Evidence for coexistence of dopamine and CCK in meso-limbic neurones

Vanderhaeghen et al. reported the occurrence of gastrin-like immunoreactivity in the mammalian brain. Subsequent studies have revealed that this immunoreactivity corresponded mainly to the COOH-terminal octapeptide of cholecystokinin (CCK-8), which has a COOH-terminal pentapeptide identical to gastrin. Also, two peptides resembling the NH- and the COOH-terminal tetrapeptide fragments of CCK-8 are present in the central nervous system (CNS). Using COOH-terminal-specific antisera raised to gastrin and/or CCK, the distribution of CCK neurones has been described with immunohistochemical techniques. Although high numbers of cells and nerve terminals are found in cortical areas, the CCK systems are also present in most other parts of the brain and spinal cord. In the CNS, true gastrin molecules, gastrin-17 and gastrin-34 have been located only in the neurohypophysis, hypothalamus and occasionally in the medulla oblongata (unpublished results). We describe here the occurrence of peptides in meso-limbic dopamine neurones in the rat brain. Evidence has also been obtained that mesencephalic dopamine neurones in the human brain contain similar peptides.     

Cholecystokinin induces a decrease in Ca2+ current in snail neurons that appears to be mediated by protein kinase C

Three distinct classes of protein kinases have been shown to regulate Ca2+ current in excitable tissues. Cyclic AMP-dependent protein kinase mediates the action of noradrenaline on the Ca2+ current of cardiac muscle cells. Cyclic GMP-dependent protein kinase mediates the serotonin-induced modulation of the Ca2+ current in identified snail neurons. The Ca2+/diacylglycerol-dependent protein kinase (protein kinase C) has also been found to regulate Ca2+ currents of neurons. However, no neurotransmitter has yet been shown to regulate Ca2+ current through the activation of protein kinase C. We now report that cholecystokinin, a widely occurring neuropeptide which is present in molluscan neuron, modulates the Ca2+ current in identified neurons of the snail Helix aspersa, and that this effect appears to be mediated by protein kinase C. Specifically, sulphated cholecystokinin octapeptide 26-33 (CCK8), activators of protein kinase C, and intracellular injection of protein kinase C, all shorten the Ca2+-dependent action potential and decrease the amplitude of the Ca2+ current in these cells. All these effects are not reversible within the duration of the experiments. Moreover, intracellular injections of low concentrations of protein kinase C, which are ineffective by themselves, enhance the effectiveness of low concentrations of CCK8 on the Ca2+ current.     

A physiological role for endogenous zinc in rat hippocampal synaptic neurotransmission.

The mammalian central nervous system (CNS) contains an abundance of the transition metal zinc, which is highly localized in the neuronal parenchyma. Zinc is actively taken up and stored in synaptic vesicles in nerve terminals, and stimulation of nerve fibre tracts that contain large amounts of zinc, such as the hippocampal mossy fibre system, can induce its release, suggesting that it may act as a neuromodulator. The known interaction of zinc with the major excitatory and inhibitory amino-acid neurotransmitter receptors in the CNS supports this notion. That zinc has a role in CNS synaptic transmission, however, has so far not been shown. Here we report a physiological role for zinc in the young rat hippocampus (postnatal, P3-P14 days). Our results indicate that naturally occurring spontaneous giant depolarizing synaptic potentials (GDPs) in young CA3 pyramidal neurones, mediated by the release of GABA (gamma-aminobutyric acid), are induced by endogenously released zinc. These synaptic potentials are inhibited by specific zinc-chelating agents. GDPs are apparently generated by an inhibitory action of zinc on both pre- and postsynaptic GABAB receptors in the hippocampus. Our study implies that zinc modulates synaptic transmission in the immature hippocampus, a finding that may have implications for understanding benign postnatal seizures in young children suffering with acute zinc deficiency.     

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.     

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.     

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.     

Selective antagonists of benzodiazepines

Benzodiazepines produce most, if not all, of their numerous effects on the central nervous system (CNS) primarily by increasing the function of those chemical synapses that use gamma-amino butyric acid (GABA) as transmitter. This specific enhancing effect on GABAergic synaptic inhibition is initiated by the interaction of benzodiazepines with membrane proteins of certain central neurones, to which drugs of this chemical class bind with high affinity and specificity. The molecular processes triggered by the interaction of these drugs with central benzodiazepine receptors, and which result in facilitation of GABAergic transmission, are still incompletely understood. Theoretically, benzodiazepines could mimic the effect of hypothetical endogenous ligands for the benzodiazepine receptors, although there is no convincing evidence for their existence; in vitro studies indicate that benzodiazepines might compete with a modulatory peptide which is present in the supramolecular assembly formed by GABA receptor, chloride ionophore and benzodiazepine receptor and which reduces the affinity of the GABA receptor for its physiological ligand. The mechanisms of action of benzodiazepines at the molecular level are likely to be better understood following our recent discovery of benzodiazepine derivatives, whose unique pharmacological activity is to prevent or abolish in a highly selective manner at the receptor level all the characteristic centrally mediated effects of active benzodiazepines. Here, we describe the main properties of a representative of this novel class of specific benzodiazepine antagonists.     

Replication of the neurochemical characteristics of Huntington's disease by quinolinic acid

Huntington's disease (HD) is an autosomal dominant neurological disorder characterized by progressive chorea, cognitive impairment and emotional disturbance. The disease usually occurs in midlife and symptoms progress inexorably to mental and physical incapacitation. It has been postulated that an excitotoxin is involved in the pathogenesis of HD. Schwarcz and colleagues have shown that quinolinic acid (QA) can produce axon-sparing lesions similar to those observed in HD. The lesions result in a depletion of neurotransmitters contained within striatal spiny neurones, for example gamma-aminobutyric acid (GABA), while dopamine is unaffected. Recently, we and others have demonstrated that in HD striatum there is a paradoxical 3-5-fold increase in both somatostatin and neuropeptide Y which is attributable to selective preservation of a subclass of striatal aspiny neurones in which these peptides are co-localized. In the present study we demonstrate that lesions due to quinolinic acid closely resemble those of HD as they result in marked depletions of both GABA and substance P, with selective sparing of somatostatin/neuropeptide Y neurones. Lesions produced by kainic acid (KA), ibotenic acid (IA) and N-methyl-D-aspartate (MeAsp) were unlike those produced by QA, as they affected all cell types without sparing somatostatin/neuropeptide Y neurones. These results suggest that QA or a similar compound could be responsible for neuronal degeneration in HD.     

琥珀酰CCK—7对小鼠记忆的影响

琥珀酰ＣＣＫ－７是对ＣＣＫ－８进行分子改构后获得的一种新药，本实验以一次被动回避反应模型观察比较了这种改构物与ＣＣＫ－８对小鼠记忆巩固过程的影响，同时探讨了ｓｕｃ－ＣＣＫ－７影响记忆过程的剂量－效应关系以及时相－效应关系。结果表明：（１）ｓｕｃ－ＣＣＫ－７促进记忆的有效剂量为１００－２００μｇ／ｋｇ，小于１００μｇ／ｋｇ无明显效应。（２）ｓｕｃ－ＣＣＫ－７增强小鼠记忆的效应在电击后４８ｈ最明显，７     

Substance P in the ascending cholinergic reticular system

The neocortex receives a major cholinergic innervation from magnocellular neurones in the basal forebrain. However, an ascending cholinergic reticular system has also been postulated to arise from acetylcholinesterase (AChE)-containing neurones in the midbrain and pontine tegmentum. Lesions of this region decrease both AChE and choline acetyltransferase (ChAT) in various forebrain areas, and recent immunohistochemical studies have identified a group of ChAT-containing cell bodies in the midbrain reticular formation and dorsolateral pontine tegmentum. Here we have combined retrograde tracing with ChAT immunohistochemistry to demonstrate that this tegmental cholinergic cell group also directly innervates the cerebral cortex. Other immunohistochemical studies have indicated that the neuropeptide substance P is also present in certain cells in the laterodorsal tegmentum, and these too appear to project to the forebrain. We have therefore performed immunohistochemistry for both ChAT and substance P and have discovered that a subpopulation of the ascending cholinergic reticular neurones contains substance P. Thus, peptide-cholinergic coexistence, previously noted in peripheral neurones, also occurs in the brain.     

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.     

Inhibitory neurones of a motor pattern generator in Xenopus revealed by antibodies to glycine

Glycine and gamma-aminobutyric acid (GABA) are inhibitory transmitters of major importance. Whereas neurones using GABA as the transmitter can be visualized by immunocytochemical methods for glutamate decarboxylase (GAD) or GABA, no comparable techniques have been available for the selective visualization of glycinergic neurones. We have now produced polyclonal antibodies which specifically recognize glycine in glutaraldehyde-fixed tissue. We used these antibodies to investigate the distribution of glycine in the simple central nervous system (CNS) of the Xenopus embryo, which contains an anatomically and physiologically defined class of reciprocal inhibitory interneurones, the commissural interneurones. These interneurones have an important role in the generation of the swimming motor pattern and are thought to be glycinergic. The glycine antibodies specifically stain these interneurones, revealing their distribution and number in the embryo CNS. This is the first demonstration of the selective localization of glycine-like immunoreactivity in a putative glycinergic class of neurone that has been characterized physiologically, pharmacologically and anatomically.     

Selective collateral elimination in early postnatal development restricts cortical distribution of rat pyramidal tract neurones

The pyramidal tract, comprising those axons which pass from the neocortex to the medulla and spinal cord, is among the most thoroughly studied projections of the mammalian cortex. Recent studies using anterograde axon tracing techniques have provided information concerning the time course of the growth of pyramidal tract fibres, yet much remains to be learned about its development. We have now begun to study the distribution of the neurones of origin of the pyramidal tract during the postnatal development of the rat neocortex using the recently introduced retrogradely transported fluorescent marker, True blue. During the first postnatal week, injections of True blue into the pyramidal decussation result inthe labelling of pyramidal tract neurones which are distributed virtually throughout the tangential extent of layer V of the neocortex, whereas after comparable injections during the fourth postnatal week the distribution of such cells is much more restricted and remains restricted into adult life. This developmental restriction is most dramatic in the occipital cortex: during the first postnatal week many pyramidal tract neurones are found throughout the visual cortex while none is seen in this area of the adult. When True blue is injected into the pyramidal decussation during the first postnatal week and the animals are allowed to survive until the fourth postnatal week, the distribution of pyramidal tract neurones is as widespread as in the immediate postnatal period and includes the entire visual cortex. This implies that many of the neurones in the occipital cortex initially send a collateral into the pyramidal tract which is later eliminated, although the neurones themselves persist. These findings, together with similar recent observations on the development of the callosal connections, indicate that the elimination of axon collaterals may be a general feature of the development of cortical projection systems, and that such transitory collaterals may traverse considerable distances.     

A subpopulation of rat dorsal root ganglion neurones is catecholaminergic

The neurotransmitters used by the sensory neurones of the dorsal root ganglia (DRG) are unknown. A proportion of these cells contain physiologically active peptides; for example, subpopulations of small-diameter neurones contain substance P or somatostatin. Although these peptides probably have some influence on synaptic transmission in the dorsal horn of the spinal cord, their status as neurotransmitters is uncertain and it is possible that they coexist with conventional neurotransmitters. In addition, the neurones containing identified peptides account for only a fraction of the DRG sensory neurones. There is evidence that the DRG contain catecholamines within fibres thought to be autonomic, but these substances have not been found within the sensory cell bodies themselves. Moreover, the apparently inappropriate, inhibitory physiological effect of catecholamines in the dorsal horn has argued against their being primary sensory neurotransmitter molecules. We have used here antisera against tyrosine hydroxylase (TH; EC 1.14.16.2) and dopamine-beta-hydroxylase (DBH; EC 1.14.17.1), two enzymes specific to catecholaminergic cells, to show that a subpopulation of rat DRG neurones is catecholaminergic and that the neurotransmitter they make is probably dopamine. We believe this to be the first report of catecholaminergic sensory neurones.     

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.     

Protein kinase C injection into hippocampal pyramidal cells elicits features of long term potentiation

Long-term potentiation (LTP) in the hippocampus is an interesting example of synaptic plasticity because of its induction by physiological discharge rates and its long duration. Of the possible biochemical mechanisms that regulate prolonged changes in cell function, protein phosphorylation is a particularly attractive candidate. We have therefore examined the effect of intracellular injection of calcium/diacylglycerol-dependent protein kinase (protein kinase C (PKC] in CA1 pyramidal neurones in hippocampal slices. Injection of the active enzyme elicited long-lasting enhancement of synaptic transmission, similar to LTP, whereas inactivated kinase failed to do so. The observed changes included an increased amplitude of the excitatory post-synaptic potential (e.p.s.p.) and an increased probability of firing and a reduced latency of the associated actin potential.     

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).     

Fetal occipital cortical neurones transplanted to the rostral cortex can extend and maintain a pyramidal tract axon

In adult rats, cortical neurones that send axons through the pyramidal tract are confined to layer V, over the rostral two-thirds of the cerebral hemisphere. However, during the first postnatal week, many neurones in layer V in the occipital cortex (including the visual cortex) also extend axon collaterals through the pyramidal tract and into the spinal cord. These occipital corticospinal collaterals are completely eliminated over the subsequent 2 weeks, although their cells of origin do not die. We now report that when portions of the occipital cortex from fetal rats are transplanted to more rostral cortical regions of newborn rats, some of the transplanted neurones not only extend axons through the pyramidal tract, but also maintain these axons beyond the stage at which they are normally eliminated. These results suggest that normally-eliminated cortical axons can be 'rescued' and, in the case of pyramidal tract neurones, the position of the neurones within the tangential plane of the cortex is a critical factor in determining which neurones retain and which lose their pyramidal tract collaterals.     

Stimulation of food intake in rats by centrally administered hypothalamic growth hormone-releasing factor

Hypothalamic growth hormone-releasing factors (GRFs) have been purified recently from human pancreatic (hp) tumours and from rat hypothalamus (rh). GRF peptides have strong homology with peptides of the glucagon, vasoactive intestinal polypeptide and PHI-27 family. Aside from their potent actions on release of somatotropin, no other biological actions of GRFs have been reported. GRF has been localized in neurones bordering the ventromedial hypothalamic nucleus, a region associated frequently with experimental analysis of feeding behaviour. We now report that intracerebroventricularly (i.c.v.)-administered rhGRF and hpGRF(1-40) in doses of 0.2, 2.0 and 20.0 pmol, produced an increase in food intake in hungry rats. This effect seemed to be specific to GRF as i.c.v. injections of a structurally related but physiologically inactive peptide in the same doses had no effect on feeding. In addition, peripheral injections of rhGRF or growth hormone had no effect on food intake, suggesting that the present effects may be mediated centrally. Injections (i.c.v.) of rhGRF (0.2, 2.0 and 20.0 pmol) had no effect on general activity, suggesting that GRF does not produce nonspecific arousal.