The carbon monoxide releasing molecule (CORM-3) inhibits expression of vascular cell adhesion molecule-1 and E-selectin independently of haem oxygenase-1 expression.

BACKGROUND AND PURPOSE: Although carbon monoxide (CO) can modulate inflammatory processes, the influence of CO on adhesion molecules is less clear. This might be due to the limited amount of CO generated by haem degradation. We therefore tested the ability of a CO releasing molecule (CORM-3), used in supra-physiological concentrations, to modulate the expression of vascular cell adhesion molecule (VCAM)-1 and E-selectin on endothelial cells and the mechanism(s) involved. EXPERIMENTAL APPROACH: Human umbilical vein endothelial cells (HUVECs) were stimulated with tumour necrosis factor (TNF)-alpha in the presence or absence of CORM-3. The influence of CORM-3 on VCAM-1 and E-selectin expression and the nuclear factor (NF)-kappaB pathway was assessed by flow cytometry, Western blotting and electrophoretic mobility shift assay. KEY RESULTS: CORM-3 inhibited the expression of VCAM-1 and E-selectin on TNF-alpha-stimulated HUVEC. VCAM-1 expression was also inhibited when CORM-3 was added 24 h after TNF-alpha stimulation or when TNF-alpha was removed. This was paralleled by deactivation of NF-kappaB and a reduction in VCAM-1 mRNA. Although TNF-alpha removal was more effective in this regard, VCAM-1 protein was down-regulated more rapidly when CORM-3 was added. CORM-3 induced haem oxygenase-1 (HO-1) in a dose- and time-dependent manner, mediated by the transcription factor, Nrf2. CORM-3 was still able to down-regulate VCAM-1 expression in HUVEC transfected with siRNA for HO-1 or Nrf2. CONCLUSIONS AND IMPLICATIONS: Down-regulation of VCAM and E-selectin expression induced by CORM-3 was independent of HO-1 up-regulation and was predominantly due to inhibition of sustained NF-kappaB activation.

Pygmy squids and giant brains: mapping the complex cephalopod CNS by phalloidin staining of vibratome sections and whole-mount preparations.

Among bilaterian invertebrates, cephalopod molluscs (e.g., squids, cuttlefish and octopuses) have a central nervous system (CNS) that rivals in complexity that of the phylogenetically distant vertebrates (e.g., mouse and human). However, this prime example of convergent evolution has rarely been the subject of recent developmental and evolutionary studies, which may partly be due to the lack of suitable neural markers and the large size of cephalopod brains. Here, we demonstrate the usefulness of fluorescence-coupled phalloidin to characterize the CNS of cephalopods using histochemistry combined with confocal laser scanning microscopy. Whole-mount preparations of developmental stages as well as vibratome sections of embryonic and adult brains were analyzed and the benefits of this technique are illustrated. Compared to classical neuroanatomical and antibody-based studies, phalloidin labeling experiments are less time-consuming and allow a high throughput of samples. Besides other advantages summarized here, phalloidin reliably labels the entire neuropil of the CNS of all squids, cuttlefish and octopuses investigated. This facilitates high-resolution in toto reconstructions of the CNS and contributes to a better understanding of the organization of neural networks. Amenable for multi-labeling experiments employing antibodies against neurotransmitters, proteins and enzymes, phalloidin constitutes an excellent neuropil marker for the complex cephalopod CNS.

FMRFamide-like immunoreactivity in the central nervous system of the cephalopod mollusc, Idiosepius notoides.

For more than a century, cephalopod molluscs have been the subject of extensive studies with respect to their complex neuroanatomy and behavior. In comparison to gastropod molluscs surprisingly little work has been carried out on the characterization of neurons in the central nervous system (CNS) of cephalopods with respect to their neurotransmitter phenotypes. This study presents preliminary results on the distribution of FMRFamide-like immunoreactive neurons within the CNS of the pygmy squid Idiosepius notoides. Its gross neuroanatomy resembles that of other cephalopods. FMRFamide-like immunoreactivity was observed in most of the brain lobes. High abundance of FMRFamidergic perikarya was found in the dorsal basal, the central palliovisceral, and the olfactory lobes, whereas none were observed in the middle suboesophageal mass. Single individual perikarya are located within the optic lobes and the vertical lobes. Although certain immunohistochemical traits are shared with other cephalopods, such as a wall-like arrangement of FMRFamide-like immunoreactive cell somata within the dorsal basal lobe, others have so far only been found in Idiosepius. However, future investigations on other species are necessary in order to broaden our knowledge on a common recruitment of certain neurotransmitters in distinct brain lobes of the highly advanced brain of cephalopods.

Immunofluorescence analysis of the internal brain anatomy of Nereis diversicolor (Polychaeta, Annelida).

Comparative analyses of neuroanatomical characters can make valuable contributions to the inference of phylogenetic relationships. Whereas investigations in this field are numerous for arthropods, in-depth studies on other protostomes are sparse. Here, we provide a survey of the internal neuroarchitecture of the brain of the aciculate ragworm Nereis diversicolor (Polychaeta, Annelida). Descriptions are based on confocal laser scanning microscope analyses of brain sections labeled with the nuclear marker DAPI and antibodies raised against FMRF-amide, serotonin, and histamine. Autofluorescence of the nervous tissue has been utilized to further elucidate the anatomical structures of the brain. The architecture of two major brain compartments, i.e., the paired mushroom bodies and the central optic neuropil, is described in detail. The findings are compared with existent literature on polychaete neuroanatomy and on arthropod neuroanatomy, and possible phylogenetic implications are outlined.

Comparative morphology of central neuropils in the brain of arthropods and its evolutionary and functional implications.

Most insects and decapod crustaceans possess an assemblage of midline neuropils, the central complex. Recent phylogenetic studies show a sister-group relationship between hexapods and decapods, suggesting that central complexes in both groups are homologous structures derived from a basal ancestral neuropil. This ancestral archetype of the central complex (lacking the protocerebral bridge) might be represented in the chilopods. Until recently, diplopods were regarded as closely related to chilopods and united within the taxon "Myriapoda". The entire lack of a midline neuropil in diplopods, however, renders the monophyletic origin of the class Myriapoda unlikely. In this study we used a palette of immunocytochemical and neuroanatomical methods to investigate mid-line neuropils in hitherto poorly examined arthropod groups. Of special interest for resolving arthropod phylogeny are onychophorans, who are believed to be an evolutionary ancient group that resembles the ancestors of modern arthropods. Striking similarities in central brain neuroarchitecture of the onychophoran Euperipatoides rowellii and of a chelicerate species, however, suggest a close phylogenetic relationship between these two groups. Our findings imply that onychophorans either represent the oldest form of the chelicerates or that extant onychophorans have developed from chelicerate-like ancestors by neoteny.

Conserved and convergent organization in the optic lobes of insects and isopods, with reference to other crustacean taxa.

The shared organization of three optic lobe neuropils-the lamina, medulla, and lobula-linked by chiasmata has been used to support arguments that insects and malacostracans are sister groups. However, in certain insects, the lobula is accompanied by a tectum-like fourth neuropil, the lobula plate, characterized by wide-field tangential neurons and linked to the medulla by uncrossed axons. The identification of a lobula plate in an isopod crustacean raises the question of whether the lobula plate of insects and isopods evolved convergently or are derived from a common ancestor. This question is here investigated by comparisons of insect and crustacean optic lobes. The basal branchiopod crustacean Triops has only two visual neuropils and no optic chiasma. This finding contrasts with the phyllocarid Nebalia pugettensis, a basal malacostracan whose lamina is linked by a chiasma to a medulla that is linked by a second chiasma to a retinotopic outswelling of the lateral protocerebrum, called the protolobula. In Nebalia, uncrossed axons from the medulla supply a minute fourth optic neuropil. Eumalacostracan crustaceans also possess two deep neuropils, one receiving crossed axons, the other uncrossed axons. However, in primitive insects, there is no separate fourth optic neuropil. Malacostracans and insects also differ in that the insect medulla comprises two nested neuropils separated by a layer of axons, called the Cuccati bundle. Comparisons suggest that neuroarchitectures of the lamina and medulla distal to the Cuccati bundle are equivalent to the eumalacostracan lamina and entire medulla. The occurrence of a second optic chiasma and protolobula are suggested to be synapomorphic for a malacostracan/insect clade.

Anatomy and physiology of neurons with processes in the accessory medulla of the cockroach Leucophaea maderae.

The accessory medulla (AMe), a small neuropil in the insect optic lobe, has been proposed to serve a circadian pacemaker function analogous to the role of the suprachiasmatic nucleus in mammals. Building upon considerable knowledge of the circadian system of the cockroach Leucophaea maderae, we investigated the properties of AMe neurons in this insect with intracellular recordings combined with dye injections. Responses of neurons with processes in the AMe to visual stimuli, including stationary white light, moving objects, and polarized light were compared with the responses of adjacent medulla tangential neurons. Neurons with processes in the AMe and additional ramifications in the medulla strongly responded to stationary light stimuli and might, therefore, be part of photic entrainment pathways to the clock. Accessory medulla neurons lacking significant processes in the medulla but with projections to the midbrain or to the contralateral optic lobe, in contrast, responded weakly or not at all to light and, thus, seem to be part of the clock's output pathway. Two types of commissural neurons with tangential arborizations in both medullae were sensitive to polarized light, suggesting a role of these neurons in celestial navigation. Sidebranches in the AMae of one of the two cell types are discussed with respect to a possible involvement of the AMe in polarization vision. Finally, neurons responding to movement stimuli did not arborize in the AMe. The results show that the AMe receives photic input and support a role of this neuropil in circadian timekeeping functions.

Candidates for extraocular photoreceptors in the cockroach suggest homology to the lamina and lobula organs in beetles.

Using light- and electron microscopic methods, we describe two novel putative extraocular photoreceptor organs in the optic lobes of the cockroaches Leucophaea maderae and Blaberus craniifer. The lamina organ is an elongated structure distal to the first optic chiasm, adjacent to the anterior edge of the lamina. The lobula organ is situated on the anterior distal surface of the lobula. In cross sections through the pigment-free organs, cell bodies are arranged in a closed or open circle and are interconnected by desmosomes. They send protrusions with rhabdom-like microvilli into a common, central space apparently filled with extracellular matrix. A different cell type gives rise to electron-dense lamellae, which also extend into the central space and partly join to form a common lamellar bundle. Axonal processes extend from the microvillar cells and run along the outer surface of the organs to the neighboring optic neuropils. The organs receive multiple efferent innervation from neurosecretory axons. Both organs show strong immunostaining with an antiserum against Arabidopsis cryptochrome 2 that is associated with the lamellated structure in the central lumen. The specific features of the organs suggest that they are homologous to similar organs in the optic lobe of beetles and may serve a role as extraocular photoreceptors for light entrainment of the circadian system.

Histamine-immunoreactive neurons in the brain of the cockroach Leucophaea maderae.

Histamine is the neurotransmitter of insect photoreceptor cells but has also been found in a small number of interneurons in the insect brain. In order to investigate whether the accessory medulla (AMe), the putative circadian pacemaker of the cockroach Leucophaea maderae receives direct visual input from histaminergic photoreceptors, we analyzed the distribution of histamine-like immunoreactivity in the optic lobe and midbrain of the cockroach. Intense immunostaining was detected in photoreceptor cells of the compound eye, which terminated in the first optic neuropil, the lamina, and in a distal layer of the medulla, the second optic neuropil. Histamine immunostaining in parts of the AMe, however, originated from a centrifugal neuron of the midbrain. Within the midbrain 21-23 bilaterally symmetric pairs of cell bodies were stained. Most areas of the brain were innervated by one or more of these neurons, but the protocerebral bridge and the mushroom bodies were devoid of histamine immunoreactivity. The branching patterns of most histamine-immunoreactive neurons could be reconstructed individually. While the majority of identified neurons arborized in both brain hemispheres, five cells were local neurons of the antennal lobe. A comparison with other insect species shows striking similarities in the position of certain histamine-immunoreactive neurons, but considerable variations in the presence and branching patterns of others. The data suggest a role for histamine in a non-photic input to the circadian system of the cockroach.