Effects of copper-aspirin complex on plasma 6-keto-prostaglandin F1α level and platelet cytosolic calcium in rabbits

Effects of copper-aspirin complex on washed platelet aggregation, thromboxane B₂ formation and 6-ketoprostaglandin F_1α level were monitored by Born's and Terashita's methods, respectively. The influence of copper-aspirin complex on cytosolic free calcium was examined using the fluorescent indicator, Fura 2-AM. Copper-aspirin complex significantly inhibited arachidonic acid-induced aggregation in washed platelets. The IC₅₀ value was 9.6 μmolL^-1. Copper-aspirin complex significantly decreased arachidonic acid-induced thromboxane B₂ formation by 87.1% in washed platelets. Ten mg kg^-1 of copper-aspirin complex given intragastrically markedly increased the plasma level of 6-keto-prostaglandin F_1α . Aspirin, however, reduced both thromboxane B₂ formation and 6-keto-prostaglandin F_1α level. In the presence of CaCl₂ 1 mmolL^-1, copper-aspirin complex (20, 40 and 80 mu mol L^-1) markedly lowered arachidonic acid-induced increase in platelet calcium from the resting level (270±36 nmolL^-1) to 213±14, 170±20 and 135±17 nmolL^-1, respectively. In the presence of ethylene glycol-bis (β-aminoethyl ether) N,N,N',N'-tetraacetic acid 1 mmolL^-1, copper-aspirin complex (20, 40 and 80 mu molL^-1) significantly suppressed the release of intracellular calcium induced by arachidonic acid from 127±23 nmolL^-1 to 108±17, 93±12 and 70±13 nmolL^-1.     

Expression of the vesicular glutamate transporter vGluT2 in a subset of cones of the mouse retina

Cone photoreceptors have a continuous release of glutamate that is modulated by light. Vesicular glutamate transporters (vGluT) play an essential role for sustaining this release by loading synaptic vesicles in the cone synapse, the so-called cone pedicle. In the present study mouse retinas were immunostained for vGluT1 and vGluT2. vGluT1 was localized to all cone pedicles and rod spherules, whereas vGluT2 was found in only 10% of the cone pedicles. The vGluT2-expressing cones were characterized in more detail. They are distributed in a regular array, suggesting they are a distinct type. Their proportion does not differ between dorsal (L-cone-dominated) and ventral (S-cone-dominated) retina, and they are not the genuine blue cones of the mouse retina. During development, vGluT1 and vGluT2 expression in cones starts at around P0 and right from the beginning vGluT2 is only expressed in a subset of cones. Bipolar cells contact the vGluT2-expressing cones and other cones nonselectively. The possible functional role of vGluT2 expression in a small fraction of cones is discussed.     

Expression of synaptic and phototransduction markers during photoreceptor development in the marmoset monkey Callithrix jacchus

Marmoset photoreceptor development was studied to determine the expression sequence for synaptic, opsin, and phototransduction proteins. All markers appear first in cones within the incipient foveal center or in rods at the foveal edge. Recoverin appears in cones across 70% of the retina at fetal day (Fd) 88, indicating that it is expressed shortly after photoreceptors are generated. Synaptic markers synaptophysin, SV2, glutamate vesicular transporter 1, and CTBP2 label foveal cones at Fd 88 and cones at the retinal edge around birth. Cones and rods have distinctly different patterns of synaptic protein and opsin expression. Synaptic markers are expressed first in cones, with a considerable delay before they appear in rods at the same eccentricity. Cones express synaptic markers 2–3 weeks before they express opsin, but rods express opsin 2–4 weeks before rod synaptic marker labeling is detected. Medium/long‐wavelength‐selective (M&L) opsin appears in foveal cones and rod opsin in rods around the fovea at Fd 100. Very few cones expressing short‐wavelength‐selective (S) opsin are found in the Fd 105 fovea. Across peripheral retina, opsin appears first in rods, followed about 1 week later by M&L cone opsin. S cone opsin appears last, and all opsins reach the retinal edge by 1 week after birth. Cone transducin and rod arrestin are expressed concurrently with opsin, but cone arrestin appears slightly later. Marmoset photoreceptor development differs from that in Macaca and humans. It starts relatively late, at 56% gestation, compared with Macaca at 32% gestation. The marmoset opsin expression sequence is also different from that of either Macaca or human. J. Comp. Neurol. 512:218–231, 2009. © 2008 Wiley‐Liss, Inc.     

Synaptic inputs onto small bistratified (blue‐ON/yellow‐OFF) ganglion cells in marmoset retina

The inner plexiform layer of the retina contains functional subdivisions, which segregate ON and OFF type light responses. Here, we studied quantitatively the ON and OFF synaptic input to small bistratified (blue‐ON/yellow‐OFF) ganglion cells in marmosets (Callithrix jacchus). Small bistratified cells display an extensive inner dendritic tier that receives blue‐ON input from short‐wavelength‐sensitive (S) cones via blue cone bipolar cells. The outer dendritic tier is sparse and is thought to receive yellow‐OFF input from medium (M)‐ and long (L)‐wavelength‐sensitive cones via OFF diffuse bipolar cells. In total, 14 small bistratified cells from different eccentricities were analyzed. The cells were retrogradely labeled from the koniocellular layers of the lateral geniculate nucleus and subsequently photofilled. Retinal preparations were processed with antibodies against the C‐terminal binding protein 2, the AMPA receptor subunit GluR4, and/or gephyrin to identify bipolar and/or amacrine input. The results show that the synaptic input is evenly distributed across the dendritic tree, with a density similar to that reported previously for other ganglion cell types. The population of cells showed a consistent pattern, where bipolar input to the inner tier is about fourfold greater than bipolar input to the outer tier. This structural asymmetry of bipolar input may help to balance the weight of cone signals from the sparse S cone array against inputs from the much denser M/L cone array. J. Comp. Neurol. 517:655–669, 2009. © 2009 Wiley‐Liss, Inc.     

Specificity of mood stabilizer action on neuronal growth cones

Objectives:  Lithium, valproic acid (VPA) and carbamazepine (CBZ) are commonly used mood stabilizers, but their therapeutic mechanism is unclear. These drugs all cause the same morphological effects on postnatal rat neuronal dorsal root ganglia (DRG) growth cones via an inositol-reversible mechanism. However, due to limitations in earlier analysis, the effects of combining drugs, drug specificity and inositol stereoisomer specificity are unknown. We devised an improved analytical method to address these issues.
Methods:  Dorsal root ganglia explants were cultured individually and incubated with combinations of psychotropic drugs and inositol stereoisomers. We recorded axonal growth cone morphology and calculated growth cone area per a modified method described by Williams et al. ( Nature 2002; 417 : 292–295). Statistically significant changes in area were calculated using non-parametric statistical testing.
Results:  (i) Lithium and VPA showed an additive effect on growth cone spreading. (ii) Among eight additional psychotropic drugs to those previously tested, only imipramine and chlorpromazine altered DRG growth cone morphology. As this effect was not reversed by myo -inositol, it arises from a different mechanism to the mood stabilizers lithium, VPA and CBZ. (iii) Myo -inositol, but not scyllo - or epi -inositol, causes a significant reversal of the lithium effect on the growth cones spreading, consistent with the inositol depletion hypothesis.
Conclusions:  These results show that lithium, VPA and CBZ are unique in causing altered neuronal morphology via myo -inositol depletion.     

Induction of BIM(EL) following growth factor withdrawal is a key event in caspase-dependent apoptosis of 661W photoreceptor cells

Apoptosis of photoreceptor cells in the early postnatal period is a normal feature of mammalian retinal development. The role of mitochondria and caspases in the process has been well established; however, the identification of key apoptotic mediators still remains elusive. Here we report that BIM(EL), a pro-apoptotic BCL-2 family member, may be one such molecule. Following growth factor deprivation, BIM(EL) was up-regulated in mouse 661W cone photoreceptors. This event correlated with the release of mitochondrial apoptogenic factors into the cytosol, the activation of caspases and apoptosis. Moreover, a similar behaviour was observed in response to UV radiation, ionomycin or H(2)O(2) treatments. We identified the PI3K-Akt-FKHRL1 signalling cascade as the main regulatory pathway of BIM(EL) expression in these cells. Finally, using RNA interference, we were able to silence BIM(EL) expression and subsequently suppress caspase-3 activation. In conclusion, we propose BIM(EL) as a critical factor in mitochondria-dependent apoptosis of 661W photoreceptors.     

CNTF induces dose-dependent alterations in retinal morphology in normal and rcd-1 canine retina

Ciliary neurotrophic factor (CNTF) provides morphologic preservation of rods in several animal models of retinitis pigmentosa (RP). However, CNTF may alter photoreceptor morphology and rod photoreceptor differentiation in vitro, as well as affecting normal retinal electrophysiology. In addition, the capacity of CNTF to support other cell types affected secondarily in RP (cones and ganglion cells) is unclear. The purposes of this study were to examine the effects of CNTF upon a canine model of RP, the rod-cone degeneration (rcd-1) dog. Archival tissue from a previous study assessing the capacity of CNTF to rescue photoreceptors in rcd-1 dogs was used. One eye was treated for 7 weeks before being explanted. The contralateral eye was untreated. A total of 23 rcd-1 dogs and seven control dogs (four untreated and three CNTF-treated) were used. Morphometric data describing outer and inner nuclear layer thickness, inner retinal thickness, cones and ganglion cells were collected at nine evenly spaced points along each retina and analysed using a mixed effects model. Immunohistochemistry was performed on a subset of 11 dogs for expression of rhodopsin, human cone arrestin (hCAR) and recoverin. CNTF protected the outer nuclear layer and increased inner retinal thickness in a dose-dependent manner (both were maximal at CNTF doses of 1-6 ng day-1). Significant cone loss or reduction of inner nuclear layer width in rcd-1 did not occur in this model, therefore we were unable to assess the protective effect of CNTF upon these parameters. CNTF did not afford significant ganglion cell protection. CNTF induced morphologic changes in rods and ganglion cells, as well as reducing expression of hCAR and rhodopsin, but not recoverin. The dose of CNTF which provided optimal outer nuclear layer protection also resulted in several other effects, including altered ganglion cell morphology, increased thickness of the entire retina, and reduced expression of some phototransduction proteins. These changes were more marked in rcd-1 retinas than in wild-type retinas. This implies that the consequences of CNTF treatment may be substantially influenced by the cellular context into which it is introduced.     

Topography of the multifocal electroretinogram

The functional topography of the human retina was characterized using the multifocal electroretinogram (ERG), with particular attention to the form of the decline in response with retinal eccentricity. Population response variability was examined and compared to standard full field ERG variability. Burian-Allen contact lens electrodes were used to record the cone multifocal ERG from 50 young eyes (28.3 years ± 5.9 years). Responses were recorded in 8 min from 103 retinal locations within the central ± 22°. The spatial distribution of local responses showed an exponential fall-off with eccentricity. The exponential slope parameter was highly similar across individuals. Excluding responses to the central element, the fall off with eccentricity approximated a power function with an exponent of -0.6, which compares to the -0.74 exponent for the human cone density profile. The inter-individual variance in response density is greatest at the central fovea, reducing towards more peripheral locations. The logarithm of response density, however, shows approximately equal variance across eccentricity, making log density a more appropriate way to view response topography. The population range (± 2 S.D.) of response density is 0.42 log unit, similar to that of standardized ganzfeld electroretinography. The response exponential decay provides a potentially useful addition to element-by-element comparison, in deciding whether an eye's response is within normal limits. This revised version was published online in July 2006 with corrections to the Cover Date.     

Cone positive off-response in normal and dystrophic cats

Light-adapted cone ERG in response to white stimuli with long duration (200 ms) was studied in seven normal cats and six cats with early to moderate, inherited retinal dystrophy. The stimuli typically elicited an ERG consisting of an a- and b-wave in response to light onset, whereas light-offset was followed by a cornea-positive d-wave and subsequent negative dip and occasionally a second positive peak in both normal and dystrophic cats, although b- and d- waves had less distinct peaks in cats with moderately advanced retinal dystrophy. Linear regression models indicated a positive correlation between d-wave amplitude and stimulus luminance, whereas a negative correlation was found between amplitude and background light luminance in normal cats. The d-wave implicit time was independent of both stimulus and background light luminance in normal cats. The d-wave amplitude was not significantly different in dystrophic cats, whereas the implicit time was increased when affected cats were compared to normal cats. The significant increase in implicit time in dystrophic cats could not be explained with a reduced sensitivity of the off-pathway to background or stimulus light. This is supported by the finding that d-wave amplitude was not significantly altered in early to moderately advanced dystrophic cats. This revised version was published online in July 2006 with corrections to the Cover Date.     

Molecular functionalization of carbon nanotubes and use as substrates for neuronal growth

Carbon nanotubes are strong, flexible, conduct electrical current, and can be functionalized with different molecules, properties that may be useful in basic and applied neuroscience research. We report the first application of carbon nanotube technology to neuroscience research. Methods were developed for growing embryonic rat-brain neurons on multiwalled carbon nanotubes. On unmodified nanotubes, neurons extend only one or two neurites, which exhibit very few branches. In contrast, neurons grown on nanotubes coated with the bioactive molecule 4-hydroxynonenal elaborate multiple neurites, which exhibit extensive branching. These findings establish the feasability of using nanotubes as substrates for nerve cell growth and as probes of neuronal function at the nanometer scale.