Neuron, Vol. 5, 373-381,September,1990,Copyright © 1990by Cell Press

VIP-Mediated Increase in cAMP Prevents Tetrodotoxin-lnduced Retinal Ganglion Cell Death In Vitro Peter K. Kaiser and Stuart A. Lipton Laboratory of Cellular and Molecular Neuroscience Departments of Neurology Children's Hospital Beth Israel Hospital Brigham and Women's Hospital Boston, Massachusetts 02115 Program in Neuroscience Harvard Medical School Boston, Massachusetts 02115

Summary Afferent influences on natural cell death were modeled in retinal cultures derived from neonatal rats. Tetrodotoxin (TTX) blockade of electrical activity produced a significant reduction in surviving retinal ganglion cell (RGC) neurons during a critical period of development, similar in magnitude to the reduction observed during natural cell death in the intact retina at a similar developmental stage. The addition of vasoactive intestinal peptide (VI P) protected the RGCs from the lethal action of TTX. This effect was specific, since the related peptides PHI-27 and secretin produced no significant increase in RGC survival. Radioimmunoassay of cyclic nucleotides showed that TTX decreased culture levels of cAMP and that this trend was reversed by VI P. Decreases in RGC survival associated with TTX electrical blockade were prevented by 8-bromo:cAMP or forskolin. Furthermore, VIPt0-28, the C-terminal fragment that inhibits VIP stimulation of adenylate cyclase, reduced the number of surviving RGCs. Thus, our results suggest that VIP, acting by increasing cAMP, has a neurotrophic effect on electrically blocked RGCs and may be an endogenous factor modulating normal cell death in the retina. Introduction An interesting facet in the normal development of the vertebrate central nervous system (CNS) is the large proportion of neurons that die naturally (Oppenheim, 1981). One example is found in the mammalian visual system. Overproduction of retinal ganglion cells (RGCs), neurons that project from the retina to deeper centers of the brain, appears to be a fundamental characteristic of development (Land and Lund, 1979; Thompson, 1979; Finlay et al., 1979; Rakic and Riley, 1983, 1984; Insausti et al., 1984; Williams et al., 1986). The death of RGCs is thought to serve at least two purposes. First, the initial overproduction of RGCs ensures that the target structures receive adequate input and that the subsequent cell death quantitatively matches the projecting neuronal population to the needs of their targets (for reviews see Oppenhelm, 1981; Hamburger and Oppenheim, 1982; Cowan et al., 1984; Cowan and O'Leary, 1984). Second, it

eliminates those cells whose axons project into inappropriate regions either in the correct target fields or in entirely incorrect areas (for reviews see Clarke, 1981; Cowan et al., 1984; O'Leary, 1987). In other words, it effectively removes certain developmental miscues that cause aberrant axonal projections or neuronal targeting errors. In adult rats, essentially all of the RGCs send axons to the superior collicutus in a highly ordered projection. The RGCs differentiate between days 12 and 16 of gestation, and the axons reach the contralateral superior colliculus as early as embryonic day 16 (E16; Morest, 1970; Bunt et al., 1983). The number of axons in the optic nerve reaches its peak at approximately E20; thereafter there is a rapid loss of RGCs (Bunt et al., 1983; Crespo et al., 1985; Williams et al., 1986). In the pigmented rat at least half of the RGCs die naturally, and this normal cell death proceeds for the first 1 1/2 weeks of postnatal development (Cunningham et al., 1982; Perry et al., 1983). Although the exact cellular sequence of events is still under debate, the loss of neurons is thought to be caused by at least two mechanisms: the intermingling and competition of RGC axonal fibers from the same eye and from the two eyes for their postsynaptic targets du ring the early stages of development (the efferent influence; Cowan et al., 1984; Fawcett et al., 1984; Rakic and Riley, 1984; O'Leary et al., 1986b; Sretavan and Shatz, 1986; Williams et al., 1986), and the effect of local factors in the retina from cells presynaptic to the RGCs (the afferent influence; Perry and Linden, 1982; Cunningham, 1982; Okado and Oppenheim, 1984; Clarke, 1985; Lipton, 1986; Furber et al., 1987). Several recent findings have shown that competition, and the consequent cell death, are centered around the blockade of electrical activity during a critical period of development (Cowan et al., 1984; Fawcett et al., 1984; Lipton, 1986; O'Leary et al., 1986b). Previous experiments in this laboratory studied the effects of electrical blockade on identified RGCs dissociated from retinas of neonatal rats (Lipton, 1986). On the second day in culture, 10%-15% of the RGCs were found to be solitary neurons; the remainder existed in small clusters with other retinal cells. Electrical recordings determined that 50% of the clustered RGCs displayed spontaneous postsynaptic potentials and action potentials, whereas the solitary RGCs did not. Furthermore, the same population of clustered RGCs that exhibited spontaneous activity died within 24 hr of exposure to either tetrodotoxin (-I-rx) or IowCa2+/high-Mg 2+ medium to block synaptic activity. Similar to natural cell death, these effects occurred only if the RGCs were at a particular stage of development (postnatal day 2 to 10 [P2-P10] animals, but not older) when they either possessed or were acquiring electrical activity. Cell death was not found in the solitary RGCs that lacked synaptic input and consequent

Neuron 374

spontaneous activity (Lipton, 1986). Therefore, when spontaneous electrical activity was prevented during a critical period of development, half of the clustered RGCs died. In contrast, there was no deleterious effect on the solitary RGCs that failed to exhibit electrical activity. Although most investigations of mechanism of natural cell death have focused on interactions between RGCs or other neurons and their targets (efferent influence), recent evidence has shown that afferent inputs may also playa prominent role in this phenomenon (Furber et al., 1987). For example, it is possible that the activity of amacrine cells, which are presynaptic to RGCs, may be necessary for the presence of a chemical trophic factor in retinal cultures (Lipton, 1986). One candidate for a protective trophic factor is vasoactive intestinal peptide (VlP), since it has been found to increase neuronal survival of cultured spinal cord neurons whose electrical activity had been blocked (Brenneman and Eiden, 1986). VlP has been detected by radioimmunoassay in the mammalian retina (Ekman and Tornquist, 1985) and has been localized by immunochemistry exclusively to a subpopulation of amacrine cells in the rat retina (Lor~n et al., 1979, 1980; Eriksen and Larssonn, 1981; Brecha and Karten, 1985). We have developed a novel in vitro model for presynaptic or afferent influences on cell death in isolation from postsynaptic or efferent target effects; such separation is not easily achieved in vivo. Natural cell death occurs in vivo in a large number of RGCs that are the same age as the clustered RGCs in our model tissue culture system (Lipton, 1986). However, whether the mechanism for cell death in culture is a true reflection on the normal process of cell death in the retina remains to be established. Nevertheless, since the period of susceptibility to impulse blockade in vitro corresponds to the interval of normal neuronal death in vivo and since the proportion of RGCs dying normally is similar to that induced by "I-I'X in this model system, it appears that the study of these cultures may be relevant to mechanisms of naturally occurring loss of RGCs in vivo (Lipton, 1986). in the present study we show that Vlp, acting via cAMP, can rescue RGCs cultured in the absence of their tectal targets from death engendered by electrical blockade. It is therefore possible that VIP and cAMP play a critical trophic role in the regulation of retinal ganglion cell death during neuronal development. Results Tetrodotoxin Increases Cell Death RGCs were identified, cultured, and scored for survival as described in Experimental Procedures (Figure 1A). Viable RGCs were counted after a 20 hr incubation period. As reported previously, the addition of 1 ~M TTX to culture dishes increased death among

clustered but not solitary RGCs almost 2-fold over that encountered in sibling control dishes. VIP Prevents Cell Death To test whether VlP could affect the TTX-mediated neuronal death, the neuropeptide was added to the culture dishes at the time of plating. A dose-response study of VlP-mediated effects on the number of living RGCs after treatment with TTX is shown in Figure lB. RGCs were counted after a 20 hr incubation period. Electrical blockade by TTX treatment resulted in a significant decrease in RGC counts from those observed in control cultures; approximately half of the clustered RGCs died. The addition of VlP to -I-I-X-treated culture dishes yielded a dose-dependent increase in neuronal survival. At low concentrations (0.1 nM to 10 nM), VlP did not prevent RGC death in the face of TTX blockade; however, as the VlP concentration rose (100 nM to I I~M), the number of living RGCs rose and was not significantly different from that found in control cultures lacking 1-I-X. In fact, the addition of 0.1 I~M VlP to electrically blocked cultures allowed approximately 90% of the RGCs to survive. The protective effect reached a plateau above 0.1 ~M VlP. These results indicated that the addition of VlP prevented death of clustered RGCs treated with TTX. The addition of VIP alone (in the absence of TTX) at concentrations ranging from 0.1 nM to 1 I~M led to no significant difference in survival of clustered RGCs compared with controls (data not shown). Furthermore, the number of solitary RGCs in the various VlP and/or TTX treatments remained almost constant. Roughly 10% of the RGCs in the culture dishes were observed to be solitary neurons. There was no significant difference in su rvival of these solitary RGCs at any concentration of VlP and/or TTX treatment compared with controls (data not shown). VIP10-28 Increases Cell Death To test further the possibility that VlP or a VlP-like substance can influence activity-dependent neuronal survival, VlP10-2~, the C-terminal fragment of Vlp, was added to culture dishes in increasing doses. The VlP fragment was chosen since recent evidence has shown that it inhibits VlP stimulation of adenylate cyclase (Bissonnette et al., 1984). As the concentration of the VlP fragment increased, the number of surviving clustered RGCs decreased until a plateau was reached (Figure 2). The addition of VlP10_28produced a significant decrease in the number of living RGCs compared with control levels. In fact, the decreased tally of living RGCs was not significantly different from counts of cultures treated with TIX (see survival value in the absence of VlP in Figure 1B). Maximal decreases in RGC survival were reached at 0.1 I~M VlP10-28. Interestingly, this was the same concentration at which VlP produced maximal neuronal survival in l-FX-treated cultures. Therefore, VlP10-28 produced a dose-dependent decrease in neuronal cell

VIP or cAMP Prevents l~X-lnduced Retinal Cell Death 375

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Figure 1. Survival of RGCs in Culture Treated with TI-X and Varying Concentrations of VIP for 20 hr 100] * . (A) Upper left: A combination of phase and epifluorescence photomicrographs (DAPt filter cube) showing two clustered RGCs that had been labeled previously in situ by retrograde ~ 80 transport of the dye granular blue. Upper right: Epifluorescence o. o photomicrograph of the same field using a fluorescein filter 6o -" ~ cube to demonstrate that the corresponding RGCs are living because of their ability to take up and cleave fluorescein diacetate ,E o X to fluorescein. Lower left: A combination of phase and epiflu.~ "~ 40 orescence photomicrographs showing a solitary RGC (marked by the blue dye). Lower right: Epifluorescence photomicrograph 2o of the same field using a fluorescein filter cube to demonstrate that this solitary RGC is viable. The solitary RGC is 20 I~m in diameter. In this manner, double staining with granular blue 0 , and fluorescein was used to score surviving solitary and clus-oo -10 -9 -8 --> -6 tered RGCs. (B) Electrical blockade by TTX (1 IIM) resulted in a significant delog [vm] (M) crease in clustered RGC viability compared with control cultu res. The addition of VIP to ]q-X-treated cultures yielded a dosedependent increase in neuronal survival. Each point is the mean value from 8 experiments with 12-16 culture dishes per experiment. The ordinate value represents the percentage of the mean of living clustered RGCs in the control dishes (average number of cells counted per treatment ,'~2500). The error bars indicate standard error of the mean; statistical comparisons were made with an ANOVA, followed by a Scheff~ multiple comparison of means. This analysis yielded the following rank order (P < 0.05): []q-X + 1 llM VIP] = [TTX + 0.1 I~M VlP] > [TI-X + 10 nM VlP] = [TTX + 1 nM VIP] = []q-X + 0,1 nM VlP] = [Trx]. Values significantly different from the TI-X value are indicated by asterisks.

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Neuron 376

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VlP102~produced a decrease in neuronal cell survival similar to that found after exposure to TFX. Furthermore, adding Trx (1 #M) to VlP10_2~did not increase the amount of death over that produced by either alone, i.e.,there was no additive effect of TTX (data not shown). Each point is the mean value from 4 experiments with 12-16dishes per experiment. The error bars indicate standard error of the mean; statistical comparisons were made with an ANOVA, followed by a Scheff# multiple comparison of means. The multiple comparison of means indicated (P < 0.01): [Control] > [1 nM VIP10-28] = [10 nM VlP10_2~]= [0.1 ~M VlP10_26] = [1 #M VIP10_28].Values significantly different from control are indicated by asterisks.

survival that was similar to that found with TTX treatment. The addition of increasing concentrations of VIP~0-28 to cultures treated with -I-TX produced clustered RGC counts that were not significantly different from those treated with ]-IX alone. Thus, the lethal effects of the VIP fragment and of TTX were not additive. Furthermore, the solitary RGC counts in cultures treated with VlP10-28 and/or -Frx were not significantly different from control levels (data not shown). Thus, VlP10-2~ increased cell death only among clustered RGCs. The effect was not additive to the lethal effects of "FIX electrical blockade and probably involved the inhibition of VlP stimulation of adenylate cyclase (see below). Related Peptides and Cell Death To test the specificity of the VlP-mediated effects on RGC survival, [D-Phe 4] peptide histidine-isoleucinamide (PHI-27) and secretin, two peptides with partial sequence h o m o l o g y to VIP (Tatemoto and Mutt, 1981; Robberecht et al., 1987), were examined for neurotrophic properties. Treatment with PHI-27 or secretin at concentrations ranging from 1 nM to 1 pM did not change the n u m b e r of surviving clustered RGCs from levels found in cultures with l - r x electrical blockade alone (Figures 3A and 3B). Furthermore, the number of surviving RGCs after addition of the peptides alone did not differ significantly from control levels. Thus, both PHI-27 and secretin did not appear to have any neurotrophic effects despite their partial sequence

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h o m o l o g y to VlP. They did not increase neuronal cell survival in the presence of -FrX electrical blockade, nor did they produce any apparent effects on neuronal Iongevitywhen administered alone, i.e., the number of viable RGCs at 20 hr was comparable to normal control cultures. VIP and cAMP Levels in Culture Attempts to measure VIP in the culture medium by radioimmunoassay were thwarted by the extremely labile nature of VIP, apparently due to endogenous peptidases. This lability was confirmed in experiments in which I I~M exogenous VIP was added to the cultures and then measured by radioimmunoassay at various incubation times (10 min to 24 hr). Even with short incubation periods, o n l y picomolar amounts of VIP could be recovered. Therefore, the cultures were tested for other factors that might be triggered by a transient rise in VIE It has been shown that Vl P potently stimulates ade-

VIP or cAMPPreventsTrXqnducedRetinalCell Death 377

nylate cyclase in the retinas of a n u m b e r of species (Longshore and Makman, 1981; Amiranoff and Rosselin, 1982; Lasater et al., 1983; Koh et al., 1984). The resulting increase in cAMP could serve as a second messenger for mechanisms involved in neuronal survival during development (Van Buskirk and Dowling, 1981; Lasater et al., 1983). Thus, it was of interest to test the effects of VlP on cAMP levels in culture with and w i t h o u t TTX electrical blockade at various incubation times (Figure 4). Electrical blockade by TTX had a prof o u n d and immediate effect (within 10 min) on the cAMP levels in the cultures. The concentration of cAMP w i t h i n 10 min of plating in the electrically blocked cultures was significantly less than control levels. Alone, exogenous VlP appeared to increase cAMP levels over control points but did not reach statistical significance. However, the addition of VIP to electrically blocked cultures significantly increased the level of cAMP within 10 min compared with that observed after TTX treatment alone. A similar trend was still evident when cAMP was assayed 6 hr after treating the cultures with VIP, TTX, or both. The related peptides, PHI-27 and secretin, did not affect cAMP levels. These findings indicate that there is an important link between stimulation with VIP and increased cAMP levels in the retinal cultures.

Forskolin Prevents Cell Death To test the hypothesis that VIP stimulation of adenylate cyclase may start the cascade of events that leads to increased neuronal survival, forskolin, a diterpene

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Figure 5. Survival of RGCs for 20 hr after Treatment with TI'X (1 pM) and/or Forskolin (100 pM), 8-Bromo:cAMP (1 mM), or 8-Bromo:cGMP (1 mM) TTX-induced electrical blockade significantly increasedcell death over control levels.The lethal effect of TIX was reversed by the addition of forskolin (FSK) or 8-bromo:cAMP (8 Br:cAMP), but not by 8-bromo:cGMP (8 Br:cGMP). Except for TI-X, the experimental drugs had no noticeable effect when administered alone. Each point is the mean value from 3 experiments with 12-16 dishes per experiment. The error bars indicate standard error of the mean; statistical comparisons were made with an ANOVA, followed by a Scheff~ multiple comparison of means.The multiple comparison of means indicated (P < 0.01):[Control] = [FSK] = [FSK + "l]-X] > [TTX]; [Control] = [8 Br:cAMP] = [8 Br:cAMP + "I]-X] > ['FFX]; [Control] = [8 Br:cGMP] > [8 Br:cGMP + Trx] = [TI-X].Valuessignificantly different from control are indicated by asterisks.

isolated from the roots of coleus forskholii (Linder et al., 1978) that directly stimulates the catalytic subunit of adenylate cyclase (Daly, 1984), was added to the cultures under similar experimental conditions (Figure 5). We found that I IIM ]-I-X potently decreased the n u m b e r of surviving clustered RGCs and that the lethal effect of TTX was completely reversed by the addition of 100 I~M forskolin. It appears that forskolin mimicked the protective effect of VIP on activityd e p e n d e n t neuronal survival (see below). The fact that the neuronal counts in cultures treated with FI-X plus forskolin were almost identical to control RGC counts supports the notion that adenylate cyclase activation plays an important role in the mechanism of neuronal survival. A d d i t i o n of forskolin alone produced no noticeable change in RGC counts from control levels (data not shown).

8-Bromo:cAMP Prevents Cell Death Since forskolin can have effects in addition to its stimulation of adenylate cyclase, a relatively membranepermeable derivative of cAMP was also tested for possible effects on neuronal survival. The blockade of electrical activity by TTX predictably decreased clustered RGC survival (Figure 5). This increased mortality was reversed by the addition of I m M 8-bromo:cAMP to the TTX-treated cultures. In fact, the n u m b e r of surviving RGCs was not significantly different from con-

Neuron 378

trol counts in the presence of 8-bromo:cAMP plus TTX. The cyclic nucleotide mimicked the action of VlP on activity-dependent neuronal survival. Application of 8-bromo:cAMP alone to the cultures produced no obvious effect, since the RGC counts were not significantly different from control levels. In addition, in these experiments the number of solitary RGCs was not affected by 8-bromo:cAMP and/or TTX treatment (data not shown). Thus, 8 bromo:cAMP could prevent neuronal cell death in electrically blocked cultures. These results support the hypothesis that the VlPstimulated increase in cAMP levels increased neuronal survival in activity-blocked cultures. 8-Bromo:cGMP Does Not Prevent Cell Death

To test the specificity of cyclic nucleotides, 8-bromo: cGMP was added with and without 1 I~M TTX to culture dishes. The addition of I mM 8-bromo:cGMP did not reverse the cytotoxic effects of TTX (Figure 5). Thus, 8-bromo:cGMP appears to have little or no protective effect against the lethal action of TTX electrical blockade in these cultures. The application of 8-bromo:cGMP alone to culture dishes also did not affect the RGCs, since the counts were not significantly different from control (data not shown). These results are consistent with the finding that VlP specifically stimulates adenylate cyclase and strengthen the hypothesis that the increased survival of RGCs in electrically blocked cultures is not generalized to all cyclic nucleotides, but is specific to cAMP. Discussion

Although the exact function of VIP in the mammalian retina remains unknown, it is present in a subpopulation of amacrine cells and is thought to act as a neurotransmitter or neuromodulator. The present study suggests that VIP acts as a neurotrophic factor for retinal ganglion cells in an in vitro model of natural cell death. As demonstrated previously, blockade of electrical activity in retinal cultures with TTX resulted in neuronal death; a subpopulation of RGCs that were either acquiring or possessed spontaneous activity died within 24 hr of exposure to TTX (Lipton, 1986). This cytotoxic effect was evident only in clustered RGCs with apparent synaptic input, and not in solitary cells that did not demonstrate spontaneous action potentials. At first these findings may appear to be at odds with those of Fawcett et al. (1984) and O'Leary et al. (1986b). They examined the effect of FIX activity block on the competition for postsynaptic tectal targets between RGC fibers from one eye (O'Leary et al., 1986b) or from both eyes (Fawcett et al., 1984); -FIX injected into the eye of a newborn rat led to increased survival of RGC subpopulations that otherwise would have died. The patterning of cell death was altered in that topographically incorrectly projecting RGCs were not eliminated in the l-rX-injected eye. Nevertheless, the

overall amount of RGC death in the developing rat retina was normal under TTX activity-block conditions, i.e., about 60% of the RGCs still died in the activityblocked eye as well as in the uninjected eye (Fawcett et al., 1984; O'Leary et al., 1986a). However, the paradigm in those experiments was substantially different from ours in that efferent influences of activity block on neuronal cell death were monitored. In the case of our studies, by removing the target tissue, an additional effect of TTX on an afferent influence on cell death was uncovered in the retinal cultures. One possible explanation for an afferent influence on natural cell death is the activity-dependent release of a trophic factor necessary for neuronal survival. This factor must be present within the retina itself, since no normal target tissue was available to the RGCs in these in vitro experiments. In the present study, VlP meets several criteria for a survival factor. VlP rescued TTX-treated RGCs that otherwise would have died. VlP, located only in amacrine cells, potently stimulates adenylate cyclase, which in turn converts ATP to cAMP in the retinas of many species (Van Buskirk and Dowling, 1981; Longshore and Makman, 1981; Amiranoff and Rosselin, 1982; Lasater et al., 1983; Watling and Dowling, 1983; Koh et al., 1984). Results presented here indicate that VlP increases the level of cAMP in retinal cultures. Since cAMP effects long-lasting changes in other retinal neurons (Dowling, 1986), VlP may be mediating its physiological action on RGCs through this second messenger. TTX treatment decreased cAMP levels significantly below control levels. One explanation for this decline may be that the electrical blockade of activity-dependent release of VlP caused decreased adenylate cyclase stimulation. This in turn lowered the concentration of cAMP within the cultures. The addition of VlP to the electrically blocked cultures nearly restored the cAMP concentration to control levels. Thus, it appears that VlP indeed stimulates an increase in cAMP in these neuronal cultures. Moreover, the application of forskolin, an adenylate cyclase activator, or 8-bromo:cAMP increased RGC survival in electrically blocked cultures. Apparently, 8-bromo:cAMP and forskolin mimicked VIP stimulation of the neurons and reversed the effects of "I-TX blockade. In contrast, experiments adding 8-bromo: cGMP to ]-IX-treated cultures did not rescue RGCs from death. These findings support the notion that VIP, acting specifically through cAMP action, plays an important role in neuronal survival. It appears that the neurotrophic action of VlP is capable of rescuing all of the clustered RGCs that are vulnerable to 1-I-X electrical blockade (Figure 1B). The nonadditive effect of applying both FIX and VlP10-2~ to the cultures supports this conclusion. Furthermore, VlP and its related peptides had little effect on the number of surviving solitary RGCs. The effect of VlP was limited to the clustered RGCs that were acquiring or possessed spontaneous electrical activity.

VIP or cAMP PreventsTTX-InducedRetinalCell Death 379

This notion is supported by the fact that isolated or solitary RGCs exhibit no spontaneous action potentials, and TTX treatment affects o n l y RGCs that are acquiring or possess spontaneous activity, which constitute 50% of the clustered RGCs (Lipton, 1986). We specu late~that RGCs acquiring electrical activity in the absence o f tectal targets become d e p e n d e n t on the subsequent increase in.VIP (and the consequent production of cAMP) as a trophic or growth factor. Conversely, RGCs that do not have spontaneous action potentials may not yet have attained the stage of differentiation at which VlP is required for viability. The neu rotrophic effect of VlP appears to be very specific. The application of two closely related peptides, PHI27 and secretin, to electrically blocked culture dishes produced no noticeable effect on neuronal survival. In fact, the n u m b e r of surviving RGCs did not differ significantly from that observed in the presence of "I-I-X. The exact route of neurotrophic action of VlP is not known. VlP may exert its influence directly on the RGCs, or indirectly through contiguous neurons or glial cells. In other preparations several lines of evidence support the notion of a glia-mediated mechanism. Functional VIP receptors have been observed on cultured astrocytes, and trophic substances have been found in the c o n d i t i o n e d medium of glial cultures (Brenneman and Eiden, 1986; Brenneman et al., 1987). Consequently, one mechanism may involve activity-dependent release of VIP from neurons that in turn interacts with glial cells. These VlP-stimulated glial cells might produce additional factors required for neuronal growth and development (Brenneman et al., 1985, 1987; Brenneman and Eiden, 1986). In this scheme, VlP w o u l d act as a releasing factor. Conversely, VIP may directly exert its influence on activityd e p e n d e n t neurons, for example, to increase their level of cAMP. Although the retinal cultures have relatively few glial cells (Lipton et al., 1988), our evidence at present cannot differentiate definitively between the two mechanisms. Therefore, further research is essential to determine the exact mode of VIP action. Studies have shown that ~50% of the RGCs in vivo die naturally at a similar age as those in culture (Perry et al., 1983; Cunningham et al., 1982; Lipton, 1986). The question remains, however, w h e t h e r results in vitro truly reflect the normal process of cell death in the CNS (see Introduction). For example, in our model system, many anatomical constraints were removed by tissue dissociation. Thus, the in vitro neuronal networks that form are based on u n k n o w n preferences that may or may not relate to networks in vivo. Nevertheless, in light of the comparable p r o p o r t i o n of RGCs dying in vivo and in -n-X-treated cultures and the similar critical period of neuronal susceptibility, it is tempting to speculate that the neurotrophic effects of VIP and cAMP observed in vitro reflect an afferent mechanism for the regulation of natural cell death in the living animal.

Experimental Procedures RGC Culture Preparation RGCs were identified and cultured as previously described (Leifer et al., 1984; Lipton and Tauck, 1987). Briefly, the RGCs were labeled in situ by retrograde transport of the fluorescent dye granular blue. Using a fine needle, the dye was injected stereotactically as an ~,2% (w/v) suspension in saline into the superior colliculi of pigmented (Long Evans) rat pups (Charles River Laboratories)under anesthesia on P4or P5. Two days after the injection, the animals were sacrificed by cervical dislocation. The retinas were enucleated and dissociated by gentle trituration following digestion with papain, as previously described (Leifer et al., 1984). The retinal cells were cultured in a mixture of Eagle'sminimum essential medium (GIBCO) with 0.7% (w/v) methylcellulose, 5% (v/v) rat serum, 2 mM glutamine (Sigma Chemical Co., St. Louis, MO), 16 mM glucose, and 1 ~,g/mlgentamicin. Approximately 2 x 104cells were plated onto glass coverslips coated with poly-L-lysine.At the time of plating, 1% of the retinal cells contained the fluorescent blue dye, indicating that they were RGCs(Lipton, 1986);this proportion of RGCsis similar to that encountered in the intact retina. Electrical blockade was achieved by the addition of 1 llM l-rx (Sigma Chemical Co.), which completely blocks the fast action potentials and Na+ current of rat RGCs (Lipton and Tauck, 1987).The vehicle for TTXwas citrate buffer (1 llM TTX contained 7 llM sodium citrate); control experiments showed that this amount of citrate buffer had no effect on RGCviability (as determined below). VIP (0.1 nM to 1 ~M), forskolin (100 llM; Sigma), 8-bromo:cAMP (1 raM), or 8-bromo:cGMP (1 mM; Boehringer Mannheim Biochemicals, Indianapolis, IN) was added in the presence and absence of I-FX to the ceil culture medium prior to the plating of the retinal cells. As controls, we tested PHI-27 and secretin, two peptides closely related to VIP, and VlP10-28 (the C-terminal fragment of VIP). All peptides were obtained from Bachem, Inc. (Torrance,CA). The concentrated stock solutions of these peptides contained water and no other vehicles. Incubation of retinal cultures with these drugs lasted for 20 hr at 37°C in a humidified atmosphere of 5% CO2/95% air, and then the RGCs were scored for viability. Viability Assay of RGCs The ability of the RGCsto take up and cleavefluorescein diacetare to fluorescein was used as an index of viability. The cell culture medium was exchangedfor physiological saline containing 0.0005% (w/v) fluorescein diacetate for 15-60 s, and the cultures were rinsed with Hanks solution (see below for composition). Some RGCsthat did not contain the fluorescein dye (and thus were not viable) remained visible under a DAPI filter cube because of the continued presence of the marker dye granular blue; other dead RGCs had simply disintegrated, and only debris remained. In contrast, the viable RGCs displayed not only a blue color under the DAPI filter, but also a yellow-green fluorescence under an FIT(] filter cube. Thus, living RGCs ap~ peared to fluoresce in yellow-green under the FIT(] filter and in blue-green under the DAPI filter, while dying ganglion cells appeared to fluoresce in blue under the DAPI filter and did not appear under the FITC filter (for color photographs of this technique, see Figure 1A). The use of the FIT(] and DAPI exchangeable filter sets permitted the rapid and positive identification of all viable RGCs in the cultures and virtually eliminated scoring errors. The cultures were scored blindly under high power (500x)on a ZeissIM35 invertedphasemicroscopeequipped with epifluorescence optics. After a 20 hr incubation, the control cultures lost approximately 20% of the viable RGCs(both solitary and clustered), as compared with cell density at the time of plating (Lipton, 1986). The Hanks saline was based upon Hanks balanced salt solution and contained 137 mM NaCI, 5.36 mM KCI, 0.34 mM Na2HPO4,0.44mM KH2PO4,5 mM HEPES-NaOH, 1 mM NaHCO3,22.2 mM glucose, 2.5 mM CaCI2,0.5 mM MgCI2,

Neuron 38O

0.5 mM MgSO4, and 0.001% (v/v) phenol red indicator. The solution was adjusted to pH 7.3 with 0.3 N NaOH and filtered before use (0.22 lira; Millipore).

Radioimmunoassay of VIP Fluid was aspirated from retinal cultures prepared as described above without any additional experimental drugs, with I I~M VIP, with I !~M TTX, or with I lIM VlP plus I lIM TTX, and immediately frozen and stored at -80°C for subsequent analysis. Radioimmunoassay, preceded by purification on a charcoal column, was carried out as described previously (Riskind et al., 1989).

Radioimmunoassay of cAMP The Rianen cAMP [12Sl] RIA kit (New England Nuclear NEK-033, Boston, MA) was used to measure cAMP levels. The tissue preparation techniques and assay were adapted from procedures described by Rosenberg and Dichter (1989). The retinal cells were prepared as described above without any additional experimental drugs, with 1 p.M VIP, with 1 llM TTX, or with 1 llM VlP plus 1 jiM ]-IX. Samples were taken at 0 min, 10 rain, and 6 hr after plating the retinal cells. At the appropriate time, the medium was aspirated off the coverslips and 0.6 M HCIO4 was added to each well. The wells were incubated at 4°C for 1 hr. Then, 6 M KHCO3 was added, and the solution was placed in Eppendorf tubes. After a 15 rain neutralization period, the Eppendorf tubes were microcentrifuged at 1200 x g for 3 rain. The supernatant was aspirated off and analyzed. Radioactivity was quantified in a gamma counter at a counting efficiency of 50%-70% for 1 rain.

Acknowledgments This work was supported in part by a Fight For Sight Student Fellowship and a Herman and Albert Mosler Memorial Award, Fight For Sight, Inc., New York City (to P. K. K.), and by NIH grants NS00879, NS07264, EY06087,and EY05477 (to S. A. L.). One of us (S. A. L.) is an Established Investigator of the American Heart Association. We are grateful to Dr. Peter Riskind of the Massachusetts General Hospital for performing the VlP assays. We thank Drs. Paul A. Rosenberg, Elias Aizenrnan, and Jin Hahn for helpful discussions and Maureen Oyola and Francis Peale for their help in the preparation of this manuscript. Correspondence should be addressed to S. A. Lipton, Children's Hospital, Enders Building, Room 350, 300 Longwood Avenue, Boston, Massachusetts 02115. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Recieved February 15, 1990; revised June 6, 1990.

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After submission of our manuscript, D. W. Pincus, E. M, DiCiccoBloom, and I. B, Black reported that VlP has other neurotrophic actions (Vasoactive intestinal peptide regulates mitosis, differentiation and survival of cultu red sympathetic neu rob lasts. Natu re, 1990, 343, 564-567).