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Effects of kynurenate and other excitatory amino acid antagonists as blockers of light- and kainate-induced retinal rod photoreceptor disc shedding.

Photoreceptor disc shedding in the retina involves detachment of discs from distal outer segments and phagocytosis of those discs by adjacent pigment epithelial cells. The disc-shedding process balances the continuous renewal of photosensitive membrane. In amphibians, rod disc shedding normally is light-stimulated. However, excitatory amino acids such as kainate stimulate disc shedding independent of a dark-light transition. Excitatory amino acid-induced disc shedding is accompanied by toxic changes within the retina. To evaluate the possible role of an endogenous excitatory amino acid in the regulation of light-evoked disc shedding, we examined the effects of excitatory amino acid antagonists on kainate-induced and light-evoked disc shedding and on retinal toxicity. Using eyecups from Rana pipiens, we found that kynurenate, D-O-phosphoserine, and cis-2,3-piperidine dicarboxylic acid (cis-PDA) all block both the neurotoxic and disc-shedding effects of kainate. Kynurenate and D-O-phosphoserine, but not cis-PDA, also block light-evoked disc shedding. Our analysis suggests that kynurenate blocks the mechanism by which light "triggers" disc shedding rather than directly inhibiting disc detachment and phagocytosis. The observation that cis-PDA antagonizes the effects of kainate, but not light, suggests that the receptor mediating the kainate effect on disc shedding may not be involved in the normal initiation of the response by light. In contrast, our data on kynurenate suggest that it antagonizes an endogenous mechanism involved in the normal control of disc shedding. Thus, analysis of the differences between cis-PDA and kynurenate as antagonists in the retina may yield important insight into the mechanism by which light initiates disc shedding.

Mg2+ reduces N-methyl-D-aspartate neurotoxicity in embryonic chick neural retina in vitro.

N-Methyl-D-aspartate (NMDA) is a potent neurotoxin that affects cells in the inner layers of the embryonic chick retina exposed in vitro. After exposure of the embryonic day 12 neural retina to 0.5-10.0 mM NMDA for 30 min, 50-80% of the cells in the inner region of the inner nuclear layer and 50-100% of the cells in the ganglion cell layer were hypochromatic. When retinas were incubated with Mg2+ (0.5-10.0 mM) for 15 min and then incubated with Mg2+ and NMDA (0.5 mM) for 30 min, the NMDA effect in the inner layers was dramatically reduced but not abolished. Removal of Mg2+ before NMDA exposure produced retinas as seriously affected as retinas not exposed to Mg2+. Studying the effects of NMDA inhibitors, such as Mg2+, may help elucidate the mechanism of the cytotoxic events that occur in the retina in response to certain excitatory acidic amino acids.

Quinolinate. A selective neurotoxin in embryonic and posthatching chicken retinas.

Quinolinate (QUIN), an endogenous dicarboxylic amino acid, structurally related to the putative retinal neurotransmitter aspartate, acts as a specific neurotoxin in the chick neural retina. Qualitative analysis of QUIN's neurotoxic effects reveals that sensitivity to the amino acid is first detected in the 9-day-old embryonic chick retina. Nuclei and cytoplasm of some cells in the inner region of the inner nuclear layer and in the ganglion cell layer appear hypochromatic or electron lucent when examined by light or electron microscopy, respectively. Between day 10 and 12, the sensitivity of the embryonic retina to QUIN increases and remains around the day 12 level throughout the remaining embryonic and initial posthatching period. Cells in the inner half of the inner nuclear layer continue to be the most severely affected throughout retinal development, ganglion cells less so. Photoreceptor and most cells in the outer region of the inner nuclear layer remain undamaged. QUIN effects are partially reversible: retinas exposed to QUIN briefly in vitro and then transferred to fresh QUIN-free medium are not as severely affected as those allowed no recovery time. In day 1 posthatching chick retinas, similar patterns of QUIN-toxicity were observed in vitro (0.5-5 mM QUIN; 5-30 min) and in vivo (200-600 micrograms QUIN/eye; 0.5-24 hr following intravitreal injection).

In vitro effects of kainate on embryonic and posthatching chick retina.

The onset and developmental pattern of kainate effects were determined in isolated embryonic and posthatching chick neural retinas exposed to KA (0.01-0.24 mM; 1-30 min) in vitro. Damage was first apparent in day 8 embryonic retinas; cells in the inner region of the inner nuclear layer and neuronal processes in the inner plexiform layer were affected. In older embryonic retinas, cells in the inner region of the inner nuclear layer continued to be the most damaged although cells in the ganglion cell layer and the outer region of the inner nuclear layer, and neurites in the outer plexiform layer were also affected. Cells in the outer nuclear layer stained normally in both embryonic and posthatching retinas. Partial reversibility of kainate effects was observed in retinas which had been exposed briefly to kainate and then allowed to recover in kainate-free medium. The results of our in vitro experiments are consistent with those reported for a variety of neonatal and adult retinas treated with kainate in vivo.

Glutamine synthetase in cultured whole retinas from the embryonic chick. Role of protein and RNA syntheses in 4 degrees C storage enhancement.

Glutamine synthetase (GS) activity is enhanced in cultured whole retinas when a 72 h incubation at 37 degrees C is preceded by storage at 4 degrees C for 2-24 h. This enhancement occurs even in the absence of glucocorticoids and is maximal in retinas from 11 to 14 d embryos. In comparison, cortisol-induced increases in retinal GS activity at 37 degrees C are optimal in retinas from 8 to 12 d embryos. This study, using cycloheximide (an inhibitor of protein synthesis) and cordycepin (an inhibitor of RNA synthesis), indicates that both protein and RNA synthesis are required for the 4 degrees C storage enhancement of GS activity. The necessary RNA synthesis occurs within the first 48 h following transfer to 37 degrees C and does not require concomitant protein synthesis. Uridine uptake, but not incorporation into trichloroacetic acid-precipitable material, is increased by initial 4 degrees C storage when compared with whole retina controls incubated at 37 degrees C for the total time. In contrast, both uptake and incorporation of amino acids are increased in 4 degrees C-stored retinas for as long as 72 h subsequent to transfer from 4 to 37 degrees C. This suggests that enhancement GS activity may arise from a combination of elevated general protein synthesis and specific messenger-RNA synthesis following 4 degrees C storage.

Effects of alpha-aminoadipate isomers on the morphology of the isolated chick embryo retina.

The glutamate analogue, alpha-aminoadipate (alpha AA), was administered in the DL-, D-, and L-forms to chick embryo retinas in vitro. Two-hour incubation of retinas, with each form of alpha AA, resulted in glial swelling of progressive severity as the concentration of the adipate increased. Damage was most severe with the L-isomer, which produced a mixed glial-neuronal lesion that affected inner neuronal structures. The effect of the D,L racemic mixture was limited to glia and was less severe than damage seen with the L-isomer. The D-isomer produced effects similar to, but less severe than, those seen with the D,L mixture. Neuronal damage was seen subsequent to extensive Müller cell swelling in longterm cultures (24 hours) with the L- and DL-isomers. In contrast, the D-isomer did not produce discernible neurotoxicity even after a 24-hour treatment with 2.4 mM. Morphologic changes resulting from 2-hour adipate treatment were, to a large extent, reversible.

Age-dependent effects of glutamate on photoreceptor cells of the isolated chick embryo retina.

Neurotoxic and gliotoxic effects of glutamate were studied in isolated chick embryo retinas of various ages. Employing a short-term incubation system, we found that initial cellular changes in retinas from 15- and 21-day chick embryos were localized to glial Müller cells as previously shown in retinas from 12-day embryos. In the older retinas, however, an additional selective lesion was consistently found in the presumptive photoreceptor cells. Similar photoreceptor damage was not seen in 12-day retinas, even after treatment with relatively high glutamate doses. Autoradiographic uptake of tritiated glutamate in retinas at 12 and 14 days was localized to the glial Müller cells; in the 14-day retinas, however, there was also uptake of the labeled amino acid into the developing inner segments. Uptake of tritiated glutamate in retinas at younger (8-day) and older (21-day) ages did not show any obvious localization of the label.

Glutamate-induced cellular injury in isolated chick embryo retina: Müller cell localization of initial effects.

The neurotoxic and gliotoxic effects of glutamate and several glutamate analogues were studied in isolated chick embryo retinas. To facilitate examination of initial pathological events, a short-term incubation system was developed and used for light microscopic and autoradiographic investigation. Low-dose, short-term glutamate treatment of 12-day retinas resulted in a glial-specific lesion in the Müller cells, characterized by extensive cellular edema; at higher concentrations and/or longer treatment times, neurotoxic as well as gliotoxic effects were seen. The early glial damage was identical in appearance to that seen after incubation with DL-alpha-aminoadipate and other reported gliotoxins. No evidence of a similar glial-specific action was seen after administration of kainic acid, although extensive neuronal degeneration did result. Incubation of retinas with tritiated glutamate (3H-glu) revealed a selective uptake of the label by Müller cells. Autoradiographic grains were localized over Müller foot processes at the inner limiting membrane, and by 30 minutes labeled the entire glial system. Prior treatment with neurotoxic levels of glutamate did not alter the autoradiographic localization to glial cells. Possible glial-neuronal interactions and their effect on cytotoxic patterns are discussed.

Effect of dissociative methods on cortisol binding and glutamyltransferase inducibility in chick embryo retina.

The ability of the isolated embryonic chick retina (12 days) to bind a steroid (cortisol) decreases when the tissue is dissociated; the extent of this decrease depends upon the method of dissociation. Trypsin and mechanical dissociation decreased cortisol binding slightly; papain dissociation essentially eliminated it. Cortisol binding decreased with time in culture in both whole retina and monolayer cultures; this decrease may reflect, in part, a similar development decrease in ovo. Inducibility of glutamine synthetase in whole retinas and retinal monolayers prepared with either trypsin or papain also decreased with time in culture. For whole and trypsin-dissociated retinas, the drop in inducibility correlates with the drop in cortisol-binding capacity. This was not the case for monolayer cultures prepared by papain dissociation.

Glutamine synthetase induction in chick embryo retina monolayers.

Induction of glutamine synthetase (GS) by cortisol has been shown to occur in monolayer cultures of cells obtained by enzymatic dissociation of retinas from 8- and 12-day-old chick embryos with papain (0.1%) or trypsin (0.25%). Although essentially single cells when plated, monolayers obtained by enzymatic dissociation show significant aggregation by 4--6 h. Monolayers prepared by mechanical dispersion (cells forced through successively smaller gage needles) are minimally inducible, perhaps owing to poor viability in such cultures. Storage at 4 degrees C for 24 h prior to treatment with cortisol significantly elevated both basal GS activity and inducibility in whole (but not in monolayer) retina cultures.

Effects of kainate during glutamine synthetase induction in chick embryo retina.

This report concerns the effect of kainate on induction of glutamine synthetase in chick embryo retina. This enzyme can be prematurely induced in such retinas at 12 days of development by cortisol. Induction is 50-70% inhibited if 2.4 mM L-glutamate is added to the medium with cortisol for the 24-h induction period. Addition of kainate, an analogue of glutamate, enhances cortisol induction of glutamine synthetase by a factor of 2 under similar conditions. Kainate or L-glutamate treatment for 5 min (followed by incubation with cortisol for 24 h) also sometimes enhances retinal glutamine synthetase compared with untreated controls.

Glutamine synthetase localization in cortisol-induced chick embryo retinas.

We report here for the first time, in chick retina, Muller cell localization of glutamine synthetase (GS) activity by an immunohistochemical technique, in agreement with previous reports of glial localization of this enzyme in rat brain and retina. Age-dependent changes in the endogenous enzyme activity as well as cortisol-induced changes in GS activity, both in ovo and in vitro, measured biochemically, reflect the changes observed by staining.