Development and specificity of inhibitory terminal arborizations in the central nervous system.

This study examined the morphological development of single inhibitory arborizations in the gerbil central auditory brain stem. Using a brain slice preparation, neurons of the medial nucleus of the trapezoid body (MNTB) were filled with horseradish peroxidase (HRP), and their complete arborizations were analyzed along the tonotopic axis of the lateral superior olive (LSO). The projections in neonatal animals displayed well-defined arbors that were ordered appropriately within the LSO. It was evident from the axonal pathways that the MNTB afferents could correct for projection errors after reaching the postsynaptic population. As development progressed, a number of arbors established diffuse or inappropriate projections within the LSO. These immature arborizations were no longer apparent by 18-25 days postnatal. The anatomical specificity of arbors at 12-13 and 18-25 days was quantified by measuring the distance that terminal boutons spread across the frequency axis. There was a significant reduction of this distance in older animals. In addition, there was a significant reduction in the mean number of boutons per arbor between 12-13 days and 18-25 days. The maximum nucleus cross-sectional area continued to increase through 15-16 days, indicating that the refined arbors occupied an even smaller fraction of the postsynaptic structure. Taken together, these observations suggest that central inhibitory arbors form exuberant contacts that must be eliminated during development.     

Development of a topographically organized auditory network in slice culture is calcium dependent

Inhibitory and excitatory connections of remarkably precise topographic order are characteristic features of the mammalian auditory system, particularly within the superior olivary complex (SOC). Little is known about the requirements for the correct development of these specific connections. Previous in vivo experiments have demonstrated a high expression of calcium-binding proteins in this system during development, pointing to the need for precise calcium regulation. Here, we have employed an organotypic slice culture from the above neuronal network and analyzed the requirements for the maintenance and development of this system in vitro. When slices from neonatal rats were incubated in standard culture medium for up to 7 days, we found no organotypic features. Only if 25 mM KCl was added to the culture medium, the cytoarchitecture of the nuclei, the neuronal morphology, and the specificity and topography of internuclear connections were indistinguishable from that in vivo. The addition of calcium channel blockers (MgCl₂ and nifedipine) to the high-KCl medium reduced organotypicity drastically, indicating that a depolarization-induced increase of intracellular calcium is indispensable. Furthermore, the temporal course of the expression of the calcium-binding protein parvalbumin in culture under high KCl mimics that in vivo, demonstrating developmental processes during incubation. The need for calcium influx into neurons of this auditory network in vitro (which is not seen in other slice culture systems) strengthens the hypothesis that an optimal calcium concentration is exceptionally important in auditory neurons. The effect of KCl in the slice cultures may substitute for input activity regulating intracellular calcium in auditory neurons in vivo. © 1998 John Wiley & Sons, Inc. J Neurobiol 34: 97–112, 1998     

Development and regeneration in the central nervous system

As part of our attempts to understand principles that underly organism development, we have been studying the development of the rat optic nerve. This simple tissue is composed of three glial cell types derived from two distinct cellular lineages. Type-1 astrocytes appear to be derived from a monopotential neuroepithelial precursor, whereas type-2 astrocytes and oligodendrocytes are derived from a common oligodendrocyte-type-2 astrocyte (O-2A) progenitor cell. Type-1 astrocytes modulate division and differentiation of O-2A progenitor cells through secretion of platelet-derived growth factor, and can themselves be stimulated to divide by peptide mitogens and through stimulation of neurotransmitter receptors. In vitro analysis indicates that many dividing O-2A progenitors derived from optic nerves of perinatal rats differentiate symmetrically and clonally to give rise to oligodendrocytes, or can be induced to differentiate into type-2 astrocytes. O-2Aperinatal progenitors can also differentiate to form a further O-2A lineage cell, the O-2Aadult progenitor, which has properties specialized for the physiological requirements of the adult nervous system. In particular, O-2Aadult progenitors have many of the features of stem cells, in that they divide slowly and asymmetrically and appear to have the capacity for extended self-renewal. The apparent derivation of a slowly and asymmetrically dividing cell, with properties appropriate for homeostatic maintenance of existing populations in the mature animal, from a rapidly dividing cell with properties suitable for the rapid population and myelination of central nervous system (CNS) axon tracts during early development, offers novel and unexpected insights into the possible origin of self-renewing stem cells and also into the role that generation of stem cells may play in helping to terminate the explosive growth of embryogenesis. Moreover, the properties of O-2Aadult progenitor cells are consistent with, and may explain, the failure of successful myelin repair in conditions such as multiple sclerosis, and thus seem to provide a cellular biological basis for understanding one of the key features of an important human disease.     

Colocalization of synaptic GABAC-receptors with GABAA-receptors and glycine-receptors in the rodent central nervous system

Fast inhibition in the nervous system is preferentially mediated by GABA- and glycine-receptors. Two types of ionotropic GABA-receptor, the GABA_A-receptor and GABA_C-receptor, have been identified; they have specific molecular compositions, different sensitivities to GABA, different kinetics, and distinct pharmacological profiles. We have studied, by immunocytochemistry, the synaptic localization of glycine-, GABA_A-, and GABA_C-receptors in rodent retina, spinal cord, midbrain, and brain-stem. Antibodies specific for the α1 subunit of the glycine-receptor, the γ2 subunit of the GABA_A-receptor, and the ρ subunits of the GABA_C-receptor have been applied. Using double-immunolabeling, we have determined whether these receptors are expressed at the same postsynaptic sites. In the retina, no such colocalization was observed. However, in the spinal cord, we found the colocalization of glycine-receptors with GABA_A- or GABA_C-receptors and the colocalization of GABA_A- and GABA_C-receptors in approximately 25% of the synapses. In the midbrain and brain-stem, GABA_A- and GABA_C-receptors were colocalized in 10%–15% of the postsynaptic sites. We discuss the possible expression of heteromeric (hybrid) receptors assembled from GABA_A- and GABA_C-receptor subunits. Our results suggest that GABA_A- and GABA_C-receptors are colocalized in a minority of synapses of the central nervous system.     

Fibroblast growth factors and their receptors in the central nervous system

Fibroblast growth factors (FGFs) and their receptors constitute an elaborate signaling system that participates in many developmental and repair processes of virtually all mammalian tissues. Among the 23 FGF members, ten have been identified in the brain. Four FGF receptors (FGFRs), receptor tyrosine kinases, are known so far. Ligand binding of these receptors greatly depends on the presence of heparan sulfate proteoglycans, which act as low affinity FGFRs. Ligand binding specificity of FGFRs depends on the third extracellular Ig-like domain, which is subject to alternative splicing. Activation of FGFRs triggers several intracellular signaling cascades. These include phosphorylation of src and PLC leading finally to activation of PKC, as well as activation of Crk and Shc. SNT/FRS2 serves as an alternative link of FGFRs to the activation of PKC and, in addition, activates the Ras signaling cascade. In the CNS, FGFs are widely expressed; FGF-2 is predominantly synthesized by astrocytes, whereas other FGF family members, e.g., FGF-5, FGF-8, and FGF-9, are primarily synthesized by neurons. During CNS development FGFs play important roles in neurogenesis, axon growth, and differentiation. In addition, FGFs are major determinants of neuronal survival both during development and during adulthood. Adult neurogenesis depends greatly on FGF-2. Finally, FGF-1 and FGF-2 seem to be involved in the regulation of synaptic plasticity and processes attributed to learning and memory.     

Cytokines that signal through the leukemia inhibitory factor receptor-beta complex in the nervous system

Cytokines that signal through the leukemia inhibitory factor (LIF) receptor, such as LIF and ciliary neuronotrophic factor, have a wide range of roles within both the developing and mature nervous system. They play a vital role in the differentiation of neural precursor cells into astrocytes and can prevent or promote neuronal differentiation. One of the conundrums regarding signalling through the LIF receptor is how it can have multiple, often conflicting roles in different cell types, such as enhancing the differentiation of astrocytes while inhibiting the differentiation of some neuronal cells. Factors that can modulate signal transduction downstream of cytokine signalling, such as "suppressor of cytokine signalling" proteins, which inhibit the JAK/STAT but not the mitogen-activated protein kinase pathway, may therefore play an important role in determining how a given cell will respond to cytokine signalling. This review discusses the general effects of cytokine signalling within the nervous system. Special emphasis is placed on differentiation of neural precursor cells and the role that regulation of cytokine signalling may play in how a given precursor cell responds to cytokine stimulation.     

Development of axon pathways in the zebrafish central nervous system

The zebrafish has a number of distinct advantages as an experimental model in developmental biology. For example, large numbers of embryos can be generated in each lay, development proceeds rapidly through a very precise temporal staging which exhibits minimal batch-to-batch variability, embryos are transparent and imaging of wholemounts negates the need for tedious histological preparation while preserving three-dimensional spatial relationships. The zebrafish nervous system is proving a convenient model for studies of axon guidance because of its small size and highly stereotypical trajectory of axons. Moreover, a simple scaffold of axon tracts and nerves is established early and provides a template for subsequent development. The ease with which this template can be visualized as well as the ability to spatially resolve individual pioneer axons enables the role of specific cell-cell and molecular interactions to be clearly deciphered. We describe here the morphology and development of the earliest axon pathways in the embryonic zebrafish central nervous system and highlight the major questions that remain to be addressed with regard to axon guidance.     

Constitutive phosphorylation of the vesicular inhibitory amino acid transporter in rat central nervous system

gamma-Aminobutyric acid (GABA) and glycine are stored into synaptic vesicles by a recently identified vesicular inhibitory amino acid transporter [VIAAT, also called vesicular GABA transporter (VGAT)]. Immunoblotting analysis revealed that rat brain VIAAT migrated as a doublet during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with a predominant slower band in all areas examined except olfactory bulb and retina. The slower band corresponded to a phosphorylated form of VIAAT as it was converted to the faster one by treating brain homogenates with alkaline phosphatase or with an endogenous phosphatase identified as type 2A protein-serine/threonine phosphatase using okadaic acid. In contrast, the recombinant protein expressed in COS-7 or PC12 cells co-migrated with the faster band of the brain doublet and was insensitive to alkaline phosphatase. To investigate the influence of VIAAT phosphorylation on vesicular neurotransmitter loading, purified synaptic vesicles were treated with alkaline phosphatase and assayed for amino acid uptake. However, neither GABA nor glycine uptake was affected by VIAAT phosphorylation. These results indicate that VIAAT is constitutively phosphorylated on cytosolic serine or threonine residues in most, but not all, regions of the rat brain. This phosphorylation does not regulate the vesicular loading of GABA or glycine, suggesting that it is involved at other stages of the synaptic vesicle life cycle.     

LGI proteins in the nervous system

The development and function of the vertebrate nervous system depend on specific interactions between different cell types. Two examples of such interactions are synaptic transmission and myelination. LGI1-4 (leucine-rich glioma inactivated proteins) play important roles in these processes. They are secreted proteins consisting of an LRR (leucine-rich repeat) domain and a so-called epilepsy-associated or EPTP (epitempin) domain. Both domains are thought to function in protein–protein interactions. The first LGI gene to be identified, LGI1, was found at a chromosomal translocation breakpoint in a glioma cell line. It was subsequently found mutated in ADLTE (autosomal dominant lateral temporal (lobe) epilepsy) also referred to as ADPEAF (autosomal dominant partial epilepsy with auditory features). LGI1 protein appears to act at synapses and antibodies against LGI1 may cause the autoimmune disorder limbic encephalitis. A similar function in synaptic remodelling has been suggested for LGI2, which is mutated in canine Benign Familial Juvenile Epilepsy. LGI4 is required for proliferation of glia in the peripheral nervous system and binds to a neuronal receptor, ADAM22, to foster ensheathment and myelination of axons by Schwann cells. Thus, LGI proteins play crucial roles in nervous system development and function and their study is highly important, both to understand their biological functions and for their therapeutic potential. Here, we review our current knowledge about this important family of proteins, and the progress made towards understanding their functions.     

Co-packaging of opposing neurotransmitters in individual synaptic vesicles in the central nervous system

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External sensilla of the locust abdomen provide the central nervous system with an interganglionic network

External mechanoreceptors and contact chemoreceptors on the cuticle of the sixth abdominal segment of locusts have divergent primary projections of their sensory neurons that form arbours in the segmental and anterior abdominal ganglia. Homologous interganglionic projections from adjacent segments converge in the neuropile of each abdominal ganglion. Of the contributing types of sensilla, three were previously unknown for locust pregenital segments: tactile mechanosensory hairs with dual innervation, external proprioceptors of the hairplate type covered by intersegmental membranes and single campaniform sensilla that monitor cuticular strain in sternites and tergites. In general, interdependence of motor coordination in the abdominal segments is based on a neural network that relies heavily on intersegmental primary afferents that cooperate to identify the location, parameters and strength of external stimuli.     

血红素加氧酶-1在中枢神经系统疾病中的作用

血红素加氧酶-1(heme oxygenase-1,HO-1)是一种应激蛋白,可将血红素降解为胆绿素、游离铁和一氧化碳。目前,国内外普遍认为HO-1在神经系统损伤后的应激反应中有至关重要的作用,其表达量与神经元凋亡和神经变性密切相关。本文对近年来HO-1在神经系统损伤性疾病中的现有研究结果进行了总结分析,旨在为相关研究的发展提供新的思路和方向。     

Analog specificity of the thyrotropin-releasing hormone receptor in the central nervous system: possible clinical implications

TRH has rapid-onset (30 sec), slow-offset (1-12 days) clinical benefit in patients with amyotrophic lateral sclerosis and other motor neuron disorders. This benefit is probably receptor-mediated and may have at least 2 components. To obtain a better understanding of the various responses to TRH of the spinal lower motor neurons (LMNs) in patients, and possibly to help guide selection of additional therapeutic agents, we utilized rat CNS (spinal-cord and brain membranes) to analyze the ability of certain molecules to inhibit specific binding of [3H]methyl TRH [( 3H]MeTRH) to the TRH receptor. We found: a) lack of high-affinity binding of the TRH-analog DN-1417 by spinal-cord and brain TRH receptor, despite its known strong TRH-like action physiologically on LMNs; b) lack of high-affinity binding of the TRH-product cyclo(His-Pro) by spinal-cord and brain TRH receptor despite its having some strong TRH-like physiologic actions on the CNS; and c) lack of any identifiable high-affinity receptor for cyclo(His-Pro) in spinal cord and brain. From these data we hypothesize that the acute transmitter-like action of DN-1417, TRH, and possibly other TRH-analogs and products on LMNs is via a non-TRH receptor, such as an amine or amino acid neurotransmitter receptor, e.g. a 5-hydroxytryptamine receptor. We further postulate that the CNS TRH-receptor may modulate a trophic-like influence of TRH on LMNs.     

The inhibitory glycine receptor: a ligand-gated chloride channel of the central nervous system

The postsynaptic glycine receptor (GlyR) is a major inhibitory chloride channel protein in the central nervous system. The affinity-purified receptor contains polypeptides of 48 kDa, 58 kDa, and 93 kDa. The 48-kDa (alpha) and 58 kDa (beta) subunits span the postsynaptic membrane in a pentameric arrangement to form the anion channel of the receptor. The 93-kDa polypeptide is cytoplasmically localized and may have an anchoring function. Molecular cloning revealed that different structural characteristics are shared by the membrane-spanning subunits of the GlyR and those of other ligand-gated ion channel proteins. Developmental regulation of the GlyR is characterized by alterations in antagonist binding, heterogeneity of alpha subunits, and increased levels of the 93-kDa polypeptide. Glycine receptor function can be reconstituted by expression of cloned alpha subunits in heterologous cell systems. Positive charges found at the presumed mouths of the GlyR channel appear to be important determinants of ion selectivity. These data establish the anion-conducting GlyR as a homolog of other ligand-gated ion channel proteins and suggest that the diversity of these channels originates from divergent evolution of a primordial channel protein early in phylogeny.     

Elevation of cyclic GMP levels in central nervous system by excitatory and inhibitory amino acids

—Guanosine 3′,5’cyclic monophosphate (cyclic GMP) levels in incubated slices of mouse cerebellum are increased 10-fold by glutamate and two-to three-fold by glycine or γ-aminobutyric acid (GABA). Glutamate also produces a 10-fold increase in adenosine 3′,5’cyclic monophosphate (cyclic AMP) in the same tissue. However, GABA decreases cyclic AMP levels 30-40 per cent, and glycine produces only a transient 50 per cent accumulation of this cyclic nucleotide. Theophylline slightly augments the accumulation of cyclic GMP produced by all three amino acids but markedly attenuates the accumulation of cyclic AMP produced by glutamate. In the absence of Ca²⁺, none of the three amino acids has any effect on cyclic GMP levels, and glutamate produces only a 50 per cent rise in cyclic AMP levels. The decrease of cyclic AMP levels produced by GABA is not affected by theophylline or by the absence of Ca²⁺. These data suggest an involvement of both cyclic GMP and cyclic AMP in the neurochemical actions of glutamate, GABA and glycine.     

Basis for the specificity of anti-5-HT-like antisera in immunocytochemistry applied to the central nervous system

Summary The neuropeptide tyrosine precursor (pre-pro-NPY) messenger RNA (mRNA) has been localised in formaldchyde-fixed human phaeochromocytoma tissue using a sensitive in situ hybridisation procedure and a novel single-stranded cDNA probe. The reaction product was revealed by avidin-biotin-peroxidase complex and streptavidin-gold complex with silver enhancement. This technique may be applied for the determination of biosynthetic activity of endocrine and neuronal cell bodies. This is largely due to its rapidity by comparison with conventional autoradiographic procedures, to the permanence of the reaction product and to the sensitivity of the visualisation steps.