Rat neurosphere cells protect axotomized rat retinal ganglion cells and facilitate their regeneration

We investigated the ability of a population of rat neural stem and precursor cells derived from rat embryonic spinal cord to protect injured neurons in the rat central nervous system (CNS). The neonatal rat optic pathway was used as a model of CNS injury, whereby retinal ganglion cells (RGCs) were axotomized by lesion of the lateral geniculate nucleus one day after birth. Neural stem and precursor cells derived from expanded neurospheres (NS) were transplanted into the lesion site at the time of injury. Application of Fast Blue tracer dye to the lesion site demonstrated that significant numbers of RGCs survived at 4 and 8 weeks in animals that received a transplant, with an average of 28% survival, though in some individual cases survival was greater than 50%. No RGCs survived in animals that received a lesion alone. Furthermore, labeled RGCs were also observed when Fast Blue was applied to the superior colliculus (SC) at 4 weeks, suggesting that neurosphere cells also facilitated RGC to regenerate to their normal target. Transplanted cells did not migrate or express neural markers after transplantation, and secreted several neurotrophic factors in vitro. We conclude that NS cells can protect injured CNS neurons and promote their regeneration. These effects are not attributable to cell replacement, and may be mediated via secretion of neurotrophic factors. Thus, neuroprotection by stem cell populations may be a more viable approach for treatment of CNS disorders than cell replacement therapy.

Hardware development of the Yorick series of active vision systems

This paper presents a review of the design and development of the Yorick series of active stereo camera platforms and their integration into real-time closed loop active vision systems, whose applications span surveillance, navigation of autonomously guided vehicles (AGVs), and inspection tasks for teleoperation, including immersive visual telepresence. The mechatronic approach adopted for the design of the first system, including head/eye platform, local controller, vision engine, gaze controller and system integration, proved to be very successful. The design team comprised researchers with experience in parallel computing, robot control, mechanical design and machine vision. The success of the project has generated sufficient interest to sanction a number of revisions of the original head design, including the design of a lightweight compact head for use on a robot arm, and the further development of a robot head to look specifically at increasing visual resolution for visual telepresence. The controller and vision processing engines have also been upgraded, to include the control of robot heads on mobile platforms and control of vergence through tracking of an operator's eye movement. This paper details the hardware development of the different active vision/telepresence systems.

Constitutive oligomerization of human D-2 dopamine receptors expressed in Spodoptera frugiperda 9 (Sf9) and in HEK293 cells - Analysis using co-immunoprecipitation and time-resolved fluorescence resonance energy transfer

Human D-2Long (D-2L) and D-2Short (D-2S) dopamine receptor isoforms were modified at their N-terminus by the addition of a human immunodeficiency virus (HIV) or a FLAG epitope tag. The receptors were then expressed in Spodoptera frugiperda 9 (Sf9) cells using the baculovirus system, and their oligomerization was investigated by means of co-immunoprecipitation and time-resolved fluorescence resonance energy transfer (FRET). [H-3] Spiperone labelled D-2 receptors in membranes prepared from Sf9 cells expressing epitope-tagged D-2L or D-2S receptors, with a pK(d) value of approximate to 10. Co-immunoprecipitation using antibodies specific for the tags showed constitutive homo-oligomerization of D-2L and D-2S receptors in Sf9 cells. When the FLAG-tagged D-2S and HIV-tagged D-2L receptors were co-expressed, co-immunoprecipitation showed that the two isoforms can also form hetero-oligomers in Sf9 cells. Time-resolved FRET with europium and XL665-labelled antibodies was applied to whole Sf9 cells and to membranes from Sf9 cells expressing epitope-tagged D-2 receptors. In both cases, constitutive homo-oligomers were revealed for D-2L and D-2S isoforms. Time-resolved FRET also revealed constitutive homo-oligomers in HEK293 cells expressing FLAG-tagged D-2S receptors. The D-2 receptor ligands dopamine, R-(-) propylnorapomorphine, and raclopride did not affect oligomerization of D-2L and D-2S in Sf9 and HEK293 cells. Human D-2 dopamine receptors can therefore form constitutive oligomers in Sf9 cells and in HEK293 cells that can be detected by different approaches, and D-2 oligomerization in these cells is not regulated by ligands.

Pharmacological analysis of a dopamine D-2Short: G(alpha o) fusion protein expressed in Sf9 cells

A dopamine D-2Short receptor:G(alphao) fusion protein was expressed in Sf9 cells using the baculovirus expression system. [H-3]Spiperone bound to D-2Short:G(alphao) with a pK(d) approximate to 10. Dopamine stimulated the binding of [S-35]guanosine-5'-O-(3-thio)triphosphate (GTPgammaS) to D-2Short:G(alphao) expressed with Gbeta(1)gamma(2) (E-max > 460%; pEC(50) 5.43 +/- 0.06). Most of the putative D-2 antagonists behaved as inverse agonists (suppressing basal [S-35]GTPgammaS binding) at D-2Short:G(alphao)/Gbeta(1)gamma(2) although (-)-suipiride and ziprasidone were neutral antagonists. Competition of [H-3]spiperone binding by dopamine and 10,11-dihydroxy-N-n-propylnorapo-morphine revealed two, binding sites of different affinities, even in the presence of GTP (100 muM). The D-2Short:G(alphao) fusion protein is therefore a good model for characterising D-2 receptors. (C) 2003 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

A modular head/eye platform for real-time reactive vision

This paper describes the design, implementation and testing of a high speed controlled stereo “head/eye��� platform which facilitates the rapid redirection of gaze in response to visual input. It details the mechanical device, which is based around geared DC motors, and describes hardware aspects of the controller and vision system, which are implemented on a reconfigurable network of general purpose parallel processors. The servo-controller is described in detail and higher level gaze and vision constructs outlined. The paper gives performance figures gained both from mechanical tests on the platform alone, and from closed loop tests on the entire system using visual feedback from a feature detector.

An eye for an eye: robust motion segmentation by applying the latest in human vision research

Automatically extracting interesting objects from videos is a very challenging task and is applicable to many research areas such robotics, medical imaging, content based indexing and visual surveillance. Automated visual surveillance is a major research area in computational vision and a commonly applied technique in an attempt to extract objects of interest is that of motion segmentation. Motion segmentation relies on the temporal changes that occur in video sequences to detect objects, but as a technique it presents many challenges that researchers have yet to surmount. Changes in real-time video sequences not only include interesting objects, environmental conditions such as wind, cloud cover, rain and snow may be present, in addition to rapid lighting changes, poor footage quality, moving shadows and reflections. The list provides only a sample of the challenges present. This thesis explores the use of motion segmentation as part of a computational vision system and provides solutions for a practical, generic approach with robust performance, using current neuro-biological, physiological and psychological research in primate vision as inspiration.

View-based approaches to spatial representation in human vision

In an immersive virtual environment, observers fail to notice the expansion of a room around them and consequently make gross errors when comparing the size of objects. This result is difficult to explain if the visual system continuously generates a 3-D model of the scene based on known baseline information from interocular separation or proprioception as the observer walks. An alternative is that observers use view-based methods to guide their actions and to represent the spatial layout of the scene. In this case, they may have an expectation of the images they will receive but be insensitive to the rate at which images arrive as they walk. We describe the way in which the eye movement strategy of animals simplifies motion processing if their goal is to move towards a desired image and discuss dorsal and ventral stream processing of moving images in that context. Although many questions about view-based approaches to scene representation remain unanswered, the solutions are likely to be highly relevant to understanding biological 3-D vision.

Investigation of cooperativity in the binding of ligands to the D-2 dopamine receptor

The D 2 dopamine receptor exists as dimers or as higher-order oligomers, as determined from data from physical experiments. In this study, we sought evidence that this oligomerization leads to cooperativity by examining the binding of three radioligands ([H-3] nemonapride, [H-3] raclopride, and [H-3] spiperone) to D 2 dopamine receptors expressed in membranes of Sf9 cells. In saturation binding experiments, the three radioligands exhibited different B-max values, and the B-max values could be altered by the addition of sodium ions to assays. Despite labeling different numbers of sites, the different ligands were able to achieve full inhibition in competition experiments. Some ligand pairs also exhibited complex inhibition curves in these experiments. In radioligand dissociation experiments, the rate of dissociation of [H-3] nemonapride or [H-3] spiperone depended on the sodium ion concentration but was independent of the competing ligand. Although some of the data in this study are consistent with the behavior of a cooperative oligomeric receptor, not all of the data are in agreement with this model. It may, therefore, be necessary to consider more complex models for the behavior of this receptor.

Oligomers of D-2 dopamine receptors - Evidence from ligand binding

There is increasing evidence that G protein-coupled receptors form oligomers and that this might be important for their function. We have studied this phenomenon for the D-2 dopamine receptor and have shown-using a variety of biochemical and biophysical techniques-that this receptor forms dimers or higher-order oligomers. Using ligand-binding studies, we have also found evidence that this oligomer formation has functional relevance. Thus, for the receptor expressed in either CHO cells or Sf 9 insect cells, the binding properties of several radioligands (in saturation, competition, and dissociation assays) do not conform to those expected for a monomeric receptor with a single binding site. We propose that the receptors exist in oligomers with homotropic and heterotropic negatively cooperative interactions between ligands.

The influence of G protein subtype on agonist action at D-2 dopamine receptors

In previous studies, we have shown that agonists influence the ability of D-2 dopamine receptors to couple to G proteins and here we extend this work. The human D-2Short dopamine receptor and a natural polymorphism of this D-2Short(Ser(311)Cys), have been studied by co-expressing the receptors in insect cells with Gbeta(1)gamma(2) and either Galpha(o), Galpha(i1), Galpha(i2) or Galpha(i3) G protein subunits. These preparations have been used to study the G protein coupling profiles of the two receptors and the influence of agonists. Receptor/G protein coupling was analysed in dopamine/[H-3]spiperone competition binding experiments and through stimulation of [S-35]GTPgammaS binding. Although the Ser(311)Cys polymorphism itself had no appreciable effect on the G protein coupling specificity of the D-2 receptor, agonist stimulation of [S-35]GTPgammaS binding, revealed that both dopamine and (+)-3PPP showed a clear preference for Galpha(o) compared to the Galpha(i) subtypes, but quinpirole did not. These results indicate that agonists are able to stabilise different receptor conformations with different abilities to couple to G proteins. (C) 2004 Elsevier Ltd. All rights reserved.

Mechanisms of agonist action at D-2 dopamine receptors

In this study, we investigated the biochemical mechanisms of agonist action at the G protein-coupled D-2 dopamine receptor expressed in Chinese hamster ovary cells. Stimulation of guanosine 5'-O-(3-[S-35]thio) triphosphate ([S-35]GTPgammaS) binding by full and partial agonists was determined at different concentrations of [S-35]GTPgammaS (0.1 and 10 nM) and in the presence of different concentrations of GDP. At both concentrations of [S-35]GTPgammaS, increasing GDP decreased the [S-35]GTPgammaS binding observed with maximally stimulating concentrations of agonist, with partial agonists exhibiting greater sensitivity to the effects of GDP than full agonists. The relative efficacy of partial agonists was greater at the lower GDP concentrations. Concentration-response experiments were performed for a range of agonists at the two [S-35]GTPgammaS concentrations and with different concentrations of GDP. At 0.1 nM [S-35]GTPgammaS, the potency of both full and partial agonists was dependent on the GDP concentration in the assays. At 10 nM [S-35]GTPgammaS, the potency of full agonists exhibited a greater dependence on the GDP concentration, whereas the potency of partial agonists was virtually independent of GDP. We concluded that at the lower [S-35]GTPgammaS concentration, the rate-determining step in G protein activation is the binding of [S-35]GTPgammaS to the G protein. At the higher [S-35]GTPgammaS concentration, for full agonists, [S-35]GTPgammaS binding remains the slowest step, whereas for partial agonists, another (GDP-independent) step, probably ternary complex breakdown, becomes rate-determining.

Functional coupling of the human dopamine D-2 receptor with G alpha(i1), G alpha(i2), G alpha(i3) and G alpha(o) G proteins: evidence for agonist regulation of G protein selectivity

1 The human dopamine D-2long (D-2L) receptor was expressed with four different G proteins in Sf9 cells using the baculovirus expression system. When co-expressed with G(i)/G(o) G proteins (G(i1)alpha, G(i2)alpha, G(i3)alpha, or G(o)alpha, plus Gbeta(1) and Ggamma(2)) the receptor displayed a high-affinity binding site for the agonists (dopamine and NPA), which was sensitive to GTP (100 mum), demonstrating interaction between the receptor and the different G proteins. 2 The receptor to G protein ratio (R: G ratio) was evaluated using [H-3]-spiperone saturation binding (R) and [S-35]-GTPgammaS saturation binding (G). R: G ratios of 1: 12, 1: 3, 1: 14 and 1: 5 were found for G(i1), G(i2), G(i3), and Go preparations, respectively. However, when R:G ratios of 1:2 and 1: 12 were compared for G(i2) and G(o), no difference was found for the stimulation of [S-35]-GTPgammaS binding. 3 Several agonists were tested for their ability to stimulate [S-35]-GTPgammaS binding to membranes co-expressing the receptor and various G proteins. All the compounds tested showed agonist activity in preparations expressing G(i3) and G(o). However, for G(i2) and G(i1) preparations, compounds such as S-(-)-3-PPP and p-tyramine were unable to stimulate [S-35]-GTPyS binding. 4 Most of the compounds showed higher relative efficacies (compared to dopamine) and higher potencies in the preparation expressing G(o). Comparison of the effects of different agonists in the different preparations showed that each agonist differentially activates the four G proteins. 5 We conclude that the degree of selectivity of G protein activation by the D-2L receptor can depend on the conformation of the receptor stabilised by an agonist.

Interaction of the D-2short dopamine receptor with G proteins: analysis of receptor G protein selectivity

The human D-2short (D-2S) dopamine receptor has been expressed together with the G proteins Gi2 and Go in insect cells using the baculovirus system. Levels of receptor were determined using [H-3]spiperone binding. Levels of G protein heterotrimer were determined using quantitative Western blot and using [S-35]GTPgammaS saturation binding experiments. Levels of the receptor and G protein and the receptor/G protein ratio were similar in the two preparations. Stimulation of [S-35]GTPgammaS binding by a range of agonists occurred with higher relative efficacy and in some cases higher potency in the preparation expressing Go, indicating that interaction of the D-2S receptor is more efficient with this G protein. The effects of various G protein-selective agents on 10,11-dihydroxy-N-n-propylnorapomorphine ([H-3]NPA) binding were used to examine the receptor/G protein complex in the two preparations. Suramin inhibited [H-3]NPA binding with slightly higher potency in the Gi2 preparation, whereas GppNHp inhibited [H-3]NPA binding with greater potency (similar to6-fold) in the Go preparation. This may imply that the G protein is more readily activated in the D-2S/Go preparation. [H-3]Spiperone binding occurred with an increased B-max in the presence of suramin in the Go preparation but not in the Gi2 preparation, suggesting a higher affinity interaction between the free receptor and this G protein. It is concluded that the higher efficiency activation of Go by the D-2S receptor may be a function of higher affinity receptor/G protein interaction as well as a greater ability to activate the G protein. (C) 2003 Elsevier Science Inc. All rights reserved.

Investigation of cooperativity in the binding of ligands to the D-2 dopamine receptor

The D 2 dopamine receptor exists as dimers or as higher-order oligomers, as determined from data from physical experiments. In this study, we sought evidence that this oligomerization leads to cooperativity by examining the binding of three radioligands ([H-3] nemonapride, [H-3] raclopride, and [H-3] spiperone) to D 2 dopamine receptors expressed in membranes of Sf9 cells. In saturation binding experiments, the three radioligands exhibited different B-max values, and the B-max values could be altered by the addition of sodium ions to assays. Despite labeling different numbers of sites, the different ligands were able to achieve full inhibition in competition experiments. Some ligand pairs also exhibited complex inhibition curves in these experiments. In radioligand dissociation experiments, the rate of dissociation of [H-3] nemonapride or [H-3] spiperone depended on the sodium ion concentration but was independent of the competing ligand. Although some of the data in this study are consistent with the behavior of a cooperative oligomeric receptor, not all of the data are in agreement with this model. It may, therefore, be necessary to consider more complex models for the behavior of this receptor.

Oligomers of D-2 dopamine receptors: evidence from ligand binding

There is increasing evidence that G protein-coupled receptors form oligomers and that this might be important for their function. We have studied this phenomenon for the D-2 dopamine receptor and have shown-using a variety of biochemical and biophysical techniques-that this receptor forms dimers or higher-order oligomers. Using ligand-binding studies, we have also found evidence that this oligomer formation has functional relevance. Thus, for the receptor expressed in either CHO cells or Sf 9 insect cells, the binding properties of several radioligands (in saturation, competition, and dissociation assays) do not conform to those expected for a monomeric receptor with a single binding site. We propose that the receptors exist in oligomers with homotropic and heterotropic negatively cooperative interactions between ligands