Ultrastructural identification of neuropeptides in the central nervous system

A number of different neuropeptides have been described within presynaptic terminals at the ultrastructural level in the central nervous system. The majority of these neuropeptides share a common morphology with one another. Terminals containing neuropeptides have a population of small, clear vesicles associated with the active zone of the synapse and a lesser number of large, granular vesicles that are located at a distance from the active site of the synapse. It is believed that the large, granular vesicles act as a mechanism for the transport/storage of the neuropeptides, while the small, clear vesicles are thought to be acting as structures responsible for the release of the neurotransmitter/neuropeptide into the synaptic cleft. The neuropeptide containing terminals most often have asymmetrical junctions associated with their presynaptic membranes, although symmetrical junctions have been described with peptide containing terminals in a number of areas in the central nervous system. Neuropeptide containing terminals contact every part of the neuronal membrane; however, the majority of synaptic contacts involve portions of the dendritic shafts. Evidence is beginning to accumulate to indicate that for certain neuropeptides there is a specific spatial arrangement to their termination along the neuronal membrane.     

Characterization of chemically defined neurons and their cellular relationships by combined immunocytochemistry and radioautographic localization of transmitter uptake sites

Radioautography and immunocytochemistry may be combined at the light and electron microscopic levels for simultaneously localizing uptake sites for exogenous transmitter molecules [such as (³H)monoamines or (³H)amino acids] and endogenous transmitter-related antigens (classical transmitters and their synthesizing enzymes as well as neuropeptides) in the central nervous system. Silver grain accumulations indicative of transmitter uptake sites are readily distinguishable from immunocytochemical labels of the peroxidase-antiperoxidase (PAP), avitin-biotin, or colloidal gold methods. The combination of uptake radioautography and immunocytochemistry may be applied to the investigation of (1) the chemical identity of (³H) transmitter-accumulating elements, (2) the coexistence of different neurotransmitters within the same neurons, and (3) the cellular basis of interactions between certain neurotransmitters, in particular monoamines, GABA, and neuropeptides. This article describes and evaluates the method and reviews the available experimental data derived from its application.     

Calcitonin gene-related peptide (CGRP)-containing nerve fibers in bone tissue and their involvement in bone remodeling

Bone remodeling is a process of bone renewal accomplished by osteoclastic bone resorption and osteoblastic bone formation. These two activities are regulated by systemic hormones and by local cytokines and growth factors. Moreover, the nervous system and certain neuropeptides seem to be involved in regulation of bone remodeling. In this paper, we focus on the distribution of CGRP-containing nerve fibers and their dynamics, and discuss the role of these fibers as a possible mechanism for nervous system involvement in regulation of bone remodeling. CGRP-immunoreactive nerve fibers are widely distributed in bone tissue, such as periosteum and bone marrow, and show apparent regional distribution with different densities. They are often associated with blood vessels and show a beaded appearance. The wide distribution of CGRP-immunoreactive nerve fibers in bone tissue and the changes in distribution during bone development and regeneration suggest the involvement of these fibers in bone remodeling. The effect of CGRP on bone remodeling could partly be through its action on blood vessels, thereby regulating local blood flow. Moreover, in vitro biochemical data and the localization of CGRP-immunoreactive nerve fibers in the vicinity of bone cells suggest that they are directly involved in local regulation of bone remodeling by elevating the concentration of CGRP in the microenvironment around bone cells, especially during bone growth or repair.     

Neurotransmitters and neuropeptides in the brain of the locust.

As part of continuous research on the neurobiology of the locust, the distribution and functions of neurotransmitter candidates in the nervous system have been analyzed particularly well. In the locust brain, acetylcholine, glutamate, gamma-aminobutyric acid (GABA), and the biogenic amines serotonin, dopamine, octopamine, and histamine most likely serve a transmitter function. Increasing evidence, furthermore, supports a signalling function for the gaseous molecule nitric oxide, but a role for neuroptides is so far suggested only by immunocytochemistry. Acetylcholine, glutamate, and GABA appear to be present in large numbers of interneurons. As in other insects, antennal sensory afferents might be cholinergic, while glutamate is the transmitter candidate of antennal motoneurons. GABA is regarded as the principle inhibitory transmitter of the brain, which is supported by physiological studies in the antennal lobe. The cellular distribution of biogenic amines has been analyzed particularly well, in some cases down to physiologically characterized neurons. Amines are present in small numbers of interneurons, often with large branching patterns, suggesting neuromodulatory roles. Histamine, furthermore, is the transmitter of photoreceptor neurons. In addition to these "classical transmitter substances," more than 60 neuropeptides were identified in the locust. Many antisera against locust neuropeptides label characteristic patterns of neurosecretory neurons and interneurons, suggesting that these peptides have neuroactive functions in addition to hormonal roles. Physiological studies supporting a neuroactive role, however, are still lacking. Nitric oxide, the latest addition to the list of neurotransmitter candidates, appears to be involved in early stages of sensory processing in the visual and olfactory systems.     

Analyzing the evolution of the opioid/orphanin gene family

Advances in molecular biology have made it possible to rapidly obtain the amino acid sequence of neuropeptide precursors-either by cloning and sequencing the cDNA that encodes the precursor, or by reconstructing the arrangement of exons and introns in a neuropeptide-coding gene through genomic approaches. The databases generated from these molecular approaches have been used to design probes to identify the cells that express the gene, or to ascertain the rate of expression of the gene, and even to predict the post-translational modifications that can generate functional neuropeptides from a biologically inert precursor. Although the power of these approaches is substantial, it is appreciated that a gene sequence or an mRNA sequence reflects the potential products that may be assembled in a secretory cell. To understand the functional capabilities of the secretory cell, the molecular genetics approaches must be combined with procedures that actually characterize the end-products generated by the secretory cell. Recent advances in two-dimensional gel electrophoresis and mass spectrometry now make it possible to analyze neuropeptides from a relatively small amount of tissue. These procedures can reveal novel end-products, tissue-specific endoproteolytic cleavage events, and developmental shifts in post-translational processing schemes. A gene family that illustrates all of these processes and the advantages of combining genomics with proteomics is the opioid/orphanin gene family.     

Neural input and neural control of the subcommissural organ

The neural control of the subcommissural organ (SCO) has been partially characterized. The best known input is an important serotonergic innervation in the SCO of several mammals. In the rat, this innervation comes from raphe nuclei and appears to exert an inhibitory effect on the SCO activity. A GABAergic innervation has also been shown in the SCO of the rat and frog Rana perezi. In the rat, GABA and the enzyme glutamate decarboxylase are involved in the SCO innervation. GABA is taken up by some secretory ependymocytes and nerve terminals, coexisting with serotonin in a population of synaptic terminals. Dopamine, noradrenaline, and different neuropeptides such as LH-RH, vasopressin, vasotocin, oxytocin, mesotocin, substance P, alpha-neoendorphin, and galanin are also involved in SCO innervation. In the bovine SCO, an important number of fibers containing tyrosine hydroxylase are present, indicating that in this species dopamine and/or noradrenaline-containing fibers are an important neural input. In Rana perezi, a GABAergic innervation of pineal origin could explain the influence of light on the SCO secretory activity in frogs. A general conclusion is that the SCO cells receive neural inputs from different neurotransmitter systems. In addition, the possibility that neurotransmitters and neuropeptides present in the cerebrospinal fluid may also affect the SCO activity, is discussed.     

Neuropeptides in the crayfish stomatogastric nervous system

Neuropeptides are peptides with profound effects on the nervous system. The function of neuropeptides can be studied in detail in the stomatogastric nervous system (STNS). Neuropeptides are ubiquitously distributed in the STNS and it contains well-studied neural circuits that are strongly modulated by neuropeptides. The STNS controls the movements of the foregut in crustaceans and has been studied intensively in a variety of decapod crustaceans including crayfish. This article reviews our knowledge of neuropeptides in the crayfish STNS. Within crayfish, peptides reach the circuits of the STNS as neurohormones released by neurohaemal organs or by putative neurohemal zones located within the STNS. As transmitters, neuropeptides are present in identified motoneurons, interneurons, and sensory neurons (mainly shown by immunocytochemistry), indicating a multiple role of peptides in the plasticity of neural networks. Neuropeptides are not only present in varicosities within the neuropil of ganglia, but also in varicosities on muscles and within small neuropil patches along nerves. This suggests that the muscles of the stomach are under a more direct modulatory control than previously thought, and that information processing can also occur within nerves. In addition to anatomical studies, biochemical and electrophysiological methods were used. For example, MALDI-TOF MS (matrix-assisted laser desorption ionization time of flight mass spectrometry) revealed the presence of four different peptides of the orcokinin family within a single neuron, and electrophysiological experiments demonstrated that the networks of the STNS are not only under excitatory but also inhibitory peptidergic influence. Comparing the similarities and differences between the STNS of crayfish and that of other decapod crustaceans has already contributed to our knowledge about peptides and will further help to unravel peptide function in the plasticity of neural circuits. For example, the identified neurons in the STNS can be used to study co-transmission because neuropeptides are co-localized with classical transmitters, biogenic amines, or other peptides in these neurons.     

ProbingNeuronalCommunicationViaNovelMassSpectralStrategies

Neuropeptides make up the largest and the most complex signaling molecules used in intercellular communication. Because of critical roles that these polypeptides play in the regulation of many physiological processes, it is of great interest to characterize these diverse assortments of chemical messengers and determine their functions in the neural circuitry. The simpler and well-characterized crustacean nervous system provides an excellent model system to facilitate analytical method development and to investigate how a rich repertoire of neuropeptides can fine tune a well-defined neural circuit that produces multiple outputs at the cellular and network levels. Using a highly sensitive mass spectrometry-based peptide profiling and de novo sequencing strategy, a large number of novel peptides have been discovered, revealing that even a relatively simple neural network contains an unexpectedly-rich diversity of neuropeptides^[1]. Furthermore, both mass spectrometric imaging techniques^[2] and in vivo microdialysis sampling tools^[3] have been implemented to follow neuropeptide distribution and secretion in unprecedented details. Towards the goal of functional discovery of bioactive neuropeptides, novel quantitative schemes based on isotopic formaldehyde labeling and multiplexed isobaric labeling based on N,N-dimethylated leucine^[4] have been developed to produce differential display of neuropeptidomes under different physiological conditions^[5]. Examples of neuropeptide regulation of feeding behavior and environmental stress will be described in this presentation. Collectively, these combined peptidomic and physiological studies will help to elucidate the functional roles that neuropeptides play in regulating neural network plasticity.     

Fine structure of the pinealopetal innervation of the mammalian pineal gland.

The mammalian pineal gland is innervated by peripheral sympathetic and parasympathetic nerve fibers as well as by nerve fibers originating in the central nervous system (central innervation). The perikarya of the sympathetic fibers are located in the superior cervical ganglia, while the fibers terminate in boutons containing small granular vesicles and a few large granular vesicles. Both noradrenaline and neuropeptide Y are contained in these neurons. The parasympathetic fibers originate from perikarya in the pterygopalatine ganglia. The neuropeptides, vasoactive intestinal peptide and peptide histidine isoleucine, are present in these fibers, the boutons of which contain small clear transmitter vesicles and larger granular vesicles. The fibers of the central innervation originate predominantly from perikarya located in hypothalamic and limbic forebrain structures as well as from perikarya in the optic system. These fibers terminate in boutons containing small clear and, in certain fibers, an abundant number of large granular vesicles. In rodents, the majority of the central fibers terminate in the deep pineal gland and the pineal stalk. From these areas impulses might be transmitted further caudally to the superficial pineal gland via neuronal structures or processes from pinealocytes. Several hypothalamic neuropeptides and monoamines might be contained in the central fibers. The intrapineal nerve fibers are located both in the perivascular spaces and intraparenchymally. The majority of the intraparenchymally located fibers terminate freely between the pinealocytes. However, some nerve terminals make synaptic contacts with the pinealocytes and in some species with intrapineal neurons. In fetal mammals, sympathetic, parasympathetic, and central fibers are also present. In addition, an unpaired nerve, connecting the caudal part of the pineal gland with the extreme rostral part of the mesencephalon, is present. This nerve is a homologue to the pineal nerve (nervus pinealis) observed in lower vertebrates.     

Innervation of the gastric mucosa

A plethora of neuronal messengers ("classical" transmitters, gaseous messengers, amino acid transmitters, and neuropeptides) are capable of mediating or modulating gastric functions. Accordingly, the stomach is richly innervated. Gastric nerves are either intrinsic to the gastric wall, i.e., they have their cell bodies in the intramural ganglia and thus belong to the enteric nervous system, or they reach the stomach from outside, originating in the brainstem, in sympathetic ganglia, or in sensory ganglia. Topographically, the nerve fibers in the stomach reach all layers from the most superficial portions of the gastric glands to the outer smooth muscle layer. This wide distribution implies that virtually all different cell types may be reached by neuronal messengers. Within the gastric mucosa endocrine and paracrine cells (e.g., gastrin cells, ECL cells, somatostatin cells), exocrine cells (parietal cells, chief cells, mucous cells), smooth muscle cells, and stromal cells are regulated by neuronal messengers. The sensory innervation, responding to capsaicin, plays an important role in mucosal protection, and in ulcer healing. Presumably also other nerves are involved and a plasticity in the neuropeptide expression has been demonstrated at the margin of gastric ulcers. Taken together, available data indicate a complex interplay between hormones, paracrine messengers and neuronal messengers, growth factors and cytokines in the regulation of gastric mucosal activities such as secretion, local blood flow, growth, and restitution after damage.     

Distribution and regulation of substance P-related peptide in the frog visual system

Modulation of visual signal activity has consequences for both signal processing and for activity-dependent structuring mechanisms. Among the neuromodulatory agents found in visual areas are substance P (SP)-related peptides. This article reviews what is known about these substances in the amphibian retina and optic tectum with special emphasis on the leopard frog, Rana pipiens. It is found that the distribution of these SP-related peptides is remarkably similar to that seen in mammals. This suggests that study of model amphibian systems may significantly enhance our understanding of how neuropeptides contribute to visual system function and organization.     

Discovering new invertebrate neuropeptides using mass spectrometry

Neuropeptides are a complex set of messenger molecules controlling a wide array of regulatory functions and behaviors within an organism. These neuromodulators are cleaved from longer protein molecules and often experience numerous post-translational modifications to achieve their bioactive form. As a result of this complexity, sensitive and versatile analysis schemes are needed to characterize neuropeptides. Mass spectrometry (MS) through a variety of approaches has fueled the discovery of hundreds of neuropeptides in invertebrate species in the last decade. Particularly successful are direct tissue and single neuron analyses by matrix-assisted laser desorption/ionization (MALDI) MS, which has been used to elucidate approximately 440 neuropeptides, and examination of neuronal homogenates by electrospray ionization techniques (ESI), also leading to the characterization of over 450 peptides. Additional MS methods with great promise for the discovery of neuropeptides are MS imaging and large-scale peptidomics studies in combination with a sequenced genome.     

Immunohistochemical distribution of enkephalin, substance P, and somatostatin in the brainstem of the leopard frog, Rana pipiens

The brainstems of frogs contain many of the neurochemicals that are found in mammals. However, the clustering of nuclei near the ventricles makes it difficult to distinguish individual cell groups. We addressed this problem by combining immunohistochemistry with tract tracing and an analysis of cell morphology to localize neuropeptides within the brainstem of Rana pipiens. We injected a retrograde tracer, Fluoro-Gold, into the spinal cord, and, in the same frog, processed adjacent sections for immunohistochemical location of antibodies to the neuropeptides enkephalin (ENK), substance P (SP), and somatostatin (SOM). SOM+ cells were more widespread than cells containing immunoreactivity (ir) to the other substances. Most reticular nuclei in frog brainstem contained ir to at least one of these chemicals. Cells with SOM ir were found in nucleus (n.) reticularis pontis oralis, n. reticularis magnocellularis, n. reticularis paragigantocellularis, n. reticularis dorsalis, the optic tectum, n. interpeduncularis, and n. solitarius. ENK-containing cell bodies were found in n. reticularis pontis oralis, n. reticularis dorsalis, the nucleus of the solitary tract, and the tectum. The midbrain contained most of the SP+ cells. Six nonreticular nuclei (griseum centrale rhombencephali, n. isthmi, n. profundus mesencephali, n. interpeduncularis, torus semicircularis laminaris, and the tectum) contained ir to one or more of the substances but did not project to the spinal cord. The descending tract of V, and the rubrospinal, reticulospinal, and solitary tracts contained all three peptides as did the n. profundus mesencephali, n. isthmi, and specific tectal layers. Because the distribution of neurochemicals within the frog brainstem is similar to that of amniotes, our results emphasize the large amount of conservation of structure, biochemistry, and possibly function that has occurred in the brainstem, and especially in the phylogenetically old reticular formation.     

Expression and regulatory role of receptors for vasoactive intestinal peptide in bone cells

An intense network of nerve fibers can be demonstrated in skeletal tissues, not only in the periosteum but also within cortical bone, growth plate, and bone marrow. This neuro-osteogenic network expresses a restricted number of signalling molecules, including neuropeptides, neurotransmitters, and neurotrophins. Several lines of evidence indicate that receptors for these molecules are present on bone cells and that activation of these receptors leads to changes in bone cell activities. In addition, deletion of signalling molecules has been shown to alter bone metabolism. In the present review, these studies are summarized with a focus on distribution and effects of vasoactive intestinal peptide.     

Magnocellular vasopressin system in ontogenesis: development and regulation.

This review is devoted to the development, functional activity, and regulation of the magnocellular vasopressin (VP) system in ontogenesis. Magnocellular VP neurons originate in embryos from the neuroepithelium of the third ventricle and migrate first to the supraoptic nucleus and then to the paraventricular nucleus and accessory nuclei. The preproVP gene and synthesis are expressed simultaneously in the newly formed neurons either during migration or just after arrival in magnocellular nuclei. Still, a number of VP-immunoreactive neurons increase in immature mammals to prepuberty, which is explained by VP expression in the initially "silent" neurons, or by an increase of VP synthesis that makes a cell distinguishable by immunocytochemistry. An enzymatic processing of preproVP is slightly delayed compared to the onset of preproVP synthesis. Axons of magnocellular neurons reach the pituitary posterior lobe before or just after the neuron arrival in magnocellular nuclei. The mechanisms of VP release from the axon terminals are developed in immature animals over the perinatal period. The VP neurons begin to react to functional (osmotic) stimulation by increased synthesis of VP mRNA and VP in immature animals from the end of fetal life. A functional maturation of the VP system is under control by neural afferents, neuropeptides, and some hormones of endocrine glands. Namely, glucocorticoids, VP, catecholamines, glutamate, and opioids provide short-term or long-lasting effects on differentiating VP neurons. Most of the intercellular signals inhibit the specific phenotype expression of differentiating VP neurons: VP gene and synthesis in normal conditions, as well as TH gene and synthesis under functional stimulation.     

The trigeminovascular system in bacterial meningitis

Headache as a cardinal symptom of acute meningitis reflects activation of trigeminal afferents from the meninges. With their perivascular endings, these fibers form the so-called trigeminovascular system (TVS), which releases proinflammatory neuropeptides upon nociceptive stimulation. In the present article, we review a role of the TVS in enhancing the early inflammatory response of bacterial meningitis. Furthermore, we discuss inhibition of neuropeptide release from the TVS using 5HT(1B/D) agonists as a potential new anti-inflammatory treatment strategy for early bacterial meningitis.     

Choroid plexus: target for polypeptides and site of their synthesis

Choroid plexus (CP) is an important target organ for polypeptides. The fenestrated phenotype of choroidal endothelium facilitates the penetration of blood-borne polypeptides across the capillary walls. Thus, both circulating and cerebrospinal fluid (CSF)-borne polypeptides can reach their receptors on choroidal epithelium. Several polypeptides have been demonstrated to regulate CSF formation by controlling blood flow to choroid plexus and/or the activity of ion transport in choroidal epithelium. However, many ligand-receptor interactions occurring in the CP are not involved in the regulation of fluid secretion. Increasing evidence suggests that the choroidal epithelium plays an important role in hormonal signaling via a receptor-mediated transport into the brain (e.g., leptin) and helps to clear certain CSF-borne polypeptides (e.g., soluble amyloid beta-protein). Thus, impaired choroidal transport or insufficient clearance of polypeptides may contribute to pathogenesis of systemic or central nervous system (CNS) disorders, such as obesity or Alzheimer's disease. CP epithelium is not only a target but is also a source of neuropeptides, growth factors, and cytokines in the CNS. These polypeptides following their release into the CSF may exert distal, endocrine-like effects on target cells in the brain due to bulk flow of this fluid. Distinct temporal patterns of choroidal expression of several polypeptides are observed during brain development and in various CNS disorders, including traumatic brain injury and ischemia. Therefore, it is proposed that the CP plays an integral role not only in normal brain functioning, but also in the recovery from the injury. This review attempts to critically analyze the available data to support the above hypothesis.     

Age-dependent changes in the nervous and endocrine control of the thymus

The immune system, especially the thymus, undergoes age-related modifications leading to structural and functional changes in the lymphoid organs and immunocompetent cells. Nevertheless, the consequences of thymic involution in the peripheral pool of T-cells are still a matter of controversy. The control of the thymic function is very complex and involves intrathymic signals, the autonomic nervous system, and the endocrine system. Both thymocytes and thymic stromal cells express receptors for a wide range of hormones, as well as for neurotransmitters and neuropeptides, thus affecting thymocytes maturation. This review summarizes the age-dependent variations in the extrathymic components of the thymic microenvironment, i.e., vegetative nerves and hormones, and the possible effects of those changes in the immune function.