Localization of neogenin protein during morphogenesis in the mouse embryo

Neogenin, a close relative of the axon guidance receptor DCC, has been shown to be a receptor for members of the Netrin and Repulsive Guidance Molecule families. Recent studies have begun to uncover a role for Neogenin in organogenesis. Here we examine the localization of Neogenin protein in the developing mouse embryo (embryonic day 14.5) when organogenesis is progressing rapidly. We observe that Neogenin protein is restricted to distinct tissue layers within a given organ. In some embryonic epithelia such as the gut and pancreas, Neogenin protein is predominantly polarized to the basal surfaces of the epithelial cells. In contrast, Neogenin is restricted to mesenchymal cells within the lung and kidney. Neogenin is also seen in differentiating skeletal muscle and condensing cartilage throughout the embryo, and in the trigeminal and dorsal root ganglia of the peripheral nervous system. This study supports the emerging role for Neogenin as a key receptor in the establishment of the morphological architecture in many developing organ systems.

Nervous and muscle system development in Phascolion strombus (Sipuncula)

Recent interpretations of developmental gene expression patterns propose that the last common metazoan ancestor was segmented, although most animal phyla show no obvious signs of segmentation. Developmental studies of non-model system trochozoan taxa may shed light on this hypothesis by assessing possible cryptic segmentation patterns. In this paper, we present the first immunocytochemical data on the ontogeny of the nervous system and the musculature in the sipunculan Phascolion strombus. Myogenesis of the first anlagen of the body wall ring muscles occurs synchronously and not subsequently from anterior to posterior as in segmented spiralian taxa (i.e. annelids). The number of ring muscles remains constant during the initial stages of body axis elongation. In the anterior-posteriorly elongated larva, newly formed ring muscles originate along the entire body axis between existing myocytes, indicating that repeated muscle bands do not form from a posterior growth zone. During neurogenesis, the Phascolion larva expresses a non-metameric, paired, ventral nerve cord that fuses in the mid-body region in the late-stage elongated larva. Contrary to other trochozoans, Phascolion lacks any larval serotonergic structures. However, two to three FMRFamide-positive cells are found in the apical organ. In addition, late larvae show commissure-like neurones interconnecting the two ventral nerve cords, while early juveniles exhibit a third, medially placed FMRFamidergic ventral nerve. Although we did not find any indications for cryptic segmentation, certain neuro-developmental traits in Phascolion resemble the conditions found in polychaetes (including echiurans) and myzostomids and support a close relationship of Sipuncula and Annelida.

Dissection of peripheral and central endogenous opioid modulation of systemic interleukin-1 beta responses using c-fos expression in the rat brain

In opiate addicts or patients receiving morphine treatment, it has been reported that the immune system is often compromised. The mechanisms responsible for the adverse effects of opioids on responses to infection are not clear but it is possible that central and/or peripheral opioid receptors may be important. We have utilised an experimental immune challenge model in rats, the systemic administration of the human pro-inflammatory cytokine interleukin-1 beta (IL-1 beta) to study the effects of selectively blocking peripheral opioid receptors only (using naloxone methiodide) or after blocking both central and peripheral opioid receptors (using naloxone). Pre-treatment with naloxone methiodide decreased (15%) IL-1 beta-induced Fos-immunoreactivity (Fos-IR) in medial parvocellular paraventricular nucleus (mPVN) corticotropin-releasing hormone (CRH) neurons but increased responses in the ventrolateral medulla (VLM) C1 (65%) and nucleus tractus solitarius (NTS) A2 (110%) catecholamine cell groups and area postrema (136%). However no effect of blocking peripheral opioid receptors was detected in the central nucleus of the amygdala (CeA) or dorsal bed nucleus of the stria terminalis (BNST). We next determined the effect of blocking both central and peripheral opioid receptors with naloxone and, when compared to the naloxone methiodide pre-treated group, a further 60% decrease in Fos-IR mPVN CRH neurons induced by IL-1 beta was detected, which was attributed to block of central opioid receptors. Similar comparisons also detected decreases in Fos-IR neurons induced by IL-1 beta in the VLM A1, VLM C1 and NTS A2 catecholamine cell groups, area postrema, and parabrachial nucleus. In contrast, pre-treatment with naloxone increased Fos-IR neurons in CeA (98%) and dorsal BNST (72%). These results provide novel evidence that endogenous opioids can influence central neural responses to systemic IL-1 beta and also suggest that the differential patterns of activation may arise because of actions at central and/or peripheral opioid receptors that might be important in regulating behavioural, hypothalamic-pituitary-adrenal axis and sympathetic nervous system responses during an immune challenge. (c) 2005 Elsevier Ltd. All rights reserved.

Convulsive ergotism: epidemics of the serotonin syndrome?

Between 1085 and 1927, epidemics of convulsive ergotism were widespread east of the Rhine in Europe due to consumption of grain contaminated with ergot, which is produced by the fungus Claviceps purpurea. West of the Rhine, consumption of ergot-contaminated food caused epidemics of gangrenous ergotism. The clinical features of convulsive ergotism-muscle twitching and spasms, changes in mental state, hallucinations, sweating, and fever lasting for several weeks-suggest serotonergic overstimulation of the CNS (ie, the serotonin syndrome). The ergot alkaloids are serotonin agonists. Dihydroergotamine binds to serotonin receptors in the dorsal horn of the spinal cord, which is the site of neuropathological changes in convulsive ergotism. Dihydroergotamine given to human beings can cause the serotonin syndrome. Ergots produced by different strains of Claviceps purpurea, and those growing in different soils, may have different ergot alkaloid compositions. An alkaloid, present in high concentrations in ergots from east of the Rhine, may have caused convulsive ergotism at a circulating concentration insufficient to produce peripheral ischaemia. The serotonin syndrome may, therefore, have been a public-health problem long before it was recognised as a complication of modem psychopharmacology.

Involvement of Islet-2 in the Slit signaling for axonal branching and defasciculation of the sensory neurons in embryonic zebrafish

In Drosophila melanogaster, Slit acts as a repulsive cue for the growth cones of the commissural axons which express a receptor for Slit, Roundabout (Robo), thus preventing the commissural axons from crossing the midline multiple times. Experiments using explant culture have shown that vertebrate Slit homologues also act repulsively for growth cone navigation and neural migration, and promote branching and elongation of sensory axons. Here, we demonstrate that overexpression of Slit2 in vivo in transgenic zebrafish embryos severely affected the behavior of the commissural reticulospinal neurons (Mauthner neurons), promoted branching of the peripheral axons of the trigeminal sensory ganglion neurons, and induced defasciculation of the medial longitudinal fascicles. In addition, Slit2 overexpression caused defasciculation and deflection of the central axons of the trigeminal sensory ganglion neurons from the hindbrain entry point. The central projection was restored by either functional repression or mutation of Robo2, supporting its role as a receptor mediating the Slit signaling in vertebrate neurons. Furthermore, we demonstrated that Islet-2, a LIM/homeodomain-type transcription factor, is essential for Slit2 to induce axonal branching of the trigeminal sensory ganglion neurons, suggesting that factors functioning downstream of Islet-2 are essential for mediating the Slit signaling for promotion of axonal branching. (C) 2004 Elsevier Ireland Ltd. All rights reserved.

The alpha(2)delta auxiliary subunit reduces affinity of omega-conotoxins for recombinant N-type (Ca(v)2.2) calcium channels

The omega-conotoxins from fish-hunting cone snails are potent inhibitors of voltage-gated calcium channels. The omega-conotoxins MVIIA and CVID are selective N-type calcium channel inhibitors with potential in the treatment of chronic pain. The beta and alpha(2)delta-1 auxiliary subunits influence the expression and characteristics of the alpha(1B) subunit of N-type channels and are differentially regulated in disease states, including pain. In this study, we examined the influence of these auxiliary subunits on the ability of the omega-conotoxins GVIA, MVIIA, CVID and analogues to inhibit peripheral and central forms of the rat N-type channels. Although the beta3 subunit had little influence on the on- and off-rates of omega-conotoxins, coexpression of alpha(2)delta with alpha(1B) significantly reduced on- rates and equilibrium inhibition at both the central and peripheral isoforms of the N-type channels. The alpha(2)delta also enhanced the selectivity of MVIIA, but not CVID, for the central isoform. Similar but less pronounced trends were also observed for N-type channels expressed in human embryonic kidney cells. The influence of alpha(2)delta was not affected by oocyte deglycosylation. The extent of recovery from the omega-conotoxin block was least for GVIA, intermediate for MVIIA, and almost complete for CVID. Application of a hyperpolarizing holding potential ( - 120 mV) did not significantly enhance the extent of CVID recovery. Interestingly, [R10K] MVIIA and [O10K] GVIA had greater recovery from the block, whereas [K10R] CVID had reduced recovery from the block, indicating that position 10 had an important influence on the extent of omega-conotoxin reversibility. Recovery from CVID block was reduced in the presence of alpha(2)delta in human embryonic kidney cells and in oocytes expressing alpha(1B-b). These results may have implications for the antinociceptive properties of omega-conotoxins, given that the alpha(2)delta subunit is up-regulated in certain pain states.

Molecular structure and function of the glycine receptor chloride channel

The glycine receptor chloride channel (GlyR) is a member of the nicotinic acetylcholine receptor family of ligand-gated ion channels. Functional receptors of this family comprise five subunits and are important targets for neuroactive drugs. The GlyR is best known for mediating inhibitory neurotransmission in the spinal cord and brain stem, although recent evidence suggests it may also have other physiological roles, including excitatory neurotransmission in embryonic neurons. To date, four alpha-subunits (alpha1 to alpha4) and one beta-subunit have been identified. The differential expression of subunits underlies a diversity in GlyR pharmacology. A developmental switch from alpha2 to alpha1beta is completed by around postnatal day 20 in the rat. The beta-subunit is responsible for anchoring GlyRs to the subsynaptic cytoskeleton via the cytoplasmic protein gephyrin. The last few years have seen a surge in interest in these receptors. Consequently, a wealth of information has recently emerged concerning Glyl? molecular structure and function. Most of the information has been obtained from homomeric alpha1 GlyRs, with the roles of the other subunits receiving relatively little attention. Heritable mutations to human GlyR genes give rise to a rare neurological disorder, hyperekplexia (or startle disease). Similar syndromes also occur in other species. A rapidly growing list of compounds has been shown to exert potent modulatory effects on this receptor. Since GlyRs are involved in motor reflex circuits of the spinal cord and provide inhibitory synapses onto pain sensory neurons, these agents may provide lead compounds for the development of muscle relaxant and peripheral analgesic drugs.

Ventilatory responses of healthy subjects to intravenous combinations of morphine and oxycodone under imposed hypercapnic and hypoxaemic conditions

Aims Previous isobolographic analysis revealed that coadministration of morphine and oxycodone produces synergistic antinociception in laboratory rodents. As both opioids can produce ventilatory depression, this study was designed to determine whether their ventilatory effects were synergistic when coadministered to healthy human subjects. Methods A placebo-controlled, randomized, crossover study was performed in 12 male volunteers. Ventilatory responses to hypoxaemia and hypercapnia were determined from 1-h intravenous infusions of saline ('placebo'), 15 mg morphine sulphate (M), 15 mg oxycodone hydrochloride (O), and their combination in the dose ratios of 1 : 2, 1 : 1, 2 : 1. Drug and metabolite concentrations in serial peripheral venous blood samples were measured by high-performance liquid chromatography-MS/MS. Results 'Placebo' treatment was without significant ventilatory effects. There were no systematic differences between active drug treatments on either the slopes or intercepts of the hypoxaemic and hypercapnia ventilation responses. During drug treatment, the mean minute ventilation at PETCO2 = 55 mmHg (V-E55) decreased to 74% of the subjects' before treatment values (95% confidence interval 62, 87), 68% (57, 80), 69% (59, 79), 68% (63, 73), and 61% (52, 69) for M15, M10/O5, M7.5/O7.5, M5/O10 and O15, respectively. Recovery was more prolonged with increasing oxycodone doses, corresponding to its greater potency and lower clearance compared with morphine. Conclusions Although adverse ventilatory effects of these drugs were found as expected, no unexpected or disproportionate effects of any of the morphine and oxycodone treatments were found that might impede their use in combination for pain management.

Distinct types of glial cells populate the Drosophila antenna

Background: The development of nervous systems involves reciprocal interactions between neurons and glia. In the Drosophila olfactory system, peripheral glial cells arise from sensory lineages specified by the basic helix- loop- helix transcription factor, Atonal. These glia wrap around the developing olfactory axons early during development and pattern the three distinct fascicles as they exit the antenna. In the moth Manduca sexta, an additional set of central glia migrate to the base of the antennal nerve where axons sort to their glomerular targets. In this work, we have investigated whether similar types of cells exist in the Drosophila antenna. Results: We have used different P( Gal4) lines to drive Green Fluorescent Protein ( GFP) in distinct populations of cells within the Drosophila antenna. Mz317:: GFP, a marker for cell body and perineural glia, labels the majority of peripheral glia. An additional similar to 30 glial cells detected by GH146:: GFP do not derive from any of the sensory lineages and appear to migrate into the antenna from the brain. Their appearance in the third antennal segment is regulated by normal function of the Epidermal Growth Factor receptor and small GTPases. We denote these distinct populations of cells as Mz317- glia and GH146- glia respectively. In the adult, processes of GH146- glial cells ensheath the olfactory receptor neurons directly, while those of the Mz317- glia form a peripheral layer. Ablation of GH146- glia does not result in any significant effects on the patterning of the olfactory receptor axons. Conclusion: We have demonstrated the presence of at least two distinct populations of glial cells within the Drosophila antenna. GH146- glial cells originate in the brain and migrate to the antenna along the newly formed olfactory axons. The number of cells populating the third segment of the antenna is regulated by signaling through the Epidermal Growth Factor receptor. These glia share several features of the sorting zone cells described in Manduca.