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.     

A dopamine- and cyclic AMP-regulated phosphoprotein enriched in dopamine-innervated brain regions

Several mammalian neurotransmitter candidates, for example, serotonin, dopamine and noradrenaline, may exert some of their synaptic effects by regulating protein phosphorylation systems. Comparison of the regional distribution of brain phosphoproteins with neurotransmitter systems may help to identify the specific phosphoproteins involved in the functions of particular neurotransmitters. Here we report the association of one such phosphoprotein with the dopamine pathways in brain. This protein, of apparent molecular weight (MW) 32,000 (32K), seems to be present only in nervous tissue. Its regional distribution within the brain is very similar to the pattern of dopamine-containing nerve terminals; more specifically, the protein appears to be enriched in those dopaminoceptive neurones which possess D-1 receptors (dopamine receptors coupled to adenylate cyclase). The state of phosphorylation of the protein in these dopaminoceptive neurones can be regulated by both dopamine and cyclic AMP. These results suggest that the phosphoprotein may mediate certain of the trans-synaptic effects of dopamine acting on dopaminoceptive neurones.     

Glutamate stimulates inositol phosphate formation in striatal neurones

The major excitatory amino acids, glutamate (Glu) and aspartate (Asp), are thought to act at three receptor subtypes in the mammalian central nervous system (CNS). These are termed quisqualate (QA), N-methyl-D-aspartate (NMDA) and kainate (KA) receptors according to the specific agonist properties of these compounds revealed by electrophysiological studies. Although Glu has been shown to stimulate cyclic GMP formation in brain slices, direct regulation of second messenger systems (cyclic AMP, Ca2+ or inositol phosphates) subsequent to activation of excitatory amino-acid receptors, has not been extensively studied. Here we demonstrate that in striatal neurones, excitatory amino acids, but not inhibitory or non-neuroactive amino acids, induce a three- to fourfold increase in inositol mono-, di- and triphosphate (IP, IP, IP) formation with the relative potency QA greater than Glu greater than NMDA, KA. The Glu-evoked formation of inositol phosphates appears to result principally from actions at QA as well as NMDA receptors on striatal neurones. Our results suggest that excitatory amino acids stimulate inositol phosphate formation directly, rather than indirectly by the evoked release and subsequent actions of adenosine or acetylcholine.     

Prostaglandins and the synergism between VIP and noradrenaline in the cerebral cortex

We have previously shown that vasoactive intestinal peptide (VIP) and noradrenaline (NA) interact synergistically to increase cyclic AMP levels in mouse cerebral cortical slices. The pharmacological mechanism of this synergism is the potentiation by NA, through alpha 1 adrenergic receptors, of the stimulatory effect of VIP on cAMP formation. A similar interaction has been confirmed in guinea pig cerebral cortex and in discrete nuclei of the rat hypothalamus. Furthermore VIP and NA interact synergistically to depress the spontaneous activity of identified neurons in rat neocortex. At the cellular level, this synergistic interaction suggests that VIP- and NA-containing neuronal systems may converge, at least in part, on the same target cells to increase cAMP levels in the cerebral cortex. At the molecular level, the interaction may occur at various steps in signal transduction, between receptors, intramembrane transduction processes or intracellular effector mechanisms. Here we report that the alpha 1-adrenergic potentiation of the increases in cAMP elicited by VIP involves the formation of arachidonic acid metabolites and is mimicked by prostglandins F2 alpha and E2.     

Auto-inhibition of brain histamine release mediated by a novel class (H3) of histamine receptor

Although histaminergic neurones have not yet been histochemically visualized, there is little doubt that histamine (HA) has a neurotransmitter role in the invertebrate and mammalian central nervous system. For example, a combination of biochemical, electrophysiological and lesion studies in rats have shown that histamine is synthesized in and released from a discrete set of neurones ascending through the lateral hypothalamic area and widely projecting in the telencephalon. Histamine acts on target cells in mammalian brain via stimulation of two classes of receptor (H1 and H2) previously characterized in peripheral organs and probably uses Ca2+ and cyclic AMP, respectively, as second messengers. It is well established that several neurotransmitters affect neuronal activity in the central nervous system through stimulation not only of postsynaptic receptors, but also of receptors located presynaptically which often display distinct pharmacological specificity and by which they may control their own release. Such 'autoreceptors' have been demonstrated (or postulated) in the case of noradrenaline, dopamine, serotonin, acetylcholine and gamma-aminobutyric acid (GABA) neurones but have never been demonstrated for histamine. We show here that histamine inhibits its own release from depolarized slices of rat cerebral cortex, an action apparently mediated by a class of receptor (H3) pharmacologically distinct from those previously characterized, that is, the H1 and H2 receptors.     

Defect in cyclic AMP phosphodiesterase due to the dunce mutation of learning in Drosophila melanogaster

Cyclic AMP is an intracellular mediator ('second messenger') in the nervous and endocrine control of cellular function, regulating different processes in different cell types. Although evidence is incomplete, it seems that cyclic AMP enhances the calcium-mediated release of neurotransmitter in some neurones. A simple form of memory in the mollusc Aplysia is probably encoded as a cyclic AMP-induced enhancement of neurotransmission at certain synapses of the central nervous system. The possibility that cyclic AMP participates in learning mechanisms may be explored using genetic mutants. For this purpose the fruitfly Drosophila is suitable as it is genetically well characterized and can learn through olfaction, vision or taste. We show here that independent searches for mutations of olfactory learning and of cyclic AMP metabolism, and for mutations causing female infertility have each led to the same gene--the dunce gene. Our evidence indicates that the normal dunce gene may specify a cyclic AMP phosphodiesterase.     

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.     

Oxytocin induces morphological plasticity in the adult hypothalamo-neurohypophysial system

The hypothalamo-neurohypophysial system offers a unique example in the adult mammalian central nervous system (CNS) of a functional and structural plasticity related to a physiological state. During lactation, oxytocin neurones evolve a synchronized electrical activation which permits pulsatile hormone release at milk ejection. At the same time, in the supraoptic (SON) and paraventricular nuclei, glial coverage of neurones diminishes, so that large portions of their surface membrane become directly juxtaposed; synaptic remodelling also associates pairs of neurones through the formation of common presynaptic terminals. These structural changes, reversible after weaning, affect exclusively oxytocinergic neurones and could facilitate their synchronized electrical activity. As several observations suggest that oxytocin itself is released centrally, we have examined the effect of prolonged intracerebroventricular infusions of oxytocin on the structure of the SON of non-lactating animals. We report here that the peptide indeed engenders the structural reorganization characteristic of the oxytocin system when it is physiologically activated. Similar infusion of vasopressin has no effect. Our observations thus demonstrate that a central neuropeptide can induce anatomical changes in the adult CNS, and suggest that oxytocin can regulate its own release by contributing to the dramatic restructuring of the nuclei containing the neurones responsible for its secretion.     

Role of cyclic AMP in a serotonin-evoked slow inward current in snail neurones

One model of synaptic transmission suggests that transmitters modify postsynaptic permeability through the intermediary of cyclic AMP. Thus, serotonin (5-hydroxytryptamine) evokes in molluscan neurones a decrease in a voltage-dependent K+ conductance which in turn generates a slow inward current when studied in steady voltage-clamp conditions. The serotonin-induced increase of the plateau phase of the spike of an Aplysia sensory neurone can be mimicked by both intracellularly injected cyclic AMP and extracellularly applied phosphodiesterase inhibitors, suggesting that cyclic AMP mediates the effect. We have tested whether a similar mechanism could account for the serotonin slow inward current in identified snail neurones and have found that the intracellular injection of cyclic AMP, but not of cyclic GMP or 5'-AMP, evokes a slow inward current showing similar voltage dependence, inversion potential and ionic properties to the serotonin slow inward current. Phosphodiesterase inhibitors at low concentrations (1-20 microM) potentiate the serotonin slow inward current and at higher concentrations evoke by themselves an inward current, partially or totally occluding the serotonin and cyclic AMP currents. Finally, we have found that in homogenates of pooled identified snail neurones serotonin stimulates the adenylate cyclase, increasing its activity by 50-100%.     

Rules for neural development revealed by chimaeric sensory systems in crickets

Many sensory systems are organized so that the afferent projection forms a topographic map of the sensory surface within the central nervous system (CNS). The information necessary to create such a map may be available to the neuronal cell body based on its position in the receptor array, and this 'positional information' is translated into an axonal arborization in the proper part of the CNS. To study how the location of a cell body within the sensory surface determines the termination pattern of its axon within the CNS, we have transplanted epidermis, containing identified sensory neurones, from a black cricket to a tan cricket. As we report here, when epidermis is transplanted to an unusual location in the receptor array, newly generated neurones are produced along the borders of the graft. These neurones arborize in locations that are appropriate neither to their new position nor to their original position in the array, but rather to a position somewhere in between. This is direct evidence for the idea that positional information guides the differentiation and ultimately the synaptic connections of insect sensory neurones.     

Axonal transport of vasoactive intestinal peptide in sciatic nerve.

Dense plexuses of neurones containing immunoreactive vasoactive intestinal peptide (VIP) have been found in discrete areas of the central nervous system and in peripheral organs, including the gastrointestinal tract, pancreas and urogenital system. In many of these locations VIP is concentrated in nerve endings, where it can be released by high K+ concentrations in a Ca2+-dependent manner. VIP release may also be provoked by electrical stimulation of nerves, for example the vagus. VIP thus shows some of the features of neurotransmitter or neuromodulator substances. The presence of immunoreactive VIP in the fine terminal varicosities as well as in the cell bodies of neurones suggests that it might be transported from the perikaryon, where it is presumably formed, to the nerve endings, through the axonal transport system. Such transport would be in keeping with a role for the peptide as a neurohumor or neurohormone. We report here that VIP accumulates in constricted rat sciatic nerves in a manner suggesting fast, anterograde axonal flow.     

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.     

A functional role for vasoactive intestinal polypeptide in anterior cingulate cortex

Vasoactive intestinal polypeptide (VIP) is present in high concentrations in the cerebral cortex, where it is the putative neurotransmitter of a major intracortical neuronal system. Homogenates of cortical tissue contain high-affinity, specific binding sites for VIP as well as an adenylate cyclase system which is sensitive to this peptide. As with many of the other peptidergic systems which have been identified in the central nervous system (CNS), it has proved extremely difficult to elucidate the nature and extent of the functional role of VIP in specific brain areas. Here, using the quantitative autoradiographic 14C-deoxyglucose technique in rats to provide insight into functional processes, we describe the increases in glucose utilization which occur locally in anterior cingulate cortex following the unilateral injection of VIP (20 pmol) into this key brain area and, additionally, the focal alterations in glucose use in CNS regions having known neuronal connections with the injected region (for example, ipsilateral mediodorsal thalamus, ventral tegmental area, nucleus accumbens, caudate nucleus and contralateral cingulate cortex). These data provide evidence that VIP may modify the processing of afferent and efferent information within the anterior cingulate cortex in the conscious rat.     

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.     

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.     

Phorbol esters block a voltage-sensitive chloride current in hippocampal pyramidal cells

The importance of second-messenger systems in controlling the excitability of neurones and other cells, through modulation of voltage- and calcium-dependent ionic conductances, has become increasingly clear. Cyclic AMP, acting via protein kinase A, has been identified as the second messenger for several neurotransmitters, and recent studies have suggested that activation of protein kinase C may have similar modulatory actions on neurones. Calcium and potassium currents have so far been shown to be the major ionic conductances modified by kinase activation. We now report that hippocampal pyramidal cells contain a previously undescribed voltage-dependent chloride current which is active at resting potential and is turned off either by membrane depolarization or by activation of protein kinase C by phorbol esters. We propose that this current may reside predominantly in the cell's dendritic membrane and thereby may regulate dendritic excitability.     

Regulation of glutamate receptors by cations.

Current evidence suggests that glutamate is a major excitatory neurotransmitter in the mammalian central nervous system (CNS); particularly, glutamate excites most neurones in the CNS. Until recently this effect was widely used to study glutamate receptors and to distinguish them from those of other excitatory amino acids. The development of ligand binding studies for many neurotransmitters has facilitated the study of receptors at the molecular level and using these methods we recently reported the existence in hippocampal membranes of pharmacologically distinct sodium-dependent and sodium-independent glutamate binding sites, the former related to high-affinity uptake and the latter exhibiting several characteristics of postsynaptic receptor sites. We now report that, as with other neurotransmitters, several ions regulate the Na-independent binding of glutamate; the monovalent cations induce a decreased binding while certain divalent cations enhance this Na-independent binding. Additionally, since some of these effects appear to be irreversible, we propose that the regulation of glutamate binding by cations might account for the extremely long-lasting potentiation of synaptic responses found in the hippocampus following bursts of repetitive electrical stimulation (see ref. 9 for a review).     

Modulation of a transient outward current in serotonergic neurones by alpha 1-adrenoceptors

The excitability of various neurones in the mammalian central nervous system (CNS), ranging from motoneurones to serotonergic neurones, is enhanced by alpha 1-adrenoceptor agonists. Excitations mediated via alpha 1-adrenoceptors are associated with a slow depolarization and an increase in input resistance, probably resulting from a decrease in resting potassium conductance. However, the involvement of voltage-dependent transient currents in mediating alpha 1 excitatory effects has not been evaluated. An early transient outward current has been described which is important in regulating the frequency of repetitive firing; it is activated by depolarizing voltage steps from potentials more negative than rest and blocked by 4-aminopyridine. This current, which has been termed 'IA', was found originally in invertebrates and subsequently in various vertebrate neurones. The present single-electrode voltage-clamp study demonstrates an early transient outward current (IA) in serotonergic neurones which is suppressed by noradrenaline and the alpha 1-agonist phenylephrine; a suppression of IA may account in part for the acceleration of pacemaker activity induced by alpha 1-agonists in serotonergic neurones.     

Autonomous chaotic behaviour of the slime mould Dictyostelium discoideum predicted by a model for cyclic AMP signalling

How sustained oscillations lose their periodicity and thus give rise to chaos was first analysed in mathematical models, then observed in chemical systems such as the Belousov-Zhabotinsky reaction where chaos is autonomous because it originates from endogenous kinetic mechanisms. In contrast, chaos can also be obtained by periodically forcing an oscillatory system, as shown, for example, in cardiac cells and yeast glycolysis. Biochemical evidence for autonomous chaos has been obtained both in vitro for the peroxidase reaction and in enzymatic models not based directly on experimental systems. We report here the occurrence of autonomous chaos in a realistic model for the cyclic AMP signalling system of the slime mould Dictyostelium discoideum, based on receptor modification. This model is also capable of bursting, a phenomenon characteristic of some pacemaker neurones such as R15 in Aplysia. Whereas bursting has not been observed in D. discoideum, our model suggests that 'aperiodic signalling' in the mutant Fr17 provides the first example of autonomous chaos occurring spontaneously at the cellular level.