaxation produced by field stimulation of the inhibitory nerves is frequency-dependent and tetrodotoxin-sensitive. The calcium-dependent potassium channel blocker apamin inhibits both the neurotensin- and nerve stimulation-induced muscle relaxation. Incubation of the circular smooth muscle preparation with a neurotensin antiserum substantially inhibited the nerve stimulation-induced relaxation, indicating a direct relationship between the effects of neurotensin and of nerve stimulation.     

Role for microsomal Ca storage in mammalian neurones?

Alterations in the intracellular concentration of calcium ions [( Ca2+]i) are increasingly being found to be associated with regulatory functions in cells of all kinds. In muscle, an elevation of [Ca2+]i is the final link in excitation-contraction coupling while at nerve endings and in secretory cells, similar rises in [Ca2+]i are thought to mediate exocytosis. The discovery of calcium-activated ion channels indicated a role for intracellular calcium in the regulation of membrane excitability. Calcium transients associated with either intracellular release or the inward movement of Ca2+ across the membrane have been recorded in molluscan neurons and more recently in neurones of bullfrog sympathetic ganglia. Here, we report the first recordings of calcium transients in single mammalian neurones. In these experiments we have found that the methylxanthine, caffeine, causes the release of calcium from a labile intracellular store which can be refilled by Ca2+ entering the cell during action potentials.     

Enkephalin-, VIP- and substance P-like immunoreactivity in the carotid body

The carotid body type I cell contains amines and has features, both morphological and cytochemical, which indicate that it may also produce a peptide. Many regulatory peptides are now known to be present in both central and peripheral tissues. In the periphery these neuropeptides occur in both classical endocrine (APUD) cells and the neurones of the autonomic nervous system. We have now investigated the possible presence of neuropeptides in the cat carotid body using both immunocytochemistry and radioimmunoassay. Met- and Leu-enkephalin-like material occurred in considerable quantities in carotid body extracts and enkephalin-like immunoreactivity was localised in type I cells. Both vasoactive intestinal polypeptide (VIP)- and substance P-like immunoreactivity was also present but was localised in nerve fibres distributed throughout the organ. These active neuropeptides are widely distributed in mammalian tissues, forming a diffuse regulatory system which now seems to include the carotid body.     

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.     

Immunohistochemical localization of endogenous nerve growth factor

Nerve growth factor (NGF) has been proposed as a trophic molecule essential for the development of sympathetic and primary sensory neurones. In newborn mice and rats, administration of nerve growth factor results in an increase in the number of surviving neurones, whereas administration of antiserum to NGF decreases neuronal survival. Thus it has been proposed that the factor is produced and secreted by the relevant target tissues to provide trophic support for the ingrowing nerves. The site of synthesis of nerve growth factor is still unknown, and it has been emphasized that a precise physiological role for the molecule cannot be ascribed until the cell types that produce it are known. I report here the use of immunohistochemistry to localize endogenous NGF in the rat iris, a tissue in which there is sound biochemical evidence for the production of NGF activity. Surprisingly, the results reveal that NGF can be detected readily in Schwann cells, but not in smooth muscle cells of the iris when it is sympathetically denervated or cultured.     

Lucifer dyes--highly fluorescent dyes for biological tracing

Lucifer dyes are intensity fluorescent 4-aminonaphthalimides which are readily visible in living cells at concentrations and levels of illumination at which they are nontoxic. Because of their low molecular weight they frequently pass from one cell to another; this widespread phenomenon, termed dye-coupling, is thought to reveal functional relationships between cells. Lucifer dyes can also be used for ultrastructural tracing by comparison of electron micrographs with light micrographs of the same thin section. In addition, they show promise for backfilling neurones through cut nerves, for visualizing the results of retrograde axonal transport and for the covalent labeling of macromolecules.     

VIP occurs in intrathyroidal nerves and stimulates thyroid hormone secretion

Vasoactive intestinal polypeptide (VIP) is known to have powerful effects on the secretion from several endocrine and exocrine glands, and occurs in nerves with a ubiquitous distribution in the body. This infers that neuronal VIP may be a regulator of such secretion, and there is evidence that it is involved in the regulation of exocrine pancreatic function. Previous studies have shown that adrenergic and cholinergic nerves participate in the regulation of thyroid hormone secretion. We describe here combined immunohistochemical and immunochemical studies which show that the thyroid of several species is supplied with VIP-containing nerve fibres that surround blood vessels and run between and along thyroid follicles and that in the mouse neuronal VIP participates in the regulation of thyroid hormone secretion through a mechanism that is mediated by cyclic AMP.     

Development of neuronal polarity: GAP-43 distinguishes axonal from dendritic growth cones

Outgrowth of distinct axonal and dendritic processes is essential for the development of the functional polarity of nerve cells. In cultures of neurons from the hippocampus, where the differential outgrowth of axons and dendrites is readily discernible, we have sought molecules that might underlie the distinct modes of elongation of these two types of processes. One particularly interesting protein is GAP-43 (also termed B-50, F1 or P-57), a neuron-specific, membrane-associated phosphoprotein whose expression is dramatically elevated during neuronal development and regeneration. GAP-43 is among the most abundant proteins in neuronal growth cones, the motile structures that form the tips of advancing neurites, but its function in neuronal growth remains unknown. Using immunofluorescence staining, we show that GAP-43 is present in axons and concentrated in axonal growth cones of hippocampal neurons in culture. Surprisingly, we could not detect GAP-43 in growing dendrites and dendritic growth cones. These results show that GAP-43 is compartmentalized in developing nerve cells and provide the first direct evidence of important molecular differences between axonal and dendritic growth cones. The sorting and selective transport of GAP-43 may give axons and axonal growth cones certain of their distinctive properties, such as the ability to grow rapidly over long distances or the manner in which they recognize and respond to cues in their environment.     

Evidence that substance P is a neurotransmitter in the myenteric plexus

Substance P (SP) is an undecapeptide originally isolated from the gut and since shown to occur within neurones in several parts of the peripheral and central nervous systems. Immunohistochemical studies indicate an exceedingly dense network of SP-containing nerves within the myenteric plexus of the guinea pig ileum. These nerves are intrinsic to the gut wall and can release SP to contract the longitudinal muscle layer. We have previously shown that SP directly depolarizes myenteric neurones and that this depolarization has a time course and ionic mechanism similar to the slow excitatory postsynaptic potential (e.p.s.p.) which can be produced by electrical stimulation of presynaptic nerves within the myenteric ganglia. We wondered whether SP might mediate this slow synaptic potential. We report here that the SP depolarization and the slow e.p.s.p. are reversibly depressed by chymotrypsin, an enzyme which degrades SP, although the responses to acetylcholine, serotonin and an unknown hyperpolarizing transmitter are unaffected. The results provide direct evidence that a peptide can mediate chemical transmission between neurones in the mammalian nervous system.     

Axoplasmic transport of muscarinic receptors

The reality of axoplasmic transport is widely accepted; various neutrotransmitters, enzymes, labelled proteins and peptides are known to move rapidly along the axons of different nerve fibres. In the terminals of sympathetic nerves, noradrenaline release is controlled by various regulatory mechanisms which imply the occurrence of presynaptic receptors. In this regard, there is considerable indirect physiological evidence for the existence of muscarinic cholinergic receptors in the sympathetic nerve endings; the stimulation by acetylcholine of such presynaptic receptors elicits an inhibitory effect on noradrenaline release. We not provide direct biochemical evidence for the occurrence in dog splenic nerve of muscarinic receptors which seem to move along the axon as suggested by their rapid accumulation on either side of a ligature.     

Neuropeptides modulate the beta-adrenergic response of purified astrocytes in vitro

Neuropeptides may have functions in the central nervous system (CNS) other than altering neuronal excitability. For example, they may act as regulators of brain metabolism by affecting glycogenolysis. Since it has been suggested that glial cells might provide metabolic support for neuronal activity, they may well be one of the targets for neuropeptide regulation of metabolism. Consistent with this view are reports that peptide-containing nerve terminals have been seen apposed to astrocytes, but it is also quite possible that peptides could act at sites lacking morphological specialization. Primary cultures containing CNS glial cells have been shown to respond to beta-adrenergic agonists with an increase in cyclic AMP and, as a result, with an increase in glycogenolysis and have also been shown to respond to a variety of peptides with changes in cyclic AMP. In the study reported here, we have examined the effects of several peptides on relatively pure cultures of rat astrocytes. We demonstrate that the increase in intracellular cyclic AMP induced by noradrenaline is markedly enhanced by somatostatin and substance P and is inhibited by enkephalin, even though these peptides on their own have little or no effect on the basal levels of cyclic AMP. Vasoactive intestinal peptide (VIP) on the other hand increases cyclic AMP in the absence of noradrenaline. These results suggest that neuropeptides influence glial cells as well as neurones in the CNS and, in the case of somatostatin and substance P, provide further examples of neuropeptides modulating the response to another chemical signal without having a detectable action on their own.     

Retrograde axonal transport of target tissue-derived macromolecules

Neurones depend on contact with their target tissues for survival and subsequent development. The protein, nerve growth factor (NGF), can be selectively taken up by sympathetic nerve terminals and reaches the neuronal perikaryon by a process of retrograde intra-axonal transport, suggesting that its role in vivo is to act as a target tissue-derived trophic factor. The development of the neurones of the chick ciliary ganglion requires the presence of structures derived from the optic cup. Several studies in vitro have shown that media conditioned by non-neuronal cells contain factors that result in the survival of neurones from ciliary ganglia. In particular, chick embryo iris, ciliary body and choroid contained large amounts of these factors indicating the presence of a target tissue-derived trophic factor for the cholinergic ciliary ganglion. This study demonstrates that neurones of the ciliary ganglion accumulate, by retrograde intra-axonal transport, proteins synthesized and released by optic tissues in culture.     

人胎基底动脉肽能神经的支配—免疫细胞化学方法研究

以16例24周龄至38周龄死亡人胎的基底动脉作材料,用免疫细胞化学方法(ABC法和PAP法)研究某些肽能神经纤维的分布。于光镜下观察到基底动脉壁的外膜中存在血管活性肠肽(VIP)样、P物质(SP)样和生长抑素(SOM)样免疫反应阳性神经纤维。这些神经纤维呈树枝样、爪样、索条样或点状,有些集合成束。在靠近中膜的外弹性膜附近,神经纤维较为密集。但中膜及内膜未发现神经纤维。不同周龄胎儿的基底动脉壁中,此三种免疫反应阳性纤维的分布区域和型式无明显差异。     

Two types of cholinergic innervation in cortex, one co-localized with vasoactive intestinal polypeptide

The existence of cholinergic neuronal cell bodies in mammalian cerebral cortex was long the subject of much controversy (see ref. 1 for review). Recently, however, a specific cholinergic marker, the acetylcholine synthesizing enzyme, choline acetyltransferase (ChAT, E.C.2.3.1.6), was demonstrated by immunohistochemical methods to be present in bipolar neurones in rat cortex. Here we show that at least 80% of these intrinsic cholinergic neurones also contain immunoreactivity for vasoactive intestinal polypeptide (VIP), a neuroactive peptide found to be present in a subpopulation of cortical neurones. On the other hand, we find that the ChAT-positive cells in the basal forebrain, which are another major source of cholinergic innervation of the cortex, contain no detectable VIP-immunoreactivity. In addition, we have observed by both light and electron microscopy that some VIP- and some ChAT-positive structures in cortex are closely associated with blood vessels.