Peptidergic transmitters in synaptic boutons of sympathetic ganglia

In sympathetic ganglia of the bullfrog, a slow synaptic potential lasting for minutes--the late slow excitatory postsynaptic potential (e.p.s.p.)--was discovered. This slow response, unlike other previously known synaptic potentials in the autonomic nervous system, is not mediated by acetylcholine or monoamines. Similar non-cholinergic, non-adrenergic slow synaptic potentials have since been found in several other vertebrate autonomic ganglia. We found that the late slow e.p.s.p. is probably mediated by a peptide that is identical to, or closely resembles, mammalian luteinizing hormone releasing hormone (LHRH), because (1) when applied directly to sympathetic neurones, LHRH and its agonists elicit a slow depolarization, associated with similar changes in membrane conductance and excitability as those occurring during the late slow e.p.s.p. Furthermore, both peptide-induced and nerve-evoked responses are blocked by antagonists of LHRH; and (2) radioimmunoassays indicate that a chain of sympathetic ganglia contains 100-800 pg of a LHRH-like peptide. Its distribution among spinal nerves, the great reduction of this substance following denervation, and its release from ganglia following isotonic KCl treatment or nerve stimulation suggest that the LHRH-like material is contained in preganglionic nerve fibres. Here we report that immunohistochemical staining of sympathetic ganglia shows that LHRH-like immunoreactivity is indeed present in synaptic boutons. We also show that the two types of ganglion cells (B cells and C cells) receive strikingly different patterns of peptidergic innervation.     

GABA affects the release of gastrin and somatostatin from rat antral mucosa

gamma-Aminobutyric acid (GABA) is regarded as the major inhibitory neurotransmitter in the central nervous system of vertebrates. GABA exerts its inhibitory actions by interacting with specific receptors on pre- and postsynaptic membranes and has been shown to inhibit somatostatin release from hypothalamic neurones in vitro. Concepts of innervation of the gastrointestinal tract have been expanded by recent studies which suggest that GABAergic neurones are not confined solely to the central nervous system but may also exist in the vertebrate peripheral autonomic nervous system. Jessen and coworkers have demonstrated the presence, synthesis and uptake of GABA by the myenteric plexus of the guinea pig taenia coli, and have documented the presence of glutamic acid decarboxylase (GAD) in isolated myenteric plexus. This enzyme is responsible for the conversion of glutamic acid to GABA in GABAergic neurones. The possibility that GABA may have a role in neurotransmission or neuromodulation in the enteric nervous system of the vertebrate gut has been suggested by several investigators. Furthermore, GABA receptors have been demonstrated on elements of the enteric nervous system. The effects of GABA on gastrointestinal endocrine cell function have not been examined. We report here the effects of GABA on gastrin and somatostatin release from isolated rat antral mucosa in short-term in vitro incubations.     

An N-methylaspartate receptor-mediated synapse in rat cerebral cortex: a site of action of ketamine?

It has been proposed that three major receptor subtypes subserve the putative transmitter role of glutamate and aspartate in the mammalian central nervous system. One subtype is classified by the specific agonist N-methylaspartate (NMA) and the specific antagonist 4-amino-2-phosphonovaleric acid. It has been shown recently that excitation of neurones by NMA is also selectively reduced by dissociative anaesthetics such as ketamine and phencyclidine and by sigma opiates, drugs of abuse with common psychotomimetic properties. Responses to NMA have an unusual voltage relation which may result from a voltage-dependent block of the activated channel by physiological concentrations of magnesium. No synaptic potential with properties similar to those of responses to NMA, however, has yet been reported. We describe here an excitatory postsynaptic potential (e.p.s.p.) evoked by electrical stimulation of the white matter and recorded intracellularly from pyramidal cells in slices of rat somatosensory cortex. This e.p.s.p. has the appropriate voltage relation and sensitivity to Mg2+ and ketamine to be an NMA receptor-mediated synapse and a potential central site for the psychotomimetic actions of ketamine.     

Role of endogenous substance P in stress-induced activation of mesocortical dopamine neurones

The dopamine (DA) innervation to the forebrain arises from subpopulations of midbrain DA neurones broadly classified as nigrostriatal, mesolimbic and mesocortical. Significant differences in the autoregulatory mechanisms and neuronal inputs of these DA pathways may account for their differences in physiological and pharmacological responsiveness. For example, footshock stress can activate rat mesocortical DA cells but does not alter nigrostriatal DA turnover, while also decreasing substance P (SP) concentrations in the midbrain interpeduncular nucleus and in the adjacent ventral tegmental area (VTA), but not in the substantia nigra (SN). This suggested that the activation of the SP input to the VTA may mediate activation of certain DA systems by footshock stress; behavioural studies also had suggested an excitatory effect of SP on DA cells in the VTA. SP antagonists now available are neurotoxic and of questionable efficacy, we therefore used monoclonal antibody against SP. Antibody microinjected into the VTA prevented normal footshock-induced activation of mesocortical DA neurones, suggesting mediation by SP input to the VTA. The in vivo application of antibodies may prove valuable in studies of neuropeptides in the central nervous system (CNS).     

Hyperpolarization following activation of K+ channels by excitatory postsynaptic potentials

We have postulated that an excitatory postsynaptic potential (e.p.s.p.) may open voltage-sensitive K+ ('M') channels, in an appropriate depolarizing range, and that this could alter the e.p.s.p. waveform. Consequently, the fast e.p.s.p. in neurones of sympathetic ganglia, elicited by a nicotinic action of acetylcholine (ACh), could be followed by a hyperpolarization, produced by the opening of M channels during the depolarizing e.p.s.p. and their subsequent slow closure (time constant-150 mg). This introduces the concept that transmitter-induced p.s.ps may trigger voltage-sensitive conductances other than those initiating action potentials, and that in the present case this could produce a true post-e.p.s.p. hyperpolarization. (Some hyperpolarizations other than inhibitory postsynaptic potentials (i.p.s.ps) have been reported to follow e.p.s.ps.) We show here that this is so.     

GABA may be a neurotransmitter in the vertebrate peripheral nervous system

gamma-Aminobutyric acid (GABA) is an inhibitory neurotransmitter in the peripheral nervous system of certain invertebrates and is thought to be a major transmitter in the vertebrate central nervous system. In this report we present evidence that GABA may also be a neurotransmitter in the vertebrate peripheral autonomic nervous system. We have used light and electron microscopic autoradiography to analyse high-affinity uptake of 3H-GABA into the myenteric plexus of the guinea pig taenia coli, both in situ and in a tissue culture preparation. In the isolated myenteric plexus, we have measured the specific activity of glutamic acid decarboxylase (GAD; EC 4.1.1.15), the enzyme responsible for conversion of glutamic acid to GABA in GABAergic neurones, and assessed the ability of this tissue to accumulate 3H-GABA newly synthesised from 3H-glutamic acid. Furthermore, we have measured the levels of endogenous GABA in strips of taenia coli containing the myenteric plexus.     

Enteric neurogenesis by neural crest-derived branchial arch mesenchymal cells

We have previously described a monoclonal antibody (E/C8) that recognizes an avian-specific epitope present in a variety of embryonic cells, including some cultured neural crest cells, both central and peripheral neurones in vivo, and apparently non-neuronal neural crest-derived mesenchymal cells of the posterior (third and fourth) branchial arches. The branchial arches are transient embryonic structures that serve as the lateral and ventral walls of the primitive pharynx of vertebrates and are contiguous with the developing gut. We report here that E/C8-positive mesenchymal cells of the arches can develop into neurones spontaneously in culture, or can migrate into aneural guts with which they are co-cultured and form enteric ganglia. In contrast, these cells do not develop into melanocytes--another derivative of the neural crest--in various permissive conditions. These results demonstrate that the mesenchymal cells of the posterior branchial arches are a developmentally restricted population of neural crest-derived cells, and some may serve as precursors for neurones of the enteric nervous system.     

A cell surface molecule distributed in a dorsoventral gradient in the perinatal rat retina

Brain topography may have its earliest expression as spatial gradients of molecules controlling the deposition of neurones and neuronal processes. In the vertebrate visual system there is evidence that the stereotyped alignment of central retinal projections relies on an initial spatially organized distribution of molecules in both the retina and its central target nuclei. We used an immunological approach to look for molecules that are so organized and produced a monoclonal antibody (JONES) which shows a pronounced dorsal to ventral gradient of binding in the rat retina throughout the period when retinal ganglion cell axons are forming topographically organized projections within the central nervous system (CNS). Binding is present throughout the radial thickness of the retinal epithelium in regions where postmitotic neurones are generated but is not associated with any consistent histological characteristic of the tissue. The antibody was shown to bind on the cell surface of freshly dissociated retinal cells, and dorsal retinal quadrants were found in vitro to have nearly twice as much antigen as ventral retinal quadrants. Initial biochemical characterization of the target epitope reveals that it is a lipid present in chloroform/methanol extracts from perinatal retina and is sensitive to neuraminidase digestion.     

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.     

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.     

Developmental regulation of NMDA receptor-mediated synaptic currents at a central synapse.

The central nervous system has extraordinary plasticity in early life. This is thought to involve N-methyl-D-aspartate (NMDA) receptors which, along with the non-NMDA receptors, mediate fast excitatory synaptic transmission. Although NMDA receptors may be transiently enhanced early in life, it has not been possible to demonstrate directly a functional change in the NMDA receptor-mediated synaptic response because of the voltage-dependence of the NMDA conductance and the overlapping inhibitory synaptic conductances. Here I report that the duration of evoked NMDA-receptor-mediated excitatory postsynaptic currents (e.p.s.cs) in the superior colliculus is several times longer at early developmental stages compared to that measured in older animals. In contrast, the amplitude of NMDA-receptor-mediated miniature e.p.s.cs does not change during development. The kinetic response of excised membrane patches to a brief activation of NMDA receptors is similar to that of the NMDA e.p.s.c, which suggests that the time course of the NMDA e.p.s.c. in the superior colliculus reflects slow NMDA channel properties as in the hippocampus. Therefore, these data indicate that the molecular properties of NMDA receptors are developmentally regulated and thus may be controlling the ability of synapses to change in early life.     

Channel kinetics determine the time course of NMDA receptor-mediated synaptic currents

Synaptic release of glutamate results in a two component excitatory postsynaptic current (e.p.s.c.) at many vertebrate central synapses. Non-N-methyl-D-aspartate receptors mediate a component that has a rapid onset and decay while the component mediated by N-methyl-D-aspartate (NMDA) receptors has a slow rise-time and a decay of several hundred milliseconds, 100 times longer than the mean open time of NMDA channels. The slow decay of the NMDA-mediated e.p.s.c. could be due to residual glutamate in the synaptic cleft resulting in repeated binding and activation of NMDA receptors. However, in cultured hippocampal neurons, we find that the NMDA receptor antagonist D-2-amino-5-phosphonopentanoate has no effect on the slow e.p.s.c. when rapidly applied after activation of the synapse, suggesting that rebinding of glutamate does not occur. In addition, a brief pulse of glutamate to an outside-out membrane patch results in openings of NMDA channels that persist for hundreds of milliseconds, indicating that glutamate can remain bound for this period. These results imply that a brief pulse of glutamate in the synaptic cleft is sufficient to account for the slow e.p.s.c.     

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.     

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%.     

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.     

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.     

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.     

Mechanisms of slow postsynaptic potentials

Classical views of synaptic transmission have to be modified to take account of slow postsynaptic potentials (p.s.ps), which are often mediated by novel ionic and molecular mechanisms and which are sometimes evoked by neuropeptides. Understanding slow p.s.ps will provide insights into the mechanisms of biological signal transduction and long-term signalling in the nervous system.     

P物质对大鼠星状神经节细胞的影响

应用细胞内生物电记录方法，观察神经肽Ｐ物质（ＳＰ）对大鼠星状神经节能细胞的影响，用灌流或用加压向细胞周围施加ＳＰ可使部分细胞发生膜除极反应，用低钙或用含河豚毒素克氏液灌流神经节，并不影响ＳＰ引起的除极反应的幅度和时程。ＳＰ引起除极反应的同时常伴有膜电阻增大，当膜电位增大时，除极化反应幅度变小，反转电位为－８０ｍＶ至－１００ｍＶ，研究表明，ＳＰ对部分星状神经节细胞具有直接兴奋作用，并且ＳＰ对细胞膜的