Axon navigation in the mammalian primary olfactory pathway: Where to next?

The process of establishing long-range neuronal connections can be divided into at least three discrete steps. First, axons need to be stimulated to grow and this growth must be towards appropriate targets. Second, after arriving at their target, axons need to be directed to their topographically appropriate position and in some cases, such as in cortical structures, they must grow radially to reach the correct laminar layer Third, axons then arborize and form synaptic connections with only a defined subpopulation of potential post-synaptic partners. Attempts to understand these mechanisms in the visual system have been ongoing since pioneer studies in the 1940s highlighted the specificity of neuronal connections in the retino-tectal pathway. These classical systems-based approaches culminated in the 1990s with the discovery that Eph-ephrin repulsive interactions were involved in topographical mapping. In marked contrast, it was the cloning of the odorant receptor family that quickly led to a better understanding of axon targeting in the olfactory system. The last 10 years have seen the olfactory pathway rise in prominence as a model system for axon guidance. Once considered to be experimentally intractable, it is now providing a wealth of information on all aspects of axon guidance and targeting with implications not only for our understanding of these mechanisms in the olfactory system but also in other regions of the nervous system.

Macrophages, myofibroblasts and neointimal hyperplasia after coronary artery injury and repair

Macrophages participate in the restenosis process through the release of cytokines, metalloproteinases and growth factors. Studies of peritoneal granulation tissue suggest that macrophages may be precursors of myofibroblasts. This study examined the contribution of monocyte/macrophage lineage cells to neointimal cellular mass in a porcine model of thermal vascular injury. Thermal coronary artery injury caused medial smooth muscle cell necrosis and transformation of the media into an extracellular matrix barrier. The neointimal hyperplasia that developed over the injury sites was evaluated by light microscopy, electron microscopy and immunohistochemistry. At day 3, blood monocytes were adhered to the vessel wall and infiltrated the fibrotic media. At day 14, 42 +/- 3.9% of neointimal cells had a monocytic nuclear morphology and expressed macrophage-specific antigen SWC3 (identified by monoclonal antibody DH59B). Moreover, 9.2+/-1.8% of neointimal cells co-expressed SWC3 and alpha-smooth muscle actin and had ultrastructural characteristics intermediate between macrophages and myofibroblasts. At day 28, 10.5 +/- 3.5%, of cells expressed SWC3 and 5.2+/-1.8% of cells co-expressed SWC3 and alpha-smooth muscle actin. This study indicates that hematopoietic cells of monocyte/macrophage lineage abundantly populate the neointima in the process of lesion formation and may be precursors of neointimal myofibroblasts after thermal vascular injury. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

Contribution of cholinergic systems to state-dependent modulation of respiratory control

Respiration is altered during different stages of the sleep-wake cycle. We review the contribution of cholinergic systems to this alteration, with particular reference to the role of muscarinic acetylcholine receptors (MAchRs) during rapid eye movement (REM) sleep. Available evidence demonstrates that MAchRs have potent excitatory effects on medullary respiratory neurones and respiratory motoneurones, and are likely to contribute to changes in central chemosensitive drive to the respiratory control system. These effects are likely to be most prominent during REM sleep, when cholinergic brainstem neurones show peak activity levels. It is possible that MAchR dysfunction is involved in sleep-disordered breathing, Such as obstructive sleep apnea. (C) 2002 Elsevier Science B.V. All rights reserved.

Neural control of the heart: developmental changes in ionic conductances in mammalian intrinsic cardiac neurons

The expression and properties of ionic channels were investigated in dissociated neurons from neonatal and adult rat intracardiac ganglia. Changes in the hyperpolarization-activated and ATP-sensitive K+ conductances during postnatal development and their role in neuronal excitability were examined. The hyperpolarization-activated nonselective cation current, I-h, was observed in all neurons studied and displayed slow time-dependent rectification. An inwardly rectifying K+ current, I-K(I), was present in a population of neurons from adult but not neonatal rats and was sensitive to block by extracellular Ba2+. Using the perforated-patch recording configuration, an ATP-sensitive K+ (K-ATP) conductance was identified in greater than or equal to 50% of intracardiac neurons from adult rats. Levcromakalim evoked membrane hyperpolarization, which was inhibited by the sulphonylurea drugs. glibenclamide and tolbutamide. Exposure to hypoxic conditions also activated a membrane current similar to that induced by levcromakalim and was inhibited by glibenclamide. Changes in the complement of ion channels during postnatal development may underlie observed differences in the function of intracardiac ganglion neurons during maturation. Furthermore, activation of hyperpolarization-activated and KATP channels in mammalian intracardiac neurons may play a role in neural regulation of the mature heart and cardiac function during ischaemia-reperfusion. (C) 2002 Elsevier Science B.V All rights reserved.

The synthesis and structure of an n-terminal dodecanoic acid conjugate of a-conotoxin MII

The alpha-conotoxin MII is a 16 amino acid long peptide toxin isolated from the marine snail, Conus magus. This toxin has been found to be a highly selective and potent inhibitor of neuronal nicotinic acetylcholine receptors of the subtype alpha3beta2. To improve the bioavailability of this peptide, we have coupled to the N-terminus of conotoxin MII, 2-amino-D,L-dodecanoic acid (Laa) creating a lipidic linear peptide which was then successfully oxidised to produce the correctly folded conotoxin MII construct.

A new level of conotoxin diversity, a non-native disulfide bond connectivity in alpha-conotoxin AuIB reduces structural definition but increases biological activity

alpha-Conotoxin AuIB and a disulfide bond variant of AuIB have been synthesized to determine the role of disulfide bond connectivity on structure and activity. Both of these peptides contain the 15 amino acid sequence GCCSYPPCFATNPDC, with the globular (native) isomer having the disulfide connectivity Cys(2-8 and 3-15) and the ribbon isomer having the disulfide connectivity Cys(2-15 and 3-8). The solution structures of the peptides were determined by NAIR spectroscopy, and their ability to block the nicotinic acetylcholine receptors on dissociated neurons of the rat parasympathetic ganglia was examined. The ribbon disulfide isomer, although having a less well defined structure, is surprisingly found to have approximately 10 times greater potency than the native peptide. To our knowledge this is the first demonstration of a non-native disulfide bond isomer of a conotoxin exhibiting greater biological activity than the native isomer.

Basal nonselective cation permeability in rat cardiac microvascular endothelial cells

The presence of a basal nonselective cation permeability was mainly investigated in primary cultures of rat cardiac microvascular endothelial cells (CMEC) by applying both the patch-clamp technique and Fura-2 microfluorimetry. With low EGTA in the pipette solution, the resting membrane potential of CMEC was -21.2 +/- 1.1 mV, and a Ca2+-activated Cl- conductance was present. When the intracellular Ca2+ was buffered with high EGTA, the membrane potential decreased to 5.5 +/- 1.2 mV. In this condition, full or partial substitution of external Na+ by NMDG(+) proportionally reduced the inward component of the basal I-V relationship. This current was dependent on extracellular monovalent cations with a permeability sequence of K+ > Cs+ > Na+ > Li+ and was inhibited by Ca2+, La3+, Gd3+, and amiloride. The K+/Na+ permeability ratio, determined using the Goldman-Hodgkin-Katz equation, was 2.01. The outward component of the basal I-V relationship was reduced when intracellular K+ was replaced by NMDG(+), but was not sensitive to substitution by Cs+. Finally, microfluorimetric experiments indicated the existence of a basal Ca2+ entry pathway, inhibited by La3+ and Gd3+. The basal nonselective cation permeability in CMEC could be involved both in the control of myocardial ionic homeostasis, according to the model of the blood-heart barrier, and in the modulation of Ca2+ -dependent processes. (C) 2002 Elsevier Science (USA).

Conserved modularity and potential for alternate splicing in mouse and human Slit genes

The vertebrate Slit gene family currently consists of three members;Slit1,Slit2 and Slit3. Each gene encodes a protein containing multiple epidermal growth factor and leucine rich repeat motifs, which are likely to have importance in cell-cell interactions. In this study, we sought to fully define and characterise the vertebrate Slit gene family. Using long distance PCR coupled with in silico mapping, we determined the genomic structure of all three Slit genes in mouse and man. Analysis of EST and genomic databases revealed no evidence of further Slit family members in either organism. All three Slit genes were encoded by 36 (Slit3) or 37 (Slit1 and Slit2) exons covering at least 143 kb or 183 kb of mouse or human genomic DNA respectively. Two additional potential leucine-rich repeat encoding exons were identified within intron 12 of Slit2. These could be inserted in frame, suggesting that alternate splicing may occur in Slit2 A search for STS sequences within human Slit3 anchored this gene to D5S2075 at the 5' end (exon 4) and SGC32449 within the 3' UTR, suggesting that Slit3 may cover greater than 693 kb. The genomic structure of all Slit genes demonstrated considerable modularity in the placement of exon-intron boundaries such that individual leucine-rich repeat motifs were encoded by individual 72 by exons. This further implies the potential generation of multiple Slit protein isoforms varying in their number of repeat units. cDNA library screening and EST database searching verified that such alternate splicing does occur.

Antagonists of protein kinase C inhibit rat retinal glutamate transport activity in situ

Neuronal and glial high-affinity transporters regulate extracellular glutamate concentration, thereby terminating synaptic transmission and preventing neuronal excitotoxicity. Glutamate transporter activity has been shown to be modulated by protein kinase C (PKC) in cell culture. This is the first study to demonstrate such modulation in situ, by following the fate of the non-metabolisable glutamate transporter substrate, D-aspartate. In the rat retina, pan-isoform PKC inhibition with chelerythrine suppressed glutamate uptake by GLAST (glutamate/aspartate transporter), the dominant excitatory amino acid transporter localized to the glial Muller cells. This effect was mimicked by rottlerin but not by Go6976, suggesting the involvement of the PKCdelta isoform, but not PKCalpha, beta or gamma. Western blotting and immunohistochemical labeling revealed that the suppression of glutamate transport was not due to a change in transporter expression. Inhibition of PKCdelta selectively suppressed GLAST but not neuronal glutamate transporter activity. These data suggest that the targeting of specific glutamate transporters with isoform-specific modulators of PKC activity may have significant implications for the understanding of neurodegenerative conditions arising from compromised glutamate homeostasis, e.g. glaucoma and amyotrophic lateral sclerosis.

No evidence for inhibition of ENaC through CFTR-mediated release of ATP

Both purinergic stimulation and activation of cystic fibrosis transmembrane conductance regulator (CFTR) increases Cl- secretion and inhibit amiloride-sensitive Na+ transport. CFTR has been suggested to conduct adenosine 5'-triphosphate (ATP) or to control ATP release to the luminal side of epithelial tissues. Therefore, a possible mechanism on how CFTR controls the activity of epithelial Na+ channels (ENaC) could be by release of ATP or uridine 5'-triphosphate (UTP), which would then bind to P2Y receptors and inhibit ENaC. We examined this question in native tissues from airways and colon and in Xenopus oocytes. Inhibition of amiloride-sensitive transport by both CFTR and extracellular nucleotides was observed in colon and trachea. However, nucleotides did not inhibit ENaC in Xenopus oocytes, even after coexpression of P2Y(2) receptors. Using different tools such as hexokinase, the P2Y inhibitor suramin or the Cl- channel blocker 4,4'diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), we did not detect any role of a putative ATP secretion in activation of Cl- transport or inhibition of amiloride sensitive short circuit currents by CFTR. In addition, N-2,2'-O-dibutyrylguanosine 3',5-cyclic monophosphate (cGMP) and protein kinase G (PKG)-dependent phosphorylation or the nucleoside diphosphate kinase (NDPK) do not seem to play a role for the inhibition of ENaC by CFTR, which, however, requires the presence of extracellular Cl-. (C) 2002 Elsevier Science B.V. All rights reserved.

Stressful animal housing conditions and their potential effect on sympathetic neurotransmission in mice

Although the sympathetic nervous system (SNS) plays a major role in mediating the peripheral stress response, due consideration is not usually given to the effects of prolonged stress on the SNS. The present study examined changes in neurotransmission in the SNS after exposure of mice (BALB/c) to stressful housing conditions. Focal extracellular recording of excitatory junction currents (EJCs) was used as a relative measure of neurotransmitter release from different regions of large surface areas of the mouse vas deferens. Mice were either group housed (control), isolation housed (social deprivation), group housed in a room containing rats (rat odor stress), or isolation housed in a room containing rats (concurrent stress). Social deprivation and concurrent stressors induced an increase of 30 and 335% in EJC amplitude, respectively. The success rate of recording EJCs from sets of varicosities in the concurrent stressor group was greater compared with all other groups. The present study has shown that some common animal housing conditions act as stressors and induce significant changes in sympathetic neurotransmission.

Colony-stimulating factor-1 suppresses responses to CpG DNA and expression of toll-like receptor 9 but enhances responses to lipopolysaccharide in murine macrophages

During bacterial infections, the balance between resolution of infection and development of sepsis is dependent upon the macrophage response to bacterial products. We show that priming of murine bone marrow-derived macrophages (BMMs) with CSF-1 differentially regulates the response to two such stimuli, LPS and immunostimulatory (CpG) DNA. CSF-1 pretreatment enhanced IL-6, IL-12, and TNF-alpha production in response to LPS but suppressed the same response to CpG DNA. CSF-1 also regulated cytokine gene expression in response to CpG DNA and LPS; CpG DNA-induced IL-12 p40, IL-12 p35, and TNF-alpha mRNAs were all suppressed by CSF-1 pretreatment. CSF-1 pretreatment enhanced LPS-induced IL-12 p40 mRNA but not TNF-alpha and IL-12 p35 mRNAs, suggesting that part of the priming effect is posttranscriptional. CSF-1 pretreatment also suppressed CpG DNA-induced nuclear translocation of NF-kappaB and phosphorylation of the mitogen-activated protein kinases p38 and extracellular signal-related kinases-1/2 in BMMs, indicating that early events in CpG DNA signaling were regulated by CSF-1. Expression of Toll-like receptor (TLR)9, which is necessary for responses to CpG DNA, was markedly suppressed by CSF-1 in both BMMs and thioglycolate-elicited peritoneal macrophages. CSF-1 also down-regulated expression of TLR1, TLR2, and TLR6, but not the LPS receptor, TLR4, or TLR5. Hence, CSF-1 may regulate host responses to pathogens through modulation of TLR expression. Furthermore, these results suggest that CSF-1 and CSF-1R antagonists may enhance the efficacy of CpG DNA in vivo.

The distribution and morphological characteristics of cholinergic cells in the brain of monotremes as revealed by ChAT immunohistochemistry

The present study employs choline acetyltransferase (ChAT) immunohistochemistry to identify the cholinergic neuronal population in the central nervous system of the monotremes. Two of the three extant species of monotreme were studied: the platypus (Omithorhynchus anatinus) and the short-beaked echidna (Tachyglossus aculeatus). The distribution of cholinergic cells in the brain of these two species was virtually identical. Distinct groups of cholinergic cells were observed in the striatum, basal forebrain, habenula, pontomesencephalon, cranial nerve motor nuclei, and spinal cord. In contrast to other tetrapods studied with this technique, we failed to find evidence for cholinergic cells in the hypothalamus, the parabigeminal nucleus (or nucleus isthmus), or the cerebral cortex. The lack of hypothalamic cholinergic neurons creates a hiatus in the continuous antero-posterior aggregation of cholinergic neurons seen in other tetrapods. This hiatus might be functionally related to the phenomenology of monotreme sleep and to the ontogeny of sleep in mammals, as juvenile placental mammals exhibit a similar combination of sleep elements to that found in adult monotremes. Copyright (C) 2002 S. Karger AG, Basel.

Direction selectivity in the retina

The neuronal circuitry underlying the generation of direction selectivity in the retina has remained elusive for almost 40 years. Recent studies indicate that direction selectivity may be established within the radial dendrites of 'starburst' amacrine cells and that retinal ganglion cells may acquire their direction selectivity by the appropriate weighting of excitatory and inhibitory inputs from starburst dendrites pointing in different directions. If so, this would require unexpected complexity and subtlety in the synaptic connectivity of these CNS neurons.

Immunohistochemical localization of fibromodulin in the periodontium during cementogenesis and root formation in the rat molar

Background: Cementum is essential for periodontal regeneration, as it provides anchorage between the root surface and the periodontal ligament. A variety of macromolecules present in the extracellular matrix of the periodontium, including proteoglycans, are likely to play a regulatory role in cementogenesis. Recently, the small leucine-rich proteoglycan, fibromodulin, has been isolated from bovine periodontal ligament and localized in bovine cementum, as well as in human periodontal ligament. Objective: The aim of this study was to examine the distribution of fibromodulin during cementogenesis and root formation. Methods: A standard indirect immunoperoxidase technique was employed, using an antifibromodulin polyclonal antibody on sections of molar teeth from rats aged 3, 5 and 8 weeks. Results: Immunoreactivity to fibromodulin was evident in the periodontal ligament in all sections. An intense positive stain was observed in the extracellular matrix where the periodontal ligament fibers insert into the alveolar bone and where the Sharpey's fibers insert into the cementum. There was no staining evident in the mineralized cellular and acellular cementum. The intensity of immunoreactivity to the antifibromodulin antibody increased proportionally with increasing tissue maturation. Conclusion: The results from this study suggest that fibromodulin is a significant component of the extracellular matrix in the periodontal ligament during development, and may play a regulatory role in the mineralization process or maintaining homeostasis at the hard-soft tissue interface during cementogenesis.