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G.L. Fain et al. – Photoreceptor dark adaptation

Dark adaptation in vertebrate photoreceptors G.L. Fain, H.R. Matthews and M.C. Cornwall Exposure of the eye to bright light bleaches a significant fraction of the photopigment in rods and cones and produces a prolonged decrease in the sensitivity of vision, which recovers slowly as the photopigment is regenerated.This sensitivity decrease is larger than would be expected merely from the decrease in the concentration of the pigment. Recent experiments have shown that the decrease in sensitivity is produced largely by an excitation of the phototransduction cascade by bleached pigment;even in darkness,it produces an equivalent background similar to that produced by real steady background illumination.Thus, excitation produced by a form of rhodopsin thought previously to be inactive has a profound effect on the physiology of the photoreceptor.This raises the possibility that forms of other G protein-coupled receptors thought to be inactive might also play an important role in signal transduction and disease. Trends Neurosci. (1996) 19, 502–507

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G.L. Fain is at the Depts of Physiological Science and Ophthalmology, University of California, Los Angeles, CA 90095, USA. H.R. Matthews is at the Physiological Laboratory, University of Cambridge, Downing Street, Cambridge, UK CB2 3EG. M.C. Cornwall is at the Dept of Physiology, Boston University School of Medicine, Boston, MA 02118, USA.

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HE ABSORPTION of a photon by the photopigment rhodopsin in a rod or a cone causes a photochemical reaction that converts the 11-cis retinal chromophore of the pigment into its all-trans stereoisomer1,2. The change in the configuration of retinal causes a series of changes in the conformation of the protein component of the pigment (called opsin), leading to a form of the pigment called photoactivated metarhodopsin II (or Rh*)3,4. This binds to the G protein transducin and initiates the signal-transduction cascade5–7 (see Fig. 1). Photoactivated metarhodopsin II has a lifetime estimated to be no longer than a few seconds24 (and possibly much shorter)18,25,26. It is soon phosphorylated and bound by the protein arrestin, which largely terminates its activity27–29. In order for a bleached rhodopsin molecule to absorb another photon, the pigment must be regenerated. In a vertebrate retina, regeneration is a complicated process30,31. The majority of phosphorylated metarhodopsin II first decays to another form of the pigment called metarhodopsin III, and the all-trans chromophore then separates from opsin and is reduced to retinol; this then initiates the removal of arrestin and the phosphate groups from the protein32. To regenerate chromophore (see Fig. 2), all-trans retinol actually exits the rod or cone and, with the assistance of an interphotoreceptor retinol binding protein (IRBP), is transported to an adjacent layer of epithelial cells, called the retinal pigment epithelium (RPE). In the RPE, the retinol is esterified mostly to palmitic acid, and retinoid isomerase then converts the ester to 11-cis retinol, which is then oxidized by an oxidoreductase to 11-cis retinal. 11-cis retinal is transported via IRBP to the photoreceptor, where the chromophore is recombined with dephosphorylated opsin to form rhodopsin. Regeneration is quite slow, requiring 30–60 min for completion when a significant percentage of the pigment has been bleached33. This slow timecourse reflects the transition of the protein from metarhodopsin II to metarhodopsin III and then to TINS Vol. 19, No. 11, 1996

dephosphorylated opsin, as well as the time necessary for the re-isomerization of the chromophore. It is mirrored by the slow return of sensitivity after exposure to bright light: in humans, sensitivity of vision remains depressed for as long as 30 – 45 min after bright bleaches, and recovers only as the photopigment is restored to its dark-adapted condition34,35.

Bleaching and dark adaptation in photoreceptors The desensitization after bright bleaches, called bleaching adaptation, and the subsequent slow recovery of sensitivity, called dark adaptation, have been the subject of much interest and speculation. Psychophysical measurements showed many years ago that the decrease in sensitivity after exposure to bright light is too great to be explained by the decrease in the concentration of the pigment produced by the bleach36,37. Bleaching adaptation has many of the features of desensitization produced by steady backgrounds, which is known as light adaptation or background adaptation. This suggests that bleaching adaptation and background adaptation are somehow related, although the nature of this relationship has been unclear38. Extensive investigation over the past 20–25 years has shown that much of the machinery for both light and dark adaptation is present within the photoreceptors themselves39–43. In both rods and cones, steady background light produces a sustained decrease in photoreceptor dark current, an acceleration in the kinetics of responses to brief flashes, and a decrease in sensitivity44–47. We now know that these effects are all due to a decrease in cytoplasmic Ca2+ concentration ([Ca2+]i), mediated by processes that have been reviewed recently in this journal48. Bleaches also produce sustained effects on photoreceptor dark current, response kinetics and sensitivity47,49,50. The experiment in Fig. 3 shows typical results from an isolated rod, which was exposed to a light that bleached about 90% of the pigment in its outer segment. Sensitivity recovered slowly, but then

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G.L. Fain et al. – Photoreceptor dark adaptation

Na+/Ca2+ – K+ exchanger

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Fig. 1. Scheme of vertebrate phototransduction. Upon absorption of a photon, rhodopsin (Rh) is converted to photoactivated metarhodopsin II (Rh*). This then binds to the G protein transducin and promotes the exchange of GTP for GDP on the transducin a subunit. Activated Ga (Ga*) then binds to one of the two inhibitory g subunits of the cGMP phosphodiesterase (PDE), relieving inhibition from one of the two catalytic subunits of PDE, a and b. The PDE then hydrolyses cGMP to GMP. This reduces the concentration of cGMP in the cytoplasm of the photoreceptor outer segment, which causes the cGMP-gated Na+ and Ca2+ channels to close8, and thus the influx of Ca2+ into the rod to decrease9. The efflux of Ca2+ occurs via a Na+/Ca2+–K+ countertransporter10,11 whose rate of activity is proportional to cytoplasmic Ca2+ concentration ([Ca2+]i ) (Ref. 12); thus, the closing of the channels leads to a decrease in [Ca2+]i and a corresponding reduction in Ca2+ efflux13. The change in [Ca2+]i causes a change in photoreceptor response kinetics and sensitivity by modulating (heavy arrows) the rate of the guanylate cyclase14,15, the channel open probability16,17, and some stage early in transduction18–22, perhaps the rate of phosphorylation of Rh* (Ref. 23).

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stabilized after 45–60 min. Since the rod had been removed from the retina, there was no RPE to regenerate the chromophore, and the slow timecourse of recovery reflects changes in the protein component of the pigment. After sensitivity stabilized, it remained

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0 Fig. 2. The visual cycle. Bleaching of rhodopsin (Rh) produces photoactivated metarhodopsin II (Rh*), which decays through a series of intermediates (see text) to opsin and all-trans retinal. All-trans retinal is reduced within the photoreceptor to all-trans retinol, which is then transported via interphotoreceptor retinol binding protein (IRBP) through the extracellular space to an adjacent layer of epithelial cells, called the retinal pigment epithelium (RPE). Within the RPE, the alltrans retinol is bound to cellular retinol binding protein (not shown) and then esterified to fatty acids, mostly palmitic acid. The ester is converted by retinoid isomerase to 11-cis retinol, which is bound to yet another binding protein and oxidized by oxidoreductase to 11-cis retinal. Interphotoreceptor binding protein then returns the 11-cis retinal to the photoreceptor, where it recombines with opsin to form darkadapted Rh. Modified from Ref. 30.

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I (photons µm–2) Fig. 3. Response-intensity curves and flash-response waveforms of a salamander rod in darkness, after exposure to bright bleaching light, and after addition of exogenous 11-cis retinal. The ordinate for the curves gives the peak amplitude of the photocurrent measured with a suction electrode for brief flashes of 520 nm light whose intensity is given on the abscissa. Curves are shown for responses in darkness (filled circles), at various times after a 16 s exposure to a light that bleached approximately 90% of the photopigment (open symbols), and at two times after addition of exogenous 11-cis retinal in phospholipid vesicles (filled triangles, filled diamonds). (Inset) Normalized small-amplitude responses to brief flashes for the same rod in darkness (Dk), at steady state 55 min after the bleach (Bl), and 36 min after adding 11-cis retinal (Rg). Modified from Ref. 43.

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Fig. 4. Photocurrent recorded from rod stepped into Li + solution in darkness, after bleaches, and after pigment regeneration. All records are from the same rod. Suction-pipette current as a function of time after the solution changes (A) in darkness, (B) 43 min after a bleach of 22% of the rhodopsin, (C) 50 min after a further light exposure that, together with the first, produced a cumulative bleach of 52% of the photopigment and (D) 99 min after exposure to phospholipid vesicles containing 11-cis retinal (total duration of the experiment was about 3 h). Uppermost traces show approximate timecourse of step from Na+ solution to Li + solution. Initial negative deflections immediately after the solution change reflect the greater permeability of the light-sensitive conductance to Li + than to Na+. Panels on the right show the negative of the currents on the left, plotted on a log scale. The nearly log–linear decline of the initial component of current indicates that current falls nearly exponentially, with a time constant that is speeded by bleaching and is restored to its dark-adapted value upon regeneration. Modified from Ref. 56.

lower than would be expected from the reduction in the concentration of pigment49,50. Sensitivity, amplitude of maximum response and response kinetics (Fig. 3, inset) were altered for the duration of the recording (up to 8 h). They recovered almost completely, however, when the rod was exposed to vesicles containing exogenous 11-cis retinal49,51,52.

Bleached pigment activates signal transduction The similarity of the effects of steady background light and exposure to bright bleaches on visual behavior led Stiles and Crawford over sixty years ago to postulate that bleached pigment desensitizes by producing an ‘equivalent background’ light53: that is, bleached pigment was proposed to activate the signaltransduction cascade in a similar way to real light. As bleached pigment was regenerated, the intensity of this ‘equivalent background’ would decline, until the rod or cone (and the visual system as a whole) returned to its dark-adapted sensitivity. If this theory is correct, it should be possible to show that bleached pigment activates the signal504

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transduction cascade. Since the effect of light is to activate cGMP phosphodiesterase (PDE; see Fig. 1), bleaches should also achieve this effect; the activity of PDE should then remain elevated until pigment is regenerated. Activation of PDE can be demonstrated with a physiological method first devised by Hodgkin and Nunn54. They observed that the cGMP concentration in the rod is determined by the balance of two reactions: a b GTP –→ cGMP –→ GMP The first reaction is mediated by guanylate cyclase and the second is mediated by PDE. At steady state, the concentration of cGMP is stable, and these two reactions proceed at the same rate. This rate can be determined either by blocking the cyclase and measuring the rate of cGMP hydrolysis by PDE, or by blocking PDE and measuring the rate of cGMP synthesis by the cyclase. The concentration of cGMP can be monitored indirectly by measuring the amplitude of the lightdependent current. Figure 4 shows typical measurements for PDE. An isolated rod was stepped suddenly into a solution for which Na+ was substituted with Li+. In Li+ solution, the rod is unable to extrude Ca2+ via the Na+/Ca2+–K+ countertransporter9,55. The turnover of Ca2+ in an outer segment is so rapid that the cessation of Ca2+ efflux causes a sudden rise in [Ca2+] in the outer segment; this then inhibits the cyclase14,15, and the current through the light-dependent channels declines nearly exponentially to zero as the PDE hydrolyses cGMP (Fig. 4A). Bleaching accelerates the rate of the current decrease after a step into Li+ solution, indicating that bleached pigment activates the PDE (Ref. 56). The amount of this acceleration is approximately proportional to the amount of bleaching. This would suggest that each bleached pigment molecule makes an equal contribution to activation of signal transduction and so might activate the cascade with equal probability42,56. If the pigment is regenerated by exogenous application of 11-cis retinal, the rate of hydrolysis by PDE returns to its dark-adapted level. Similar measurements using IBMX (3-isobutyl-1-methylxanthine) to inhibit the PDE show that the activity of the cyclase is also accelerated by bleached pigment, in cones57 as well as in rods56. Apparently, bleached pigment in an isolated photoreceptor produces a prolonged increase in the turnover of cGMP, which declines only when the photopigment is regenerated to its dark-adapted state.

Activation proceeds by way of the G protein transducin How does this persistent excitation occur? The simplest explanation would be that bleached pigment activates signal transduction via the same cascade of reactions as Rh* (see Fig. 1). Recent experiments incorporating the poorly hydrolysed GTP analogue GTPgS into rods indicate that this is probably the case58. In a dark-adapted rod, the introduction of GTPgS causes a slow decline in the dark current; this is probably partly because GTPgS binds to transducin spontaneously in darkness59,60. The quasi-irreversible activation of transducin by GTPgS activates PDE; as activated transducin accumulates, the rate of PDE

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G.L. Fain et al. – Photoreceptor dark adaptation

Bleaching adaptation requires a change in free [Ca2+]i Extensive investigation has shown that desensitization in steady backgrounds does not occur without a change in free [Ca2+]i: Ca2+ as a second messenger is both necessary47,62–64 and sufficient22,65 for the change in sensitivity during light adaptation. It is also necessary for the changes in sensitivity and response waveform after a bleach66. One demonstration of this is given in Fig. 5. Here, the response in a dark-adapted cone (Fig. 5A) is compared with the response of the same cone after a bleach (Fig. 5B) under conditions in which changes in [Ca2+] in the outer segment were greatly retarded or prevented altogether. The waveform was little altered (compare Fig. 3, inset), and there was little change in sensitivity. If, for the same cell, the [Ca2+]i was now allowed to fall (Fig. 5C), this resulted in a greatly accelerated waveform and a desensitized response.

The mechanism of bleaching adaptation in a photoreceptor These results indicate that bleached photopigment activates transducin, which in turn stimulates PDE. Hydrolysis of cGMP by PDE lowers the cGMP concentration and decreases the light-dependent conductance; this reduces the influx of Ca2+ into the cell and lowers the free [Ca2+]i (Refs 67–69). Recent results show that bleaching produces a maintained decrease in [Ca2+]i (Ref. 70), consistent with the maintained decrease in inward current. This decrease in [Ca2+]i is necessary for the desensitization of the photoreceptor after bleaching66 and is probably responsible for the activation of the guanylate cyclase. Therefore, it would appear that desensitization after a bleach is quite similar in mechanism to desensitization in steady background light. However, there are some differences. Bleached pigment is less effective at stimulating the signaltransduction cascade than Rh*. The efficacy of transduction can be estimated from the ratio of PDE excitation produced by bleached pigment to that produced by real light. At steady state (after a bleach or in background light), this ratio is 10 –6–10 –7 for rods56 and 10 –4–10 –5 for cones57. One reflection of this difference in gain is the difference in the noise produced by backgrounds and bleaching. In a dark-adapted rod, a single Rh* molecule can produce a decrease of perhaps 105–106 molecules of cGMP (Ref. 71), enough to produce a detectable change in the cGMP-gated current and a single photon event72. In steady backgrounds, photon events produce random noise45,

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activity increases and eventually brings the cGMP concentration and the circulating current to zero. This decline of current after GTPgS incorporation is much faster in a bleached rod than in a dark-adapted rod, which is most simply explained if transducin activation occurs at a higher rate in a bleached rod than in a dark-adapted rod58. This is consistent with the acceleration of PDE and cyclase activity that occurs after bleaching56,57, and indicates that bleached pigment molecules are likely to activate the signal-transduction cascade by the same pathway as Rh*, though with a greatly reduced gain. This point is reinforced by the recent demonstration that free opsin can activate guanine-nucleotide exchange by transducin in vitro61.

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t (s) Fig. 5. Changes in [Ca2+]i are necessary for desensitization after bleaching. Comparison of response waveform of a cone to dim flashes before and after bleaching. Responses were recorded in a solution of low Ca2+ and zero Na+, made by buffering the free Ca2+ concentration to 10 –7 M with EGTA and replacing NaCl with guanidinium chloride62–64. Since guanidinium substitutes little if at all for Na+ in Na+/Ca2+–K + exchange, this solution minimizes both Ca2+ influx and efflux and tends to hold the free intracellular Ca2+ concentration ([Ca2+]i ) near to its value just preceding the solution change. (A) Dark-adapted response in a solution of low Ca2+ and zero Na+. (B) Responses recorded from the same cone exposed to a solution of low Ca2+ and zero Na+ just before delivery of a 44% bleach, so that [Ca2+]i was held near to the darkadapted level. 500 mM IBMX (3-isobutyl-1-methylxanthine) was used to inhibit the PDE, in order to bring the cone out of saturation so that the light response could be recorded. (C) Cone returned to Ringer after the bleach to permit [Ca2+]i to reach its normal bleach-adapted level, and response then recorded in a solution of low Ca2+ and zero Na+, with [Ca2+]i now held near the value normally produced by bleaching instead of the dark level. Note the greatly accelerated response kinetics, typical of a light-adapted response. Modified from Ref. 66.

though individual events are smaller than in a darkadapted photoreceptor as a result of the desensitization produced by light adaptation. After bleaches, single events have also been detected, particularly when the bleach is relatively small73,74, but recent measurements show that there are far fewer detected events than would be expected for a comparable desensitization produced by a background light75. Furthermore, in experiments like those in Fig. 3 in which a large bleach is given and the rod then allowed to come to steady state, the level of noise present is also insufficient to account for the observed desensitization43,75. This means that the gain of single interactions between bleached pigment and transducin is much smaller than for Rh*: if a single Rh* molecule can activate 500 –1000 transducins6,71, a single bleached pigment molecule might be able to activate only one or a few. TINS Vol. 19, No. 11, 1996

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We do not know how bleached pigment interacts with transducin to produce this activation. It is possible that phosphorylated metarhodopsin II, metarhodopsin III and opsin all are capable of activating the cascade. However, in an isolated rod as in the experiment of Fig. 3, it is likely that most of the equivalent background at steady state is produced by free opsin in some form, that is, by rhodopsin lacking chromophore. There are two lines of evidence for this. First, the persistent activation of the cascade remains even after 8 h (Refs 49,51,52); after this time, all of the pigment is likely to have lost its chromophore. Second, activation of the signal-transduction cascade can be terminated not only by 11-cis retinal but also by compounds such as 9-cis C17 retinal and b-ionone76; these compounds have ring structures similar to that of retinal and so probably fit in the chromophore pocket of opsin, but have conjugated tails shorter than retinal and do not make covalent bonds with opsin or form actual pigments. Remarkably, b-ionone can be perfused on and off the photoreceptor, and activation disappears and returns repeatedly57. It is unlikely that the original chromophore could still remain in place under these conditions.

Spontaneous activation by G protein-coupled receptors We are accustomed to assuming that receptors exist in two forms, one active and one inactive. This is an oversimplification for rhodopsin and perhaps also for other G protein-coupled receptors. Even dark-adapted rhodopsin is spontaneously active, producing events indistinguishable from photon events, but with a rate so low that the half-life of the pigment is measured in hundreds of years77. Bleached forms of free opsin appear to exhibit a much higher level of spontaneous activity, which is unlikely to be evolutionarily adaptive since it produces a large decrease in sensitivity. It might be an unavoidable consequence of other design considerations, proceeding from the necessity for low noise and high gain in darkness. Spontaneous activity has been easier to measure for rhodopsin than for other G protein-coupled receptors, but might prove to be a general feature of this class of proteins. Persistent stimulation after activation might explain in part why so many G protein-coupled receptors are internalized and recycled78. Excess activity (for example, from mutant forms of rhodopsin79,80 or other G protein-coupled receptors81,82) might produce deleterious effects, including photoreceptor degeneration83 and other forms of malfunction84. The abundance of rhodopsin in a rod or cone might make this preparation uniquely accessible as a system to study how spontaneous activation occurs. Selected references 1 2 3 4 5

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9 Hodgkin, A.L., McNaughton, P.A. and Nunn, B.J. (1985) J. Physiol. 358, 447– 468 10 Cervetto, L. et al. (1989) Nature 337, 740–743 11 Schnetkamp, P.P., Basu, D.K. and Szerencsei, R.T. (1989) Am. J. Physiol. 257, C153– C157 12 Lagnado, L., Cervetto, L. and McNaughton, P.A. (1992) J. Physiol. 455, 111–142 13 Yau, K-W. and Nakatani, K. (1985) Nature 313, 579–582 14 Koch, K-W. and Stryer, L. (1988) Nature 334, 64–66 15 Koutalos, Y. et al. (1995) J. Gen. Physiol. 106, 863–890 16 Hsu, Y-T. and Molday, R.S. (1993) Nature 361, 76–79 17 Nakatani, K., Koutalos, Y. and Yau, K.W. (1995) J. Physiol. 484, 69–76 18 Matthews, H.R. (1996) J. Physiol. 490, 1–15 19 Kawamura, S. and Murakami, M. (1991) Nature 349, 420 – 423 20 Lagnado, L. and Baylor, D.A. (1994) Nature 367, 273–277 21 Jones, G.J. (1995) J. Physiol. 487, 441–451 22 Koutalos, Y., Nakatani, K. and Yau, K-W. (1995) J. Gen. Physiol. 106, 891–921 23 Kawamura, S. (1993) Nature 362, 855–857 24 Pepperberg, D.R. et al. (1992) Visual Neurosci. 8, 9–18 25 Matthews, H.R. (1996) J. Physiol. 494, 15P 26 Lyubarsky, A., Nikonov, S. and Pugh, E.N. (1996) J. Gen. Physiol. 107, 19–34 27 Wilden, U., Hall, S.W. and Kuhn, H. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 1174–1178 28 Wilden, U. and Kuhn, H. (1982) Biochemistry 21, 3014–3022 29 Hurley, J.B. (1994) Curr. Opin. Neurobiol. 4, 481–487 30 Bok, D. (1993) J. Cell Sci. 17, 189–195 31 Saari, J.C. (1990) in Progress in Retinal Research (Osbourne, N. and Chader, G., eds), pp. 363–382, Pergamon 32 Hofmann, K.P. et al. (1992) J. Biol. Chem. 267, 15701–15706 33 Hecht, S., Haig, C. and Chase, A.M. (1937) J. Gen. Physiol. 20, 831–850 34 Aguilar, M. and Stiles, W.S. (1954) Opt. Acta 1, 59–65 35 Campbell, F.W. and Rushton, W.A.H. (1955) J. Physiol. 130, 131–147 36 Wald, G. (1961) in Light and Life (McElroy, W.D. and Glass, B., eds), pp. 724–749, Johns Hopkins Press 37 Rushton, W.A.H. (1965) Proc. R. Soc. London Ser. B 162, 20– 46 38 Dowling, J.E. (1987) The Retina, Harvard University Press 39 Pugh, E.N. and Cobbs, W.H. (1986) Vis. Res. 26, 1613–1643 40 Fain, G.L. and Matthews, H.R. (1990) Trends Neurosci. 13, 378–384 41 Pugh, E.N. and Lamb, T.D. (1990) Vis. Res. 30, 1923–1948 42 Lamb, T.D. (1990) in Night Vision (Hess, R.F., Nordby, K. and Sharp, L.T., eds), pp. 171–216, Cambridge University Press 43 Fain, G.L. and Cornwall, M.C. (1993) in Contrast Sensitivity: From Receptors to Clinic (Shapley, R. and Lam, D-K., eds), pp. 3–32, MIT Press 44 Baylor, D.A. and Hodgkin, A.L. (1974) J. Physiol. 242, 729–758 45 Baylor, D.A., Matthews, G. and Yau, K-W. (1980) J. Physiol. 309, 591–621 46 Fain, G.L. (1976) J. Physiol. 261, 71–101 47 Matthews, H.R. et al. (1990) J. Physiol. 420, 447– 469 48 Koutalos, Y. and Yau, K-W. (1996) Trends Neurosci. 19, 73–80 49 Cornwall, M.C., Fein, A. and MacNichol, E.F. (1990) J. Gen. Physiol. 96, 345–372 50 Jones, G.J. et al. (1993) J. Gen. Physiol. 102, 483–502 51 Jones, G.J. et al. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 9606–9610 52 Corson, D.W. et al. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 6823–6827 53 Stiles, W.S. and Crawford, B.H. (1932) in Report of a Joint Discussion of Vision, pp. 194–211, Imperial College of Science, Physical Society 54 Hodgkin, A.L. and Nunn, B.J. (1988) J. Physiol. 403, 439–471 55 Yau, K-W. and Nakatani, K. (1984) Nature 311, 661–663 56 Cornwall, M.C. and Fain, G.L. (1994) J. Physiol. 480, 261–279 57 Cornwall, M.C. et al. (1995) J. Gen. Physiol. 106, 543–557 58 Matthews, H.R., Cornwall, M.C. and Fain, G.L. J. Gen. Physiol. (in press) 59 Lamb, T.D. and Matthews, H.R. (1988) J. Physiol. 407, 463– 487 60 Sather, W.A. and Detwiler, P.B. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 9290–9294 61 Surya, A., Foster, K.W. and Knox, B.E. (1995) J. Biol. Chem. 270, 5024–5031 62 Matthews, H.R. et al. (1988) Nature 334, 67–69 63 Nakatani, K. and Yau, K-W. (1988) Nature 334, 69–71

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Growth cones and the cues that repel them Alex L. Kolodkin Neuronal growth cones establish appropriate connections with their targets during development by responding to both positive and negative guidance cues. The importance of repulsive and inhibitory cues in pathfinding and target selection has now been firmly established at the cellular and molecular levels.Observations in vitro have demonstrated developmentally significant repulsive interactions among various neuronal populations, providing the basis for molecular and functional characterization of several families of molecules that can mediate these guidance events. Analysis of both the expression and function of these molecules in vivo suggests how they, together with positive guidance cues, participate in the dynamic process of growth-cone guidance during both development and axonal regeneration. Trends Neurosci. (1996) 19, 507–513

I

N ORDER to form the complex organization found in a functioning nervous system, it is essential that axons establish their correct pathways and find their appropriate targets during development. These early activity-independent events provide the underlying scaffold upon which the mature nervous system is built, setting the stage for later activity-dependent processes that direct synapse formation, refinement and plasticity. Axon pathfinding and target recognition rely on the ability of the growth cone (the leading edge of the extending axon) to sample its environment and integrate multiple guidance cues in order to affect steering events. Pioneering studies on the generation of precise connections during neuronal development have underscored the importance of positive, or attractive, interactions in mediating axon guidance1–3. The molecular characterization of several attractive cues shows that they belong to different families of molecules and that they are similar in invertebrates and vertebrates, supporting the notion that both the molecules and the mechanisms of growth-cone guidance are conserved phylogenetically4,5. Over the past decade, however, several types of negative growth-cone-guidance cues have been observed in a variety of experimental paradigms, and the molecular characterization of these cues is now taking place at a furious pace6–8. Negative guidance cues can be repulsive, changing the direction of axon outgrowth through alterations of steering, branching or synapse formation. Negative guidance cues can also be inhibitory, preventing any Copyright © 1996, Elsevier Science Ltd. All rights reserved. 0166 - 2236/96/$15.00

of these events from occurring at all9,10. Here, the term repulsive will be used to describe this range of activities of negative guidance cues, and will be qualified when actual inhibition, not repulsion, is observed. The distinction between repulsion and inhibition can, at times, become blurred, since the assay used to assess growth-cone guidance often dictates the type of guidance events that a specific cue can impart. A common link among the negative cues, both repulsive and inhibitory, considered here is that in some context they can either induce repulsive events in growing axons or, as is seen in the case of certain inhibitory cues, they can induce collapse of the overall structure of the growth cone. In this context, negative inhibitory cues can be distinguished from apparent inhibition that might result from the strengthening of contacts between the growth cone and its substrate, resulting in a concomitant cessation of growth-cone motility. Like attractive cues, repulsive cues can act over long or short distances. Again, context is important, since a cue that can act as a chemorepellent in one assay might be tethered in vivo to a particular substrate that a growth cone must first contact in order to then be repelled. Biochemical and molecular characterization of guidance cues that can function as chemorepellents shows that these molecules in vivo are likely to be associated with the cell surface or extracellular matrix (ECM). This raises the possibility that secreted guidance cues use haptotactic mechanisms to guide neurons, requiring contact with a substrate that displays PII: S0166-2236(96)10057-6

TINS Vol. 19, No. 11, 1996

Alex L. Kolodkin is at the Dept of Neuroscience, The Johns Hopkins University School of Medicine, 725 N. Wolfe St, Baltimore, MD 21205, USA.

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