Nicholas J. Gibson

Expression of nitric oxide synthase and soluble guanylyl cyclase in the developing olfactory system of Manduca sexta.

The gaseous messenger nitric oxide (NO), with its ability to mediate both intercellular and intracellular communication, can play important roles in mediating cellular communication in both the development and the function of the nervous system. The authors investigated the possible role of NO signaling in the developing olfactory system (antennal lobe) of the moth Manduca sexta. NO synthase (NOS), the enzyme that generates NO, was localized by using immunocytochemistry, in situ hybridization, and nicotinamide adenine dinucleotide phosphate‐diaphorase (NADPH‐d) histochemistry. Although NADPH‐d staining appears to be a poor indicator of the presence of NOS in this system, immunocytochemistry and in situ hybridization reveal that NOS is expressed in the axons of olfactory receptor neurons throughout development and in the perineurial sheath that covers the brain early in development. NOS is present in axon terminals as they form protoglomeruli, raising the possibility that NO mediates cell‐cell interactions during antennal lobe development. NO‐sensitive soluble guanylyl cyclase (sGC), one of the best characterized targets of NO, was localized in the developing olfactory system by using in situ hybridization and immunocytochemistry for the Manduca sexta sGCα1 subunit. The ability of the developing olfactory system to respond to exogenous NO also was examined by using cyclic guanosine monophosphate immunocytochemistry. sGC is expressed in mechanosensory neurons in the developing antenna and in many antennal lobe neurons in both the medial and lateral cell body clusters. Thus, NOS and sGC are expressed in a pattern that suggests that this signaling pathway may mediate intercellular communication during development of the olfactory system in Manduca sexta. J. Comp. Neurol. 422:191–205, 2000.

MEMBRANE LIPID INFLUENCES ON THE ENERGETICS OF THE METARHODOPSIN I and METARHODOPSIN II CONFORMATIONAL STATES OF RHODOPSIN PROBED BY FLASH PHOTOLYSIS

Abstract– We have investigated the relationship between rhodopsin photochemical function and the retinal rod outer segment (ROS) disk membrane lipid composition using flash photolysis techniques. Bovine rhodopsin was combined with various phospholipids to form recombinant membrane vesicles, in which the lipid acyl chain composition was maintained at that of egg phosphatidylcholine (PC), while the nature of the headgroups was varied. The ratio of metarhodopsin II (MII)/metarhodopsin I (MI) in these recombinants produced by an actinic flash was investigated as a function of pH, and compared with the photochemical activity observed for rhodopsin in native ROS membranes and dimyristoylphosphatidylcholine recombinants. In recombinants made with lipids derived from egg PC, as well as in native ROS membranes, MI and Mil were found to be present in a pH‐dependent, acid‐base equilibrium on the millisecond timescale. The recombinants made with phospholipids containing unsaturated acyl chains were capable of full native‐like Mil production, but each demonstrated a titration curve with a different pK. In addition, some of the recombinants exhibited apparent deviations from the Henderson‐Hasselbalch curve shape. The presence of either phosphatidylethanola‐mine (PE) or phosphatidylserine (PS) headgroups appeared to increase the amount of Mil produced. This may result from alteration of the curvature free energy, in the case of PE, and from the influence of the membrane surface potential in the case of PS. An investigation of the effects of temperature on the MI‐MII transition in native ROS membranes and the recombinants was also carried out. The results suggest that the thermodynamic parameters characterizing the MI and Mil conformational states of rhodopsin are influenced by the membrane bilayer environment, indicating a possible role of lipids in the visual process.

Role of phosphatidylserine in the MI-MIII equilibrium of rhodopsin*

Bovine rhodopsin was recombined with various phospholipids in which the lipid acyl chain composition was held constant at that of egg phosphatidylcholine (PC), while the identity of the headgroups was varied. The ratio of MII/MI produced in the recombinant membrane vesicles by an actinic flash was studied as a function of pH, and compared to the photochemical activity observed for rhodopsin in native ROS membranes. MI and MII were found to coexist in a pH-dependent, acid-base equilibrium on the millisecond timescale. Recombinants made with phospholipids containing unsaturated acyl chains were capable of full native-like MII production, but demonstrated titration curves with different pK values. The presence of phosphoethanolamine or phosphoserine headgroups increased the amount of MII produced. In the case of phosphatidylserine this may result from alteration of the membrane surface potential, leading to an increase in the local H+ activity. The results indicate that the Gibbs free energies of the MI and MII conformational states are influenced by the membrane bilayer environment, suggesting a possible role of lipids in visual excitation.

Activation of epidermal growth factor receptor mediates receptor axon sorting and extension in the developing olfactory system of the moth Manduca sexta.

During development of the adult olfactory system of the moth Manduca sexta, olfactory receptor neurons extend axons from the olfactory epithelium in the antenna into the brain. As they arrive at the brain, interactions with centrally derived glial cells cause axons to sort and fasciculate with other axons destined to innervate the same glomeruli. Here we report studies indicating that activation of the epidermal growth factor receptor (EGFR) is involved in axon ingrowth and targeting. Blocking the EGFR kinase domain pharmacologically leads to stalling of many axons in the sorting zone and nerve layer as well as abnormal axonal fasciculation in the sorting zone. We also find that neuroglian, an IgCAM known to activate the EGFR through homophilic interactions in other systems, is transiently present on olfactory receptor neuron axons and on glia during the critical stages of the sorting process. The neuroglian is resistant to extraction with Triton X‐100 in the sorting zone and nerve layer, possibly indicating its stabilization by homophilic binding in these regions. Our results suggest a mechanism whereby neuroglian molecules on axons and possibly sorting zone glia bind homophilically, leading to activation of EGFRs, with subsequent effects on axon sorting, pathfinding, and extension, and glomerulus development. J. Comp. Neurol. 495:554–572, 2006.

Influence of pH on the MI-MII equilibrium of rhodopsin in recombinant membranes

Rhodopsin in native rod membranes and incorporated into egg phosphatidylcholine (egg PC) vesicles was studied at pH 5 and 7 at 28 degrees C. Rhodopsin function, as monitored by the formation of metarhodopsin II (MII) from metarhodopsin I (MI) following an actinic flash, was found to be largely blocked in egg PC vesicles at pH 7. When the pH was lowered to 5, however, rhodopsin showed essentially equal activity in both native and egg PC membranes. This activity exceeded that found for rhodopsin in native membranes at pH 7. Phospholipid composition is thus shown to directly affect the MI in equilibrium MII equilibrium, which in turn is linked to visual function.

Activation of Glial FGFRs Is Essential in Glial Migration, Proliferation, and Survival and in Glia-Neuron Signaling during Olfactory System Development

Development of the adult olfactory system of the moth Manduca sexta depends on reciprocal interactions between olfactory receptor neuron (ORN) axons growing in from the periphery and centrally-derived glial cells. Early-arriving ORN axons induce a subset of glial cells to proliferate and migrate to form an axon-sorting zone, in which later-arriving ORN axons will change their axonal neighbors and change their direction of outgrowth in order to travel with like axons to their target areas in the olfactory (antennal) lobe. These newly fasciculated axon bundles will terminate in protoglomeruli, the formation of which induces other glial cells to migrate to surround them. Glial cells do not migrate unless ORN axons are present, axons fail to fasciculate and target correctly without sufficient glial cells, and protoglomeruli are not maintained without a glial surround. We have shown previously that Epidermal Growth Factor receptors and the IgCAMs Neuroglian and Fasciclin II play a role in the ORN responses to glial cells. In the present work, we present evidence for the importance of glial Fibroblast Growth Factor receptors in glial migration, proliferation, and survival in this developing pathway. We also report changes in growth patterns of ORN axons and of the dendrites of olfactory (antennal lobe) neurons following blockade of glial FGFR activation that suggest that glial FGFR activation is important in reciprocal communication between neurons and glial cells.

Glycosylation patterns are sexually dimorphic throughout development of the olfactory system in Manduca sexta

In the moth Manduca sexta, development of the adult olfactory system depends on complex interactions between olfactory receptor neurons in the antenna, antennal‐lobe neurons in the brain, and several classes of glial cells. As one approach to characterizing molecules that may play roles in these interactions, we used lectins to screen antennae and antennal lobes at different stages of adult development. We find that each of the major neural cell types has a distinct pattern of labeling by lectins. Effects of enzymatic and other treatments on lectin labeling lead us to conclude that the predominant lectin ligands are: glycosphingolipids and an O‐linked, fucose‐containing glycoprotein on axons of olfactory receptor neurons, O‐linked glycoproteins on antennal‐lobe neurons, and N‐linked glycoproteins on all classes of glial cells in the primary olfactory pathway. Wheat germ agglutinin labels all olfactory axons uniformly during much of development, but labeling becomes restricted to the pheromone‐responsive olfactory receptor neurons in the adult male. Succinylated WGA reveals differences in these axon classes earlier, as glomerului develop from protoglomeruli. The adult female displays a less pronounced difference in labeling of axons targeting ordinary and sexually dimorphic glomeruli. Differences in labeling of receptor axons targeted to ordinary and sexually dimorphic glomeruli may be correlated with differences in function or connectivity in different regions of the antennal lobe. J. Comp. Neurol. 476:1–18, 2004.

Dehydration-Induced Molecular Structural Changes of Purple Membrane of Halobacterium Halobium

The nature and extent of dehydration-induced molecular structural changes of the purple membrane of Halobacterium halobium have been studied by absorption and circular dichroism spectra in solution and in oriented membrane films. High glycerol concentrations, exhaustive dry nitrogen gas flushing, and exhaustive high-vacuum pumping were employed as dehydrants. The effect of these dehydrants on the spectra were reversible, similar, and additive. Analysis of the spectral changes observed at maximal dehydration revealed: (a) at least two additional optical states of the bacteriorhodopsin, one at higher energy and another at lower energy than the characteristic dark- and light-adapted states; (b) no change in the dichroic ratio at the visible absorption maximum within experimental error; (c) no change in the polarity of the visible monomeric retinylidene circular dichroic bands; (d) pronounced reduction in the characteristic excitonic interactions among the retinals in the hexagonal crystalline lattice of the membrane; (e) no changes in the native structural anisotropism of the membrane in respect to the orientation of the amino acid aromatic rings of the bacteriorhodopsin; (f) no changes in the secondary structure of the bacteriorhodopsin; and (g) a net tilting of approximately 20.5 degrees per segment of the helical polypeptide segments of the bacteriorhodopsin away from the membrane normal. A molecular model of the structural changes of the membrane resulting from water removal consistent with these findings can be constructed. Dehydration results in only subtle localized tertiary structural changes of the protein which do not significantly alter its shape or size. However, there are pronounced global supramolecular structural changes of the membrane. Water removal, which is most likely to be from the lipid headgroups of the membrane, disrupts the interactions responsible for maintaining the native crystalline lattice of the membrane resulting in pronounced randomization of the positions of the proteins in the membrane.

Localization of a GABA transporter to glial cells in the developing and adult olfactory pathway of the moth Manduca sexta

Glial cells have several critical roles in the developing and adult olfactory (antennal) lobe of the moth Manduca sexta. Early in development, glial cells occupy discrete regions of the developing olfactory pathway and processes of γ‐aminobutyric acid (GABA)ergic neurons extend into some of these regions. Because GABA is known to have developmental effects in a variety of systems, we explored the possibility that the glial cells express a GABA transporter that could regulate GABA levels to which olfactory neurons and glial cells are exposed. By using an antibody raised against a characterized high‐affinity M. sexta GABA transporter with high sequence homology to known mammalian GABA transporters (Mbungu et al. [ 1995 ] Arch. Biochem. Biophys. 318:489–497; Umesh and Gill [ 2002 ] J. Comp. Neurol. 448:388–398), we found that the GABA transporter is localized to subsets of centrally derived glial cells during metamorphic adult development. The transporter persists into adulthood in a subset of the neuropil‐associated glial cells, but its distribution pattern as determined by light‐and electron‐microscopic‐level immunocytochemistry indicates that it could not serve to regulate GABA concentration in the synaptic cleft. Instead, its role is more likely to regulate extracellular GABA levels within the glomerular neuropil. Expression in the sorting zone glial cells disappears after the period of olfactory receptor axon ingrowth, but may be important during ingrowth if GABA regulates axon growth. Glial cells take up GABA, and that uptake can be blocked by L‐2,4‐diaminobutyric acid (DABA). This is the first molecular evidence that the central glial cell population in this pathway is heterogeneous. J. Comp. Neurol. 518:815–838, 2010.

Roles of specific membrane lipid domains in EGF receptor activation and cell adhesion molecule stabilization in a developing olfactory system.

Background Reciprocal interactions between glial cells and olfactory receptor neurons (ORNs) cause ORN axons entering the brain to sort, to fasciculate into bundles destined for specific glomeruli, and to form stable protoglomeruli in the developing olfactory system of an experimentally advantageous animal species, the moth Manduca sexta. Epidermal growth factor receptors (EGFRs) and the cell adhesion molecules (IgCAMs) neuroglian and fasciclin II are known to be important players in these processes. Methodology/Principal Findings We report in situ and cell-culture studies that suggest a role for glycosphingolipid-rich membrane subdomains in neuron-glia interactions. Disruption of these subdomains by the use of methyl-β-cyclodextrin results in loss of EGFR activation, depletion of fasciclin II in ORN axons, and loss of neuroglian stabilization in the membrane. At the cellular level, disruption leads to aberrant ORN axon trajectories, small antennal lobes, abnormal arrays of olfactory glomerul, and loss of normal glial cell migration. Conclusions/Significance We propose that glycosphingolipid-rich membrane subdomains (possible membrane rafts or platforms) are essential for IgCAM-mediated EGFR activation and for anchoring of neuroglian to the cytoskeleton, both required for normal extension and sorting of ORN axons.