The expression of neuropeptide Y immunoreactivity in the avian sympathoadrenal system conforms with two models of coexpression development for neurons and chromaffin cells.

We have studied the expression and development of neuropeptide Y-like immunoreactivity (NPY-LI) in the sympathoadrenal system of the chicken using single and double immunocytochemical techniques and radioimmunoassay. NPY-LI is expressed by neurons of the paravertebral sympathetic ganglia and by chromaffin cells of the adrenal gland in embryonic and adult chickens. The peptide is coexpressed with catecholaminergic properties in neurons. In chromaffin cells, it is also expressed with immunoreactivity to somatostatin and serotonin. We have used the expression of NPY-LI to analyze how cells that coexpress two or more neuroactive substances arrive at their final phenotype. Our results suggest that the ontogeny of coexpression in neurons of the avian paravertebral sympathetic ganglia occurs in a sequential pattern, where the expression of the peptide follows the initial expression of the "classical neurotransmitter". In contrast, in chromaffin cells, expression of the peptides occurs concomitantly with expression of catecholaminergic properties or soon after. Initially, coexpression of several neuroactive substances occurs, but this is followed by further specialization where the expression of one peptide prevails over the other. We believe that the two models of coexpression shown by our results can be used to describe the ontogeny of coexpression in other cells of the nervous system.     

Neuropeptide signaling near and far: how localized and timed is the action of neuropeptides in brain circuits?

Neuropeptide signaling is functionally very diverse and one and the same neuropeptide may act as a circulating neurohormone, as a locally released neuromodulator or even as a cotransmitter of classical fast-acting neurotransmitters. Thus, neuropeptides are produced by a huge variety of neuron types in different parts of the nervous system. Within the central nervous system (CNS) there are numerous types of peptidergic interneurons, some with strictly localized and patterned branching morphologies, others with widespread and diffuse arborizations. From morphology alone it is often difficult to predict the sphere of influence of a peptidergic interneuron, especially since it has been shown that neuropeptides can diffuse over tens of micrometers within neuropils, and that peptides probably are released exclusively in perisynaptic (or non-synaptic) regions. This review addresses some questions related to peptidergic signaling in the insect CNS. How diverse are the spatial relations between peptidergic neurons and their target neurons and what determines the sphere of functional influence? At one extreme there is volume transmission and at the other targeted cotransmission at synapses. Also temporal aspects of peptidergic signaling are of interest: how transient are peptidergic messages? Factors important for these spatial and temporal aspects of peptidergic signaling are proximity between release sites and cognate receptors, distribution of peptidase activity that can terminate peptide action and colocalization of other neuroactive compounds in the presynaptic peptidergic neuron (and corresponding receptors in target neurons). Other factors such as expression of different channel types, receptor inactivation mechanisms and second messenger systems probably also contribute to the diversity in temporal properties of peptide signaling.     

Peptidergic pathways in the central nervous system

Before detailed studies of the physiology and pharmacology of central peptidergic neurons can be undertaken, the location of these neurons must be determined and the mechanism(s) by which they synthesize their peptide products must be explored. In the previous paper, Dr H?kfelt described his elegant immunohistochemical studies, which are designed to answer the questions: Where are peptidergic perikarya?; Where do these perikarya send their processes?; Do these processes branch extensively and innervate several structures?; and Do peptidergic cells contain more than one active product? By studying the effects of lesions on peptide levels in microdissected tissue samples, immunocytochemical data can be confirmed and extended. The microanalytical approach also allows one to determine the nature of the immunologically active substances in a tissue extract, and in vivo or in vitro pulse--chase studies provide the ultimate validation of immunohistological localization of peptidergic perikarya and new information about the biosynthesis of peptides and regulation of this biosynthetic process. Our recent studies of central proopiocortin- and neurophysin/vasopressin-producing neurons will illustrate the above points.     

Colocalization of ChAT, DβH and NADPH-d in the pancreatic neurons of the newborn guinea pig

Choline acetyltransferase (ChAT) as a rate-limiting enzyme in the biosynthetic pathway of acetylcholine is thought to be present in all cholinergic neurons. However, its immunoreactivity has not been successfully applied to the study of cholinergic neurons in the pancreas. In a previous study in the pancreas of newborn guinea pig we reported the colocalization of nicotinamide adenine dinucleotide hydrogen phosphate-diaphorase (NADPH-d), a marker for nitric oxide synthase (NOS) with various neuropeptides as well as dopamine-β-hydroxylase (DβH), the enzyme responsible for converting dopamine to noradrenaline. Whether NADPH-d is colocalized with ChAT in the pancreatic neurons is not known. Also it would be interesting to find out whether noradrenaline and acetylcholine could be colocalized in the same pancreatic neurons. In the present study, a method for triple labelling of ChAT, DβH and NADPH-d was used to answer the above questions. Colocalization of ChAT, DβH and NADPH-d was constantly demonstrated in the same neurons in the same sections. It is concluded that some of the pancreatic neurons may utilize more than one neurotransmitter such as nitric oxide (NO), acetylcholine and noradrenaline to achieve their function. The possible cotransmission of acetylcholine and noradrenaline was extremely intriguing, and its mechanism and significance needs to be further investigated.     

Segregation of the classical transmitters norepinephrine and acetylcholine and the neuropeptide Y in sympathetic neurons: Modulation by ciliary neurotrophic factor or prolonged growth in culture

Recent evidence has demonstrated that cotransmission from mammalian neurons is not uniquely achieved by costorage and corelease of transmitters and cotransmitters from single varicosities, but also by the concurrent release of mediators segregated in separate synapses of individual neurons. An important question to be addressed is whether neurons show defined patterns of segregation or whether this is a plastic feature. We addressed this question by exploring the segregation pattern of the classical sympathetic transmitters norepinephrine (NE) and acetylcholine (ACh) and the cotransmitter neuropeptide Y (NPY) in sympathetic ganglionic neurons cocultured with cardiac myocytes. Using antibodies against NPY and the vesicular NE and ACh transporters VMAT2 and vesicular acetylcholine transporter (VAChT), we investigated the effect of ciliary neurotrophic factor (CNTF) or long (three weeks) culture periods on the segregation of VMAT2, VAChT, and NPY to separate varicosities. We found that although ganglionic neurons showed cell body coexpression of all the markers examined after three days, VMAT2 was segregated from VAChT in 43% of the VAChT‐positive varicosities. In contrast, VMAT2 was only segregated from NPY in 16.3% of the NPY‐positive varicosities. Cotransmitter segregation and VAChT expression was potentiated by both CNTF and longer times in culture. We also found two types of varicosities: one was smaller and located further from neuronal somata, and the other was larger, proximal to neuronal somata and had a higher level of segregation. These data demonstrate segregation of classical transmitters in sympathetic neurons and plasticity of neurotransmitter segregation. Finally, we discuss a possible functional correlate of segregation in sympathetic neurons. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 70: 913–928, 2010     

多种神经活性物质在幼年猫和鸡视网膜神经细胞中的共存(简报)

视网膜起源于前脑泡,结构简单层次分明,因此常被视为脑的简化模型。但近期工作证明,视网膜包含的神经活性物质(神经递质、调质和神经肽)种类之多、分布之复杂并不亚于脑。尤其是无长突细胞不仅包含多种递质和肽类,而且还有递质与肽类或肽类与肽类在一个细胞中的共存。对视网膜神经递质、调质和神经肽的研究将是探索脑奥秘的有效途径。     

多种神经活性物质在幼年猫和鸡视网膜神经...

An improved post-embedding immunocytochemical technique was used to examine the coexistence of multi-neuroactive substances in neurons of young cat and newly hatched chicken retinas. We found that two classical neurotransmitters-glycine (Gly) and r-aminobutyric acid (GABA) and one neuropeptide, neurotensin (NT) were located in the amacrine cells of cat and chicken retinas. Among them, some cells contain Gly and GABA (Gly/GABA AC), some cells contain Gly and NT (Gly/NT AC) or GABA and NT (GABA/NT AC), and the other cells, a few of them contain Gly, GABA and NT (Gly/GABA/NT AC). In addition, two classical neurotransmitters Gly and GABA were also located or coexisted in the horizontal cells of cat retina. These results indicated that the coexistence of two neurotransmitters or one neurotransmitter and one neuropeptide or two neurotransmitters and one neuropeptide was presented in neurons of developing can and chicken retinas.     

The majority of dorsal spinal cord gastrin releasing peptide is synthesized locally whereas neuromedin B is highly expressed in pain- and itch-sensing somatosensory neurons

ABSTRACT: BACKGROUND: Itch is one of the major somatosensory modalities. Some recent findings have proposed that gastrin releasing peptide (Grp) is expressed in a subset of dorsal root ganglion (DRG) neurons and functions as a selective neurotransmitter for transferring itch information to spinal cord interneurons. However, expression data from public databases and earlier literatures indicate that Grp mRNA is only detected in dorsal spinal cord (dSC) whereas its family member neuromedin B (Nmb) is highly expressed in DRG neurons. These contradictory results argue that a thorough characterization of the expression of Grp and Nmb is warranted. FINDINGS: Grp mRNA is highly expressed in dSC but is barely detectable in DRGs of juvenile and adult mice. Anti-bombesin serum specifically recognizes Grp but not Nmb. Grp is present in a small number of small-diameter DRG neurons and in abundance in layers I and II of the spinal cord. The reduction of dSC Grp after dorsal root rhizotomy is significantly different from those of DRG derived markers but similar to that of a spinal cord neuronal marker. Double fluorescent in situ of Nmb and other molecular markers indicate that Nmb is highly and selectively expressed in nociceptive and itch-sensitive DRG neurons. CONCLUSION: The majority of dSC Grp is synthesized locally in dorsal spinal cord neurons. On the other hand, Nmb is highly expressed in pain- and itch-sensing DRG neurons. Our findings provide direct anatomic evidence that Grp could function locally in the dorsal spinal cord in addition to its roles in DRG neurons and that Nmb has potential roles in nociceptive and itch-sensitive neurons. These results will improve our understanding about roles of Grp and Nmb in mediating itch sensation.     

GABA is an endogenous ligand for synaptic glycine receptors

Experimental evidence refuting Dale's principle, the notion that each neuron synthesizes and releases only one neurotransmitter, has accumulated in the past four decades, and cotransmission by multiple neurotransmitters from the same axon terminal (and even from the same vesicle) now is well documented. Heretofore, in all examples of cotransmission, each released neurotransmitter acted on a different receptor. Lu, Rubio, and Trussell, however, demonstrate in this issue of Neuron the first instance of cotransmission in which two neurotransmitters act on the same postsynaptic receptor.     

Gene expression and chemical diversity in hypothalamic neurosecretory neurons

Hypothalamic neurosecretory neurons transcribe, translate, store, and secrete a large number of chemical messengers. The neurons contain hypothalamic signal substances that regulate the secretion of anterior pituitary hormones as well as the neurohypophysial peptides vasopressin and oxytocin. In addition to the classical hypophysiotropic hormones, a large number of neuropeptides and classical transmitters of amine and amino acid nature are present in the same cells. This is particularly evident in the magnocellular neurons of the supraoptic and paraventricular nuclei, and in parvocellular neurons of the arcuate and paraventricular nuclei. The changes in gene expression induced by experimental manipulations and the colocalization chemical messengers in hypothalamic neurosecretory neurons and its possible significance is summarized in this review.     

Astrocytes as GABA-ergic and GABA-ceptive Cells

GABA (gamma-aminobutyric acid) is considered to be the major inhibitory neurotransmitter that is synthesized in and released from GABA-ergic neurons in the brain. However, recent studies have shown that not only neurons but astrocytes contain a considerable amount of GABA, which can be released and activate the receptors responsive to GABA. In addition, astrocytes are themselves responsive to GABA by expressing GABA receptors. These exciting new findings raise more questions about the origin of GABA, whether it is synthesized or taken up, and about the role of astrocytic GABA and GABA receptors. In this review, we propose several potential pathways for astrocytes to accumulate GABA and discuss the evidence for functional expression of GABA receptors in astrocytes.     

Non-neuronal cell conditioned medium stimulates peptidergic expression in sympathetic and sensory neurons in vitro

Regulation of peptide neurotransmitter metabolism was examined in dissociated cell cultures of neonatal rat sympathetic and sensory ganglia. Previous studies have shown that pineal gland conditioned medium (PCM) influences substance P (SP) and somatostatin (SS) metabolism in sympathetic neurons in vitro. The present study examines mechanisms mediating these effects, and compares the actions of PCM on sympathetic and sensory neurons. PCM treatment increased SP levels in a dose-dependent manner without altering SS content of sympathetic neurons cultured in the presence of ganglion non-neuronal cells. Conversely, treatment of pure sympathetic neuron cultures resulted in a dose-dependent increase in SS, while SP was virtually undetectable at all doses. By contrast, dorsal root ganglion, trigeminal ganglion, and nondose ganglion sensory neurons contained SP both in the presence and absence of ganglion non-neuronal cells. Moreover, in each of these neuronal populations treatment with PCM increased SP levels both in the presence and in the absence of ganglion non-neuronal cells. These observations suggest that ganglion non-neuronal cells are necessary for sympathetic but not sensory neuron expression of SP. Moreover, PCM apparently stimulates SP in neurons which already contain the peptide, but the factor cannot foster de novo expression of the phenotype. PCM also influenced other transmitter traits in sympathetic neurons, suggesting linkage between mechanisms regulating peptides and other transmitters. In cultures containing both sympathetic neurons and non-neuronal cells, PCM treatment increased cholineacetyltransferase (CHAC) activity as well as SP, and decreased tyrosine hydroxylase (TOH) activity. By contrast, PCM treatment of pure sympathetic neuron cultures led to parallel increases in SS and TOH activity with negligible levels of SP and CHAC. These observations suggest that in sympathetic neurons, SS may be linked with noradrenergic expression, while SP is associated with cholinergic development, although more data are required to confirm this relationship. Moreover, there may be a reciprocal relationship between SP and SS expression by sympathetic neurons analogous to previous observations regarding cholinergic-noradrenergic expression (P. H. Patterson and L. L. Y. Chun, Proc. Natl. Acad. Sci. USA 71, 3607-3610, 1974; Dev. Biol. 56, 263-280, 1977). Consequently, neurotransmitter phenotypic expression is a complex process in which the environment regulates a balance among multiple transmitters.     

Developmental changes in the brain-stem serotonergic nuclei of teleost fish and neural plasticity

Summary 1. During early ontogeny, the serotonergic neurons in the brain stem of the three-spined stickleback shows a temporal and spatial developmental pattern that closely resembles that of amniotes.2. However, in the adult fish, only the midline nuclei of the rostral group (dorsal and median raphe nuclei) and the dorsal lateral tegmental nucleus are consistently serotonin-immunoreactive (5-HTir), whereas the groups of the upper and lower rhombencephalon (raphe pontis, raphe magnus, and raphe pallidus/obscurus nuclei) are variable and, when present, contain relatively small numbers of 5-HTir neurons.3. Using specific antisera against tryptophan 5-hydroxylase and aromaticl-amino acid decarboxylase, we have shown that the lateral B9 group and the groups of the upper and lower rhombencephalon are consistently present in adult sticklebacks. The results are discussed in relation to other known instances of neurotransmitter plasticity or transient neurotransmitter expression in teleost fish.4. While there are several instances of transient expression of neurotransmitter markers by discrete neuronal populations, there is so far no evidence of changes from one neurotransmitter phenotype to another in the brain of teleost fish. However, there are indications of plasticity of expression of catecholamines and indoleamines, and their respective synthesizing enzymes, as reflected in age-dependent changes and variation between individuals of different physiological status.5. As the brain grows continuously in teleost fish, and new neurons are added from proliferative regions, synaptic connections may be expected to undergo remodeling in all brain regions throughout life. Thus, the teleostean brain may be considered a suitable model for experimental studies of different aspects of neural plasticity.     

Homeostatic activity-dependent paradigm for neurotransmitter specification

Calcium-signaling plays a central role in specification of the chemical transmitters neurons express, adjusting the numbers of cells that express excitatory and inhibitory transmitters as if to achieve homeostatic regulation of excitability. Here we review the extent to which this activity-dependent regulation is observed for a range of different transmitters. Strikingly the homeostatic paradigm is observed both for classical and for peptide transmitters and in mature as well as in embryonic nervous systems. Transmitter homeostasis adds another dimension to homeostatic regulation of function in the nervous system that includes regulation of levels of voltage-gated ion channels, densities of neurotransmitter receptors, and synapse numbers and strength.     

Neurotransmitter phenotype plasticity: an unexpected mechanism in the toolbox of network activity homeostasis

The transmitter phenotype of a neuron has long been thought to be stable for the lifespan. Much as eyes have one color and do not change it over time, neurons have been thought to have one neurotransmitter and retain it for their lifetime. Both principles, exclusivity and stability, are challenged by recent data. More and more neurons in different regions of the brain appear to coexpress two or more neurotransmitters. Moreover, the profile of neurotransmitter expression of a given neuron has been shown to change over time, both during development and in response to changes in activity. The present review summarizes recent studies of this neurotransmitter phenotype plasticity (NPP). Homeostatic mechanisms of plasticity are aimed at maintaining the system within a functional range. They appear to be critical for optimal network operations and have been thought to operate largely by regulating intrinsic excitability, synapse number and synaptic strength. NPP provides a new and unexpected level of regulation of network homeostasis. We propose that it provides the basis for NT coexpression and discuss emerging issues and new questions for further studies in coming years.     

Probabilistic computation by neuromine networks

In this paper, we address the question, can biologically feasible neural nets compute more than can be computed by deterministic polynomial time algorithms? Since we want to maintain a claim of plausibility and reasonableness we restrict ourselves to algorithmically easy to construct nets and we rule out infinite precision in parameters and in any analog parts of the computation. Our approach is to consider the recent advances in randomized algorithms and see if such randomized computations can be described by neural nets. We start with a pair of neurons and show that by connecting them with reciprocal inhibition and some tonic input, then the steady-state will be one neuron ON and one neuron OFF, but which neuron will be ON and which neuron will be OFF will be chosen at random (perhaps, it would be better to say that microscopic noise in the analog computation will be turned into a megascale random bit). We then show that we can build a small network that uses this random bit process to generate repeatedly random bits. This random bit generator can then be connected with a neural net representing the deterministic part of randomized algorithm. We, therefore, demonstrate that these neural nets can carry out probabilistic computation and thus be less limited than classical neural nets.     

Minireview: Nicotinic Acetylcholine Receptors on Hippocampal Neurons: Distribution on the Neuronal Surface and Modulation of Receptor Activity

Abstract

The recent development of a technique that uses infrared microscopy for the visualization of well-defined areas on the surface of neurons, and a computerized system of micromanipulators led to the discovery that functional nicotinic acetylcholine receptors (nAChRs) are expressed at higher density on the dendrites than on the soma of rat hippocampal neurons. The finding that the expression of α-bungarotoxin-sensitive, α7-bearing, nAChRs and dihydro-β-erythroidine-sensitive,α4β2 nAChRs tends to increase along the dendritic length suggests that these receptors may be highly involved in the integration of synaptic functions in hippocampal neurons. The present report also discusses the finding that ligands such as the anticholinesterase galanthamine can modulate the nAChR activity by binding to a novel receptor site, and that 5-hydroxytryptamine (5-HT) may serve as an endogenous ligand for this site. The ability of 5-HT to modulate the nAChR function in vivo supports the concept that the overall CNS function is determined not only by the neuronal network established by the neuronal wiring, but also by a chemical network established by the ability of a single substance to act as the primary neurotransmitter in one system and as a co-transmitter in another system.     

Physiological and chemical analysis of neurotransmitter candidates at a fast excitatory synapse in the jellyfish Cyanea capillata (Cnidaria, Scyphozoa)

Motor nerve net (MNN) neurons in the jellyfish Cyanea capillata communicate with one another by way of fast, bidirectional excitatory chemical synapses. As is the case with almost all identified chemical synapses in cnidarians, the identity of the neurotransmitter at these synapses is unclear. MNN neurons are large enough for stable intracellular recordings. This, together with the fact that they can be exposed, providing unlimited access to them and to their synapses, prompted a study of the action of a variety of neurotransmitter candidates, including those typically associated with fast synapses in higher animals. Only the amino acids taurine and β-alanine produced physiological responses consistent with those of the normal EPSP in these cells. Moreover, chemical analysis revealed that both taurine and β-alanine are present in the neurons and released by depolarization. These various findings strongly suggest that either or both of these amino acids, or a closely related compound is the neurotransmitter at the fast chemical synapses between MNN neurons.     

Synapse formation and mRNA localization in cultured Aplysia neurons

mRNA localization and regulated translation provide a means of spatially restricting gene expression within neurons during axon guidance and long-term synaptic plasticity. Here we show that synapse formation specifically alters the localization of the mRNA encoding sensorin, a peptide neurotransmitter with neurotrophin-like properties. In isolated Aplysia sensory neurons, which do not form chemical synapses, sensorin mRNA is diffusely distributed throughout distal neurites. Upon contact with a target motor neuron, sensorin mRNA rapidly concentrates at synapses. This redistribution only occurs in the presence of a target motor neuron and parallels the distribution of sensorin protein. Reduction of sensorin mRNA, but not protein, with dsRNA inhibits synapse formation. Our results indicate that synapse formation can alter mRNA localization within individual neurons. They further suggest that translation of a specific localized mRNA, encoding the neuropeptide sensorin, is required for synapse formation between sensory and motor neurons.     

Target influences on transmitter choice by sympathetic neurons developing in the anterior chamber of the eye

In contrast to the majority of sympathetic neurons which are noradrenergic, the sympathetic neurons which innervate sweat glands are cholinergic. Previous studies have demonstrated that during development the sweat gland innervation initially contains catecholamines which are lost as cholinergic function appears. The neurotransmitter phenotype of sweat gland neurons further differs from the majority in that they contain vasoactive intestinal peptide (VIP) rather than neuropeptide Y (NPY). In the experiments described here, we addressed the question of whether sympathetic targets influence the neurotransmitter-related properties of the neurons which innervate them; in particular, do sweat glands play a role in reducing the expression of noradrenergic properties and inducing the expression of cholinergic properties and VIP in sympathetic neurons? This was accomplished by cotransplanting to the anterior chamber of the eye of host rats the superior cervical ganglia (SCG) which contains neurons that normally innervate targets other than the sweat glands and differentiate noradrenergically and footpad tissue from neonatal rats. Sweat glands developed in the transplanted footpad tissue and became innervated by the cotransplanted SCG neurons. The transplanted neurons and sweat gland innervation initially exhibited catecholamine histofluorescence which declined with further development in the anterior chamber. After 4 weeks, choline acetyltransferase (ChAT) and VIP immunoreactivities were evident. These observations suggest that as in the neurons which innervate the glands in situ, noradrenergic properties were suppressed and cholinergic function was induced in the neurons which innervated the glands in oculo. To distinguish a specific influence of the sweat glands on transmitter choice, SCG were also cotransplanted with the pineal gland, a normal target of the ganglion. Neurons cotransplanted with the pineal gland continued to exhibit catecholamine histofluorescence and contained NPY immunoreactivity. At least some neurons in SCG/pineal cotransplants, however, developed ChAT immunoreactivity. The target-appropriate expression of catecholamines and peptides in these experiments is consistent with the hypothesis that some transmitter properties are influenced by target tissues. The indiscriminant expression of ChAT, however, suggests that at least in oculo, additional factors can influence transmitter choice.