Sensory transmitters regulate intracellular calcium in dorsal horn neurons

Primary afferent terminals in the dorsal horn of the spinal cord release excitatory amino acid and peptide transmitters that initiate the central processing of nociceptive information. The postsynaptic actions of amino acid transmitters on spinal neurons have been well characterized, but the cellular basis of peptide actions remains unclear. Substance P is the best characterized of the peptides present in sensory neurons and has been shown to depolarize dorsal horn neurons and to facilitate nociceptive reflexes. To determine the mechanisms by which substance P contributes to afferent synaptic transmission, we have monitored the levels of intracellular calcium in single isolated rat dorsal horn neurons and report that substance P can produce a prolonged elevation in calcium concentration by mobilizing its release from intracellular stores. This elevation may contribute to the long-term changes in the excitable properties of dorsal horn neurons that occur following afferent fibre stimulation. We have also found that L-glutamate elevates intracellular calcium in substance P-sensitive dorsal horn neurons by increasing calcium influx. These results provide a direct demonstration of intracellular calcium changes in response to neuropeptides in mammalian central neurons. They also indicate that there is convergent regulation of intracellular calcium in dorsal horn neurons by two different classes of sensory transmitters that are co-released from the same afferent terminals.     

ATP excites a subpopulation of rat dorsal horn neurones

The peripheral receptive properties and central projections of different classes of dorsal root ganglion neurones are well characterized. Much less is known about the transmitters used by these neurones. Excitatory amino acids have been proposed as sensory transmitters but the sensitivity of virtually all central neurones to those compounds has made it difficult to assess their precise role in sensory transmission. Several neuropeptides have been localized within discrete subclasses of primary sensory neurones that project to the superficial dorsal horn of the spinal cord and may be afferent transmitters. However, only about one-third of spinal sensory neurones have been shown to contain neuropeptides. We have recently described the presence of a 5'-nucleotide hydrolysing acid phosphatase in a separate subpopulation of dorsal root ganglion neurones that project to the superficial dorsal horn. This enzyme also appears in certain autonomic and endocrine cells that contain high concentrations of releasable nucleotides in their storage granules. It is possible that the presence of this enzyme in sensory neurones is also associated with a releasable pool of nucleotides. Holton and Holton have provided evidence that ATP is released from the peripheral terminals of unmyelinated sensory fibres and have suggested that release of ATP might also occur from central sensory terminals. To investigate the possibility that nucleotides act as central sensory transmitters we have examined their actions on rat dorsal horn and dorsal root ganglion neurones maintained in dissociated cell culture. We report here a selective and potent excitation of subpopulations of both neuronal types by ATP.     

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.     

Monoclonal antibodies against carbohydrate differentiation antigens identify subsets of primary sensory neurones

Dorsal root ganglion (DRG) neurones transmit cutaneous sensory information from the periphery to the spinal cord. Within the dorsal horn of the spinal cord, classes of sensory fibres that are activated by different cutaneous stimuli terminate in separate and highly restricted laminae. Although the developmental events resulting in the laminar organization of sensory afferent terminals have not been defined, it is likely that interactions between surface molecules on DRG and dorsal horn neurones are involved in the generation of afferent synaptic connections. The identification of surface antigens that distinguish functional subclasses of DRG neurones would represent a first step in establishing the existence and nature of such molecules. We report here that monoclonal antibodies directed against carbohydrate differentiation antigens identify cytoplasmic and cell surface molecules expressed selectively by functional subsets of DRG neurons.     

Stimulation of connective tissue cell growth by substance P and substance K

Connective tissue cells proliferate actively when cultured in the presence of serum. Platelet-derived growth factor (PDGF), a basic protein of relative molecular mass approximately 30,000, has been identified as the major serum mitogen for these cells; its main physiological/pathophysiological role may be to initiate wound healing in connection with tissue injury. However, growth of cultured cells is also influenced by several other factors, including epidermal growth factor, fibroblast growth factor, insulin and somatomedins. Furthermore, Rozengurt and Sinnett-Smith recently showed that bombesin, a neuroendocrine peptide isolated from frog skin, stimulates DNA synthesis and cell division in cultures of a specific subtype of 3T3 cells. Substance P and substance K (also known as neurokinin A or neuromedin L) are mammalian peptides belonging to the tachykinin family. Substance P has been studied extensively; it is distributed widely throughout the central and peripheral nervous system, including primary sensory neurones, and can be released in the periphery from axon collaterals of stimulated pain fibres and contribute to the inflammatory response. Substance K is a member of the tachykinin family isolated from mammalian spinal cord; Nawa et al. determined the primary structure of two types of substance P precursors, one of which contained a sequence homologous to substance K, as well as the sequence of substance P. We report here that substance P and substance K stimulate DNA synthesis in cultured arterial smooth muscle cells and human skin fibroblasts, and that this stimulation is inhibited by the substance P-antagonist spantide.     

Substance P raises neuronal membrane excitability by reducing inward rectification

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

A monoclonal antibody defining antigenic determinants on subpopulations of mammalian neurones and Trypanosoma cruzi parasites

An IgM lambda class monoclonal antibody raised against membranes from rat dorsal root ganglia defines a novel antigenic determinant expressed by subpopulations of mammalian central and peripheral neurones. In the presence of complement the antibody is cytotoxic to mammalian neurones in vitro. The same antibody labels Trypanosoma cruzi, the protozoan responsible for Chagas' disease. Classes of mammalian neurones and cardiac muscle that are labelled by the antibody are known to degenerate in Chagas' disease. The common neuronal and trypanosomal antigens recognized by the antibody may therefore be important in pathogenic events underlying this disorder.     

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     

Evidence that disinhibition of brain stem neurones contributes to morphine analgesia

Analgesia results when opiates are microinjected into the rostral ventromedial medulla (RVM). This region, which includes the nucleus raphe magnus and the adjacent reticular formation, is rich in immunoreactive enkephalin-containing neurones and terminals, and contains neurones that project to the spinal cord dorsal horn where they inhibit identified nociceptive spinothalamic tract neurones. Although opiates have previously been reported either to excite or inhibit RVM cells, the possibility of an opiate effect being consistent within a physiologically defined subclass has not been examined. Recently we described a class of neurone in the RVM (the off-cell) that abruptly pauses just before a heat-evoked tail-flick reflex. If off-cells are made to fire continuously by direct electrical stimulation of the RVM, the tail-flick reflex does not occur. We report here that analgesic doses of morphine completely eliminate the pause in firing that precedes the tail-flick reflex. We propose that this disinhibition of off-cells in the RVM is a primary process contributing to opiate inhibition of nociceptor-induced reflexes.     

Central nervous system and peripheral nerve growth factor provide trophic support critical to mature sensory neuronal survival

Primary sensory neurones in cranial and dorsal root ganglia (DRG) of adult animals are generally thought to be maintained through connections with their peripheral (but not central) targets by trophic factor(s) other than nerve growth factor (NGF). Damage to the peripheral process of sensory neurones results in a dramatic response or even death of the neurones, whereas axotomy (cutting) of the central process does not initiate profound reaction in these neurones. The development and maintenance of neurones are highly dependent on a supply of trophic agents produced by targets and retrogradely transported via the peripheral process to the cell body. NGF deprivation in fetal rodents produced either by exogenously administered antibodies or by those of maternal origin, results in death of DRG and of some cranial sensory neurones. However, as chronic NGF deprivation in neonatal or adult rodents produces little or no cell death, it has been assumed that some other trophic factor(s) derived from the peripheral target sustains sensory neurones in postnatal life. By inducing NGF deprivation by autoimmunizing guinea pigs with mouse NGF and/or by cutting the central root (process) of a DRG, we demonstrate here that under certain conditions DRG neurones require NGF and centrally derived trophic support. Our results indicate that sensory neurones are maintained by the trophic support provided by both peripheral and central targets. This support is mediated by NGF and other as yet unidentified trophic factors. The relative importance of the two target fields and NGF compared with other trophic factors changes during development.     

Varicella-zoster virus DNA in human sensory ganglia

Varicella-zoster virus (VZV) causes chickenpox and shingles. Clinical and epidemiological evidence indicates that following an episode of childhood chickenpox (varicella), VZV becomes latent, presumably in dorsal root ganglia, and is reactivated many years later to produce shingles (zoster) in adults. VZV has been demonstrated in ganglia by electron microscopy and by indirect immunofluorescence, and infectious viral particles have been isolated from acutely infected ganglia of patients who died of disseminated VZV infection. However, VZV has not been detected in the ganglia of humans without recent exposure to VZV. Tissue culture explant methods that have been successful in the isolation of herpes simplex virus from ganglia have so far failed in the isolation or reactivation of VZV from trigeminal and other dorsal root ganglia. We describe here the detection of VZV DNA sequences in an acutely infected human sacral ganglion and in normal trigeminal ganglia. These findings support the hypothesis that VZV is latent in normal human ganglia.     

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.     

Substance P-immunoreactive retinal ganglion cells and their central axon terminals in the rabbit

Retinal ganglion cells are the projection neurons that link the retina to the brain. Peptide immunoreactive cells in the ganglion cell layer (GCL) of the mammalian retina have been noted but their identity has not been determined. We now report that, in the rabbit, 25-35% of all retinal ganglion cells contain substance P-like (SP) immunoreactivity. They were identified by either retrograde transport of fluorescent tracers injected into the superior colliculus, or by retrograde degeneration after optic nerve section. SP immunoreactive cells are present in all parts of the retina and have medium to large cell bodies with dendrites that ramify extensively in the proximal inner plexiform layer. Their axons terminate in the dorsal lateral geniculate nucleus, superior colliculus and accessory optic nuclei, and these terminals disappear completely after contralateral optic nerve section and/or eye enucleation. In the dorsal lateral geniculate nucleus large, beaded, immunoreactive axons and varicosities make up a narrow plexus just below the optic tract, where they define a new geniculate lamina. The varicosities make multiple synaptic contacts with dendrites of dorsal lateral geniculate nucleus projection neurons and presumptive interneurons in complex glomerular neuropil. This is direct evidence that some mammalian retinal ganglion cells contain substance P-like peptides and strongly suggests that, in the rabbit, substance P (or related tachykinins) may be a transmitter or modulator in a specific population or populations of retinal ganglion cells.     

Dynamic receptive field plasticity in rat spinal cord dorsal horn following C-primary afferent input

The central terminals of cutaneous primary afferent neurons are spatially ordered in the dorsal horn in a highly organized fashion such that a point-to-point map represents the body surface. This afferent terminal somatotopic map correlates with the map of the receptive fields of the cells on which they terminate. The location, size and modality of the cutaneous receptive fields of dorsal horn neurons necessarily depend upon the anatomical presence of afferent nerve fibres which deliver information from the periphery, directly or indirectly, to the cells. However the receptive field size and modality of a cell do not depend only on anatomical connections. Excitatory and inhibitory interneurons, descending influences and facilitations or depressions of synaptic contacts can alter receptive field properties. Here we show that prolonged and substantial cutaneous receptive field changes can be produced by brief inputs from peripheral unmyelinated afferent fibres.     

Autoradiographic distribution of substance P receptors in rat central nervous system

Among various neuropeptides present in the central nervous system (CNS), substance P, an undecapeptide, is of great interest as a putative pain neurotransmitter. Substance P is present within numerous intrinsic neural pathways throughout the CNS. Several groups have attempted to label substance P receptors on brain membranes by ligand binding techniques; only one study used native 3H-labelled substance P as the ligand and the precise anatomical distribution of substance P receptors has not yet been described. Here we report the autoradiographic localization of 3H-labelled substance P receptors in rat brain using the in vitro autoradiographic technique developed recently. 3H-substance P binds specifically to an apparently single class of sites on slide-mounted brain sections (Kd = 0.52 nM; Bmax = 21.6 fmol per mg protein). The ligand selectivity pattern suggests that 3H-substance P binding sites are similar to those found in other assays. 3H-substance P receptors are highly concentrated in the external layers of the olfactory bulb, medial amygdala, dentate gyrus, superior colliculus, dorsal parabrachial nucleus and locus coeruleus, with moderate densities being found in the nucleus accumbens, striatum, periaqueductal grey and subiculum. The distribution of 3H-substance P receptors suggests that substance P is probably involved in the control of sensory processes such as pain, vision, audition and olfaction.     

Corticotropin regulates transsynaptically the activity of septo-hippocampal cholinergic neurones

The activity of septo-hippocampal neurones is affected by the action on cholinergic perikarya in the septum of a variety of putative neurotransmitters, including substance P and beta-endorphin. (The latter is released in the septal region from neurones which originate in the medial basal hypothalamus.) It has also been reported that two other neuropeptides, corticotropin (ACTH1-24) and alpha-melanotropin (alpha-MSH), affect acetylcholine turnover in septo-hippocampal neurones in a manner that is not blocked by transection of the afferents to the hippocampus, from which it has been inferred that the neurotransmitters act directly on the hippocampus. We now describe experiments with corticotropin which show that the effect is rather the influence on septo-hippocampal cholinergic neurones of peptidergic neurones within the septum.     

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.     

Peripheral nerve injury triggers central sprouting of myelinated afferents.

The central terminals of primary afferent neurons are topographically highly ordered in the spinal cord. Peripheral receptor sensitivity is reflected by dorsal horn laminar location: low-threshold mechanoreceptors terminate in laminae III and IV (refs 2, 3) and high-threshold nociceptors in laminae I, II and V (refs 4,5). Unmyelinated C fibres, most of which are nociceptors, terminate predominantly in lamina II (refs 5, 7). There is therefore an anatomical framework for the transfer of specific inputs to localized subsets of dorsal horn neurons. This specificity must contribute to the relationship between a low-intensity stimulus and an innocuous sensation and a noxious stimulus and pain. We now show that after peripheral nerve injury the central terminals of axotomized myelinated afferents, including the large A beta fibres, sprout into lamina II. This structural reorganization in the adult central nervous system may contribute to the development of the pain mediated by A-fibres that can follow nerve lesions in humans.     

Induction of c-fos-like protein in spinal cord neurons following sensory stimulation

It has been suggested that the proto-oncogenes c-fos and c-myc participate in the control of genetic events which lead to the establishment of prolonged functional changes in neurons. Expression of c-fos and c-myc are among the earliest genetic events induced in cultured fibroblast and phaeochromocytoma cell lines by various stimuli including growth factors, peptides and the intracellular second messengers diacylglycerol, cAMP and Ca2+. We report here that physiological stimulation of rat primary sensory neurons causes the expression of c-fos-protein-like immunoreactivity in nuclei of postsynaptic neurons of the dorsal horn of the spinal cord. Activation of small-diameter cutaneous sensory afferents by noxious heat or chemical stimuli results in the rapid appearance of c-fos-protein-like immunoreactivity in the superficial layers of the dorsal horn. However, activation of low-threshold cutaneous afferents results in fewer labelled cells with a different laminar distribution. No c-fos induction was seen in the dorsal root ganglia, gracile nucleus or ventral horn. Thus, synaptic transmission may induce rapid changes in gene expression in certain postsynaptic neurons.     

鸡脑P物质的制取及分析

P物质是一种广泛存在于哺乳动物中枢及外周神经元系统中的11肽。采用有机溶剂分级法提纯鸡脑P物质,紫外分光光度计测定含量,聚酰胺薄膜层析分析氨基酸组成。结果表明:鸡脑P物质的氨基酸组成和N端氨基酸与资料中完全一致。