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Photoreceptor plasticity in the albino rat retina following unilateral optic nerve section
Exp. Eye Res. (1992) 55. 393-399
Photoreceptor
Plasticity Unilateral
JERI-LYNN Institute
of Molecular
(Received
SCHREMSER
Biophysics,
Houston
in the Albino Optic Nerve
15 July
AND
Rat Retina Section
THEODORE
Florida State University, 1997 and accepted
Following
P. WILLIAMS
Tallahassee,
FL 32303-3015,
in revised form 14 November
U.S.A.
79?!)
Rhodopsin content of the retina increased when an albino rat was moved to a lower intensity cyclic light, and decreased when moved to a higher intensity. Unilateral optic nerve section was employed to test if intact optic nerves are necessary to maintain regulation of rhodopsin when albino rats are switched between two intensities, 3 lx and 600 lx (12 hr/12 hr. cyclic light). The ability to regulate rhodopsin content was altered but not lost in the eye which had the optic nerve transected. This alteration was in the direction of incomplete up-regulation when the intensity change was from high to low, and was in the direction of excessive down-regulation when the intensity change was from low to high. This indicates that efferent fibers from the brain to the retina may be involved in the regulation of rhodopsin content. Using histological techniques, cell number and rod outer segment (ROS) length were measured: surgery alone had no effect on ROS length or outer nuclear layer thickness and in all cases ROS lengths were inversely related to habitat illuminance. Key words: rhodopsin : rat : rod photoreceptors : optic nerve : plasticity.
1. Introduction
Optic Nerve and Retina
Regulation of Light-absorption
In addition to the well-characterized afferent fibers which transfer both visual and non-visual information to the brain, the optic nerve of a number of species has been shown to contain efferent fibers. There is evidence for centrifugal fibers to the retina in invertebrates (Chamberlain and Barlow, 1979), in rats (Itaya, 1980; Ohno, 1980; Drager et al., 1984: ltaya and Itaya, 1984; Hoogland et al., 1985; Muller and Hollander, 1988), and in other vertebrate species including primates (Polyak, 1941; Brooke et al., 1965) and humans (Honrubia and Elliot, 1968). It appears that these centrifugal fibers are not abundant, constituting less than 2% of the optic nerve fibers in the rat (Molotchnikoff and Tremblay, 1986) ; however, there is evidence indicating that these fibers have
Light history, the record of all light exposure an animal receives throughout its lifetime, is a contributing factor to the regulation of photon-catch. Photon-catching ability in the rat is adjusted by changing rod outer segment (ROS) length, rod cell number, rhodopsin packing per disc, rod cell diameter, disc packing density and regeneration rate (Organisciak and Noell, 1977: Battelle and LaVail, 1978 ; Penn and Williams, 19 8 6 ; Penn and Anderson, 19 8 7 : Tolman, Koutz and Rapp, 1989). Animals raised in different intensities, therefore, have different light histories. Rod photoreceptor outer
segment length and rhodopsin content are regulated according to the light intensity of the environment within constraints determined by the animal’s light history (Battelle and LaVail, 1978 ; Penn and Williams,
1986: Williams et al., 1988). Photostasis is the regulation of photon-catch (Penn and Williams, 1986). This phenomenon derives its name from the observation that groups of albino rats raised in a range of cyclic intensities, 3-400 lx, will
absorb statistically the same number of photons per rat per day. This phenomenon has also been demonstrated in Limulus polyphemus(Hoenig and Chamberlain, 1989). The fact that photostasis occurs in such diverse speciesindicates that it may play a significant
role at a fundamental yet to be determined.
level. What this role may be is
* For correspondence. 00144835/92/090393+07
508.00/0
extensive arborizations and are involved in modu-
lation of retinal function. To observe the influence of efferent fibers on the retina, studies have been done in which the optic nerve has been ligated (Tsang, Yew and Lam, 1985) or transected (Barlow, Chamberlain and Levinson, 1980; Teirstein, Goldman and O’Brien, 1980; Barlow et al., 1985; Bush and Williams, 1991). For example, Barlow et al. (1980) observed in Limulus that when the efferent input to the retina is cut the circadian rhythms in both the structure and physiology of the retina were abolished. Teirstein et al. (1980) investigated whether the process of disc shedding is affected by optic nerve section and concluded that intact optic nerves are not necessary for maintenance of the shedding rhythm, but may be necessary to synchronize and phase shift the disc shedding rhythm. Bush 0 1992 AcademicPressLimited
J.-L.
394
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T. P. WILLIAMS
600 Ix natlves
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immigrants
Fro. 1. Light-history paradigm.Four groups,named accordingto their lighting regimes:3 lx natives; 3 lx immigrants: 600 lx natives: and 600 Ix immigrants.Nativeswere thoseanimalsthat remainedin their rearing intensity and immigrants (arrows) were thoseanimalsthat were moved to the new intensity.
and Williams ( 199 1) found that retinas with cut optic nerves suffered substantially less damage from light than did those with intact optic nerves. Williams et al. (1988) demonstrated that rats have the ability to up- and down-regulate rhodopsin content when they are moved to a new intensity. To study another possible influence of efferent fibers on the retina we asked the question: can the retina of an optic nerve sectioned eye still adjust rod outer segment (ROS) length and rhodopsin content when an animal is moved to a new lighting intensity? 2. Materials and Methods
Sprague-Dawley albino rats (breeder pairs from Charles River, Wilmington, MA) were born, raised and maintained in our laboratory, in controlled cyclic lighting, 12 hr/12 hr, light/dark. Two light intensities were used : a low-intensity condition of 3 lx; and a high-intensity condition of 600 lx. Special chambers were designed and built so that illumination could be set for 600 lx and delivered evenly to all rats. Illuminance was measured using both a Tektronix J16 digital photometer with a J6501 illuminance probe and a United Detector Technology Mode 350 Optometer. All intensities are reported in units of lx. One-hundred and eight rats were used in this experiment according to the NIH guidelines for the care and use of animals. All animals were 12 weeks of age at the time of unilateral optic nerve section (UONS) or sham surgery and 16 weeks when killed. Following surgery the animals were returned to their original intensities for a recovery period of 2 weeks and then some were switched to a ‘new intensity ’ for 2 weeks and others remained in their original intensity (Fig. 1). Natives were those animals that remained in their rearing intensity throughout their lifetime and TABLE I
Time of exposureto high intensity (600 Ix)
immigrants were those animals that were moved from their rearing intensity to the ‘new intensity ’ for 2 weeks. At 16 weeks of age, dark-adapted whole-eye rhodopsin levels, rod outer segment (ROS) length and outer nuclear layer (ONL) thickness were measured. There were four groups, named according to their lighting regimes : 3 lx natives ; 3 lx immigrants : 600 lx natives ; and 600 lx immigrants (Fig. 1). Each of the four groups was divided into three subgroups: sham eyes ; sectioned eyes (those eyes which have a severed optic nerve); and intact eyes. Table I condenses the following information but since this is an essential point to understand we will describe each group in detail. Six-hundred lux natives were born and raised in 600 lx for 16 weeks while 3 lx natives were born and raised in 3 lx for 16 weeks. A 600 lx immigrant was an animal that was raised in 3 lx until 14 weeks of age, then switched to 600 lx for 2 weeks ; therefore, a 600 lx immigrant had 2 weeks’ exposure to the high intensity. Finally, a 3 lx immi-
grant was raised in 600 lx for 14 to 3 lx for 2 weeks ; therefore, exposure to the high intensity. animals except 3 lx natives had
weeks then moved it had 14 weeks’ In summary, all at least 2 weeks’
exposure to the high intensity. UnilateraI Optic Nerve Section (UONS)
Rats were anesthetized with 0.40 cc per 100 g body weight of a mixture of 3.9 g chloral hydrate, 1.625 g pentobarbital and 3.9 g magnesium sulfate in a volume of 102.8 ml containing ethanol, propylene glycol and water. A ventral approach to the optic nerve was used because it results in IittIe or no trauma to neural tissue other than the optic nerve. For a detailed description of the surgery see Bush and Williams (199 1). Briefly, the rat was placed on its back on a surgical board and tissue covering both the hard and soft palates was removed. The exposed bone of the hard palate was cut away using a dental drill and a 1.5 mm round burr exposing the crania-pharyngeal canal as well as the presphenoid bone on the ventral
Exposure(weeks) 0
2 14 16
Group
__.---~
3 Ix natives 600 Ix immigrants 3 lx immigrants 600 lx natives
surface of the skull. A hole was drilled through the presphenoid bone exposing the optic nerves. If the dura was not penetrated by the drill, it was cut with a needle or sharp hook. One nerve was grasped with a
blunt hook (0.5 mm diameter) and was completely transected by the removal of a small section of the
PHOTORECEPTOR
PLASTICITY TABLE
395
II
Dark-adapted rhodopsin levels* for all groups Group
Sham
Intact
3 lx native 3 Ix immigrant 600 Ix native 600 lx immigrant
1.12fO.03 0~75+0~05 04 3 + 0.03 0.32 +0.04
l.Ol&-0.05 0.69+0.05 0.40 & 0.06 0.30+_0.03
*
arereportedasmolecules x 10 I’ perretina.
Mean
-f:S.F.M.
Vahcs
Sectioned 1.03+0.04 0.53$0.04 0.2 5 f 0.04 0.22+_0.01
was rinsed in buffer and dehydrated in a graded series of ethanol. Once dehydration was completed, the tissue was infiltrated with JB-4 embedding medium (Polysciences, Inc.) and embedded in JB-4/catalyst media. Four-micrometer thick sections were taken through the vertical meridian of each eye (centre of the optic disc) and were stained either with Sudan Black B (Allied Chemicals, Morristown, NJ) or hematoxylin and eosin (Polysciences, Inc.). Sudan Black B is an excellent lipophilic dye which stains ROS very well; it was used at a concentration of 0.3 g per 100 ml 70% EtOH for 40 set to 1 min and then cleared with xylene. Using a Zeiss standard microscope ( x 10 ocular) and a x 40 objective, the thickness of the ONL was measured with an ocular micrometer. The average thickness was used as an estimate of photoreceptor cell number. Beginning at the optic nerve, measurements were taken every 250 pm along both the inferior and superior halves of the retina. In a similar manner, ROS length measurements were taken using a x 100 objective with oil. The micrometer was positioned from baseto tip of the outer segmentsand followed the
angle of the outer segments to get an accurate length measurement. No oblique sectionswere usedfor either
measurement. 0.0
0.2
0.4
0.6 t?, fmolecules
0.8
I.0
I.2
I.4
x 10mi5)
Dark-adaptedRhodopsinLevels
FIG. 2. Dark-adapted whole-eye rhodopsin content of sectionedeye (K,) vs. that of intact eye(Ri) of sameanimal. Eachpoint on the graph representsone animal. If pointslie on the line, there are equal amountsof rhodopsinin each eye.Most points lie abovethe line indicating that the intact eye has more rhodopsinthan doesthe sectionedeye (P < 0.05). As expectedfrom earlier work, there is no significant difference between R, and R, of the 3 Ix native group. (A) 600 lx native: ( q ) 600 Ix immigrant: ( n ) 3 Ix native: (0) 3 Ix immigrant.
light. The animals were killed between 0800 and 1000 hr using CO, asphyxiation. Then rhodopsin was extracted as described elsewhere (Fulton et al., 1990).
nerve. The hole in the skull was closed using bone wax
was discarded and 0.7 ml of 1 “/” CTAB (cetyltri-
(Ethicon). To keep the air passageopen, an 8 mm to
methylammonium bromide, Sigma) was added to each pellet. The pellets were disrupted and mixed with the detergent so as to solubilize the ROS membranes and
Rats were dark-adapted for 16 hr prior to the assay and all subsequent procedures were done in dim red
Briefly, retinas were extruded through a slit in the
cornea and put into approximately
8 ml of distilled
water. Each retina was disrupted with a spatula and
centrifuged at 12 000 g for 15 min. The supernatant
1 cm length of Intramedic PE tubing (outer diameter 2.08 mm, inner diameter 1.57 mm) was inserted into the crania-pharyngeal canal. The palate was then repaired with bone wax. The completeness of the surgery was confirmed by observing the absence of the pupillary response and later by necropsy. Sham operations consisted of penetration of the palate and insertion of the PE tubing. Histology Histology was performed using the technique
reported by Penn, Howard and Williams (198 5). Rats were removed from their home cages and asphyxiated with CO,. Eyes were enucleated and fixed in a preparation of 1’2: paraformaldehyde and 2.5 % glutar-
aldehyde in 2 M Pipes at pH 7.2. After the eyes were fixed, both the lens and nasal portion of each eyecup were removed. The remaining portion of each eyecup
were centrifuged again for 15 min. Extraction volumes were measured and each supernatant was scanned
from 350 to 700 nm using an HP-8452A Diode Array Spectrophotometer to generate an absorbance spectrum before and after complete bleaching. Once a difference spectrum was obtained, rhodopsin concentration was calculated using the Beer-Lambert equation, assuming an absorbance coefficient of 42 000 K’ cm-’ at h max. This concentration was then converted to units of molecules per eye. Statistical Analysis Both Student’s t-tests and paired t-tests were performed using the data from the rhodopsin. ONL and ROS length measurements. Average rhodopsin levels shown in Table II were not used in calculating
J.-L.
396
significance for intact vs. sectioned eyes. Kather, the rhodopsin data in Fig. 2 were used for this comparison using paired t-tests. The level of significance was set at 0.05 for rhodopsin measurements and 0.01 for ONI, and ROS measurements.
3. Results There was no significant difference in rhodopsin content between sham and intact eyes of all groups (Table II); therefore, within each animal the eye with the optic nerve intact is used as a control for the eye with the optic nerve sectioned. For all three subgroups. sham, sectioned and intact, rhodopsin content of the retina in the immigrant groups is up-regulated when the animal is moved from a high-intensity light (600 lx) to a low-intensity light (3 lx) and is downregulated when the animal is moved from a lowintensity light to a high-intensity one. Dark-adapted rhodopsin levels are also significantly reduced (P < 0.01) in the high light intensity natives compared to those of the low intensity natives (Table II). The results given in Fig. 2 show that the retina’s response to optic nerve section depends upon the lighting environment to which the retina had been exposed. The sectioned eye had significantly less(P < 0.05) rhodopsin than did the intact eye in the same animal in the following lighting regimes: 3 lx immigrants, 600 lx immigrants and 600 lx natives (Fig. 2). Only in the 3 lx native case were the rhodopsin values the same [intact 1.02 (+ 0.04) x lOI and sectioned 1.03 * (0.04) x 101” (mean? S.E.M.)]. in agreement with Bush, Young and Williams (198 7). No ROS length differences were found within a given lighting environment among sham, sectioned or intact eyes. However, if lighting was changed, ROS
(B)
(A)
2.5:fold 1.7:fold
decrease
increase
600
Ix
native 13.4
+ I.2 ,unl
i
FIG. 3. Average rod outer segmentlengthsin micronsfor all groups (meank~.~.~.). A, Rod outer segment lengths decrease when the animal is moved to i higher intensity of
light, from 3 to 600 lx. B, Rod outer segment lengths increase when the animal light, from 600 to 3 lx.
is moved to a lower intensity
of
SCHREMSER
AND
T. P. WILLIAMS
lengths changed inversely with intensity. r.,gardless of whether the optic nerve was intact or :*\c +-el (Fig. 3). When animals were switched from :X ii, : ? lx. ROS length increased 1.7-fold. When animals were switched from 3 to 600 lx, ROS length decreased2.5fold. Average ROS length in /r.m for each lighting environment is shown in Fig. 3. Cell number, as measured by ONL thickness, was not significantly different among sham, sectioned or intact eyes for each lighting regime. Differences did arise between 3 lx natives and all other lighting regimes. Three lx immigrants, 600 lx immigrants and 600 Ix natives, all of which had at least 2 weeks’ exposure to 600 lx (Table I), had statistically the same number of cells. Average ONL thickness (mm) for these three groups was 0.024 kO.003 mm (mean + S.D.), which was significantly less(P < 0.01) than that found in the 3 Ix native group. 0.034 _+0.001 mm (mean * s.n.). 4. Discussion In the 3 lx native group (low intensity for their entire lifetime) the optic nerve sectioned retinas and the optic nerve intact retinas have equal rhodopsin content. In all other groups, the retinas with the optic nerve sectioned maintained lower rhodopsin concentrations than did those with intact optic nerves (Fig. 2). One explanation for why all retinas with a sectioned optic nerve, except 3 lx natives, have lessrhodopsin is that only those eyes that had been exposed to highintensty light, 600 lx, for at least 2 weeks (Table I) were challenged by that intensity and responded by partial regulation of rhodopsin. The 3 lx native animals were never required to readjust and perhaps this is how they are able to maintain their high levels in both eyes. That the regulation of rhodopsin levels was incomplete clearly shows that the regulatory mechanism is altered by ONS. Some ideas about how it may be altered will be discussedbelow. Note that even though the sectioned eye has less rhodopsin it still follows the same general pattern of regulation that a sham or non-surgery eye does (Williams et al., 1988). The sectioned eye is able to upregulate its rhodopsin content when moved from a high-intensity light to a low-intensity light (seeTable II and Fig. 2, 600 lx native vs. 3 lx immigrant); however. not to the same extent as occurs in a nonsurgical control animal (Williams et al., 1988). The sectioned eye is also able to down-regulate its rhodopsin content when moved from a low- to a highintensity light (see Table II and Fig. 2, 3 Ix native vs. 600 lx immigrant). In this case, however, the sectioned eye down-regulates to a level lower than anticipated, based on measurements for a 600 lx native. Given these results, we speculate that efferent input is involved in making fine adjustments in adaptation
PHOTORECEPTOR
PLASTICITY
of rhodopsin content to a new lighting environment but coarse adjustments to a new environment are controlled locally. Another possibility is that cutting the efferent input to the retina may upset the balance between up- and down-regulation of rhodopsin and deregulate the system so that it would resort to a default mode of down-regulation when challenged by light. A third possible explanation is that all regulation is local and that the efferent pathway is not involved. If this were the case, optic nerve section may disrupt normal local regulation not through interruption of efferent pathways but possibly through intra-retinal release of growth factors (Faktorovich et al., 1990). This last possibility is problematic since one effect of basic iibroblast growth factor (bFGF), the growth factor used in the Faktorovich et al. (1990) study, is to stabilize RNA and therefore contribute to an increase in translational activity and ultimately increased protein synthesis (Zeytin et al., 1988). Since it may be expected that growth factors would be released in the retina with the optic nerve sectioned, one may hypothesize that protein synthesis would increase. However, as regards rhodopsin, we find the opposite result, the retina with the optic nerve sectioned has less of the pigment. A role for growth factors cannot be fully excluded, however, given their diverse actions in other systems. The sectioned eye was able to regulate ROS length in the same way as did the intact eye, indicating that control of length changes may be a local retinal event. ROS disc shedding, which is one factor involved in modifying ROS length, has been shown to be both locally and centrally controlled (Teirstein et al., 1980). IaVail ( 19 76) demonstrated that ROS disc shedding follows a circadian rhythm. The regulatory mechanism of the disc shedding rhythm was thought to involve the pineal gland, humoral factors or be endogenous to the eye itself. LaVail (19 76) showed that the pineal gland may be involved by making reserpine injections into rats and finding that the burst of disc shedding was blocked. Reserpine abolishes some circadian rhythms by depletion of noradrenaline in the afferent nerve terminals in the pineal gland (Axelrod and Zatz, 1977). Later studies (Tamai et al., 1978: LaVail and Ward, 1978) showed that pinealectomy had no effect on disc shedding rhythms in rat. thereby eliminating the possible role of the pineal gland in the regulatory mechanism. Teirstein et al. (I 980) demonstrated, using monocularly occluded rats, that humoral factors, which would be expected to affect both eyes, cannot be responsible for the direct regulation of ROS disc shedding. The regulatory mechanism may therefore be endogenous to the eye. The data of the present study support the hypothesis that the disc shedding regulatory mechanism is endogenous to the eye since the ability to change ROS length was not affected by IJONS. This study as well as others (Battelle and LaVail, 1978 : Penn and Williams. 1986 ; Penn and Anderson,
397
1987; Williams et al., 1988) clearly shows that ROS Iength is not a constant in the rat eye if the animal is exposed to an intensity different from that to which it is accustomed. To summarize, ROS length is inversely related to habitat illuminance. This dynamic quality of the photoreceptor rod outer segments allows it to be a vehicle for adaptation to a new lighting environment (Williams et al., 1988). We tend to favor the hypothesis that fine adjustments in adaptation are controlled through efferent input. There is growing evidence that the loss of the efferent pathway may change the neurochemistry of the retina (for review see Bush and Williams, 1991). These efferent fibers which are thought to be GABAergic (Ohno, 1980) tonically inhibit dopaminergic (DA) neurons of the retina (Kamp and Morgan 1981: Marshburn and Iuvone, 1981). There is a category of amacrine cells located in the inner nuclear layer of the retina which is dopaminergic. Cutting the optic nerve which contains these efferent fibers may disinhibit the dopaminergic neurons and increase dopamine release, For example, a reduction in DA receptor binding in the rat retina has been shown to occur following a lesion of the pretectum, a potential site of centrifugal fiber origin (Itaya and Itaya, 1984 ; Plummer, Harris and Phillipson, 1986). This reduction in receptor binding may reflect an increase in dopamine release. Dopamine receptors have been found on photoreceptor cells (Brann and jelsema, 1985; Tran and Zhu, 1991). Changes in levels of dopamine may therefore influence second-messenger systems in the photoreceptors via dopamine receptors. We speculate that these second-messenger systems could affect gene expression and thereby change opsin content of the retina. As dopamine functions as a neuromodulator in the retina, many changes in related receptor systems and other transmitters may occur in relation to the alterations in retinal dopamine levels. These changes in dopamine and other transmitters have been shown to affect the physiology of the retinal cells. specifically photoreceptor cells (Pierce and Besharse. 198 5 ; Dearry and Burnside, 1986; Eder, Drake and Williams, 1991). The alteration in the physiology of the photoreceptors could be the factor linking optic nerve section to altered rhodopsin regulation. In invertebrates, both structure and physiology of the photoreceptors are affected following ONS (Fleissner and Fleissner, 1978; Barlow et al., 1980; Yamashita and Teteda, 1981). IJsing the albino rat as a model we have found that UONS may influence the physiology of vertebrate photoreceptors. In conclusion, the ability of &heoptic nerve sectioned eye to regulate rhodopsin levels is altered but not lost. The retina with the optic nerve sectioned is not able to up-regulate rhodopsin levels fully compared to the companion retina with an intact optic nerve. The ONS retina down-regulates rhodopsin more than does the intact optic nerve retina. It is possible that both local
J.-L.
and central regulatory mechanisms may play roles in the overall regulation of rhodopsin levels: the former regulating coarse adaptations and the latter regulating tine adaptations.
Acknowledgement This work was supported
by NIH grant ROI EY07753
References Axelrod, J. and Zatz, M. (1977). The p-adrenergic receptor and the regulation of circadian rhythms in the pineal gland. In Biochemicul Actions of Hormones. Vol. IV [Ed. Litwack, G.). Academic Press: New York, U.S.A. Barlow, R. B. Jr (1983). Circadian rhythms in the Limulus visual system. 1. Neurosci. 3, 856-70. Barlow. R. B. Jr. Chamberlain, S. C. and Levinson. J. 2. (1980). Limulus brain modulates the structure and function of the lateral eyes. Science 210, 1037-g. Barlow, R. B. Jr, Kaplan. E., Renninger. G. H. and Saito. T. ( 198 5). Efferent control of circadian rhythms in the Limulus lateral eye. Neurosci. Res. 2 (Suppl.), S65-S78. Battelle. B.-A. and LaVail, M. M. (19 78). Rhodopsin content and rod outer segment length in albino rat eyes: modification by dark adaptation. Exp. Eye Res. 26. 487-97. Brann. M. R. and Jelsema, C. L. (198 5 ). Dopamine receptors on photoreceptor membranes couple to a GTP-binding protein which is sensitive to both pertussis and cholera toxin. Biochem. Biophys. Res. Commun. 133. 222-7. Brooke, R. N. L., deDowner. D. J. and Powell, T. P. S. (1965). Centrifugal fibers to the retina in the monkey and cat. Nature 207. 1365-7. Bush, R. A. and Williams, T. P. (1991). The effect of unilateral optic nerve section on retinal light damage in rats. Exp. Eye Res. 52, 139-53. Bush. R. A., Young, C. T. and Williams, T. P. (1987). Rhodopsin decreases in rat retina following optic nerve section. Invest. Ophthalmol. Vis. Sci. 28 (Suppl.), 142. Chamberlain, S. C. and Barlow, R. B. (1979). Light and efferent activity control rhabdom turnover in Limulus photoreceptors. Science 206, 361-3. Dearry, A. and Burnside, B. (1986). Dopaminergic regulation of cone retinomotor movement. I. Induction of cone contraction is mediated by D2 receptors. 1. Neurochem. 46, 1022-3 I. Drager. U. C.. Edwards, D. I,. and Barnstable. C. J. (1984). Antibodies against filamentous components in discrete cell types of the mouse retina. 1. Neurosci. 4, 202 542. Eder, D. J., Drake, M. A. and Williams, T. P. (1991). Compression of the photoreceptor cell responsivity in the isolated rat retina by physiological concentrations of dopamine. Invest. Ophthalmol. Vis. Sci. 32 (Suppl.). 1260. Faktorovich, E. G., Steinberg, R. H., Yasumura. D., Matthes. M. T. and LaVail, M. M. (1990). Photoreceptor degeneration in inherited retinal dystrophy delayed by basic fibroblast growth factor. Nature 347. 83-6. Fleissner. G. and Fleissner. G. (1978). The optic nerve mediates the circadian pigment migration in the median eyes of the scorpion. Camp. Biochem. Physiol. 61A. 69-71. F&on, A. B., Dodge, J., Schremser, J.-L.. Armstrong, A.. Lanier, F., Dawson, W. W. and Williams. T. P. (1990). The quantity of rhodopsin in human eyes. Curr. Eye Res. 9, 1211-6.
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Hoenig, J. and Chamberlain, S. C. (1989). Testing the ‘photostasis’ hypothesis in the Limufus lateral eye. Invest. Ophthalmol. Vis. Sci. 30 (Suppl.), 291. Honrubia, F. M. and Elliot, J. H. (1968). merent innervation of the retina: morphologic study of the human retina. Arch. Ophthalmol. 80. 98-103. Hoogland, P. V., Vanderkrans, A., Koole, F. D. and Groenefrom the wegen, H. J, (1985). A direct projection nucleus oculomotorius to the retina in rats. .Neurosci. Lett. 56. 323-8. Itaya. S. K. (I 980). Retinal efferents from the pretectai area in the rat. Brain Res. 201, 436-41. Itaya. S. K. and Itaya, P. W. (1984). Centrifugal fibers to the rat eye from the medial pretectal area and locus coeruleus. Anat. Rec. 208, 79A. Kamp. C. W. and Morgan, W. W. (198 1). GABA antagonists enhance dopamine turnover in the rat retina in viva. Eur. JQPharmacol. 69. 2i3-9. LaVail, M. M. (1976). Rod outer segment disk shedding in rat retina : relationship to cyclic lighting. Science 194. 1071-4. LaVail. M. M. and Ward, P. A. (1978). Studies on the hormonal control of circadian outer segment disc shedding in the rat retina. Invest. Ophthalmol. Vis. SC-i. 17. 1189-92. Marshburn. P. B. and Iuvone. P. M. ( 198 1). The role of GABA in the regulation of the dopamine/tyrosine hydroxylase-containing neurons of the rat. Brain Res. 214. 335-47. Molotchnikoff. S. and Tremblay, F. (1986). Visual cortex controls retinal output in the rat. Brain Res. Bull. 17. 21-32. Muller, M. and Hoflander. H. (1988). A small population of retinal ganglion cells projecting to the retina of the other eye. Exp. Brain Res. 71, 611-17. Ohno, T. (1980). The possibility of centrifugal projections to the retina in the rat. Experientia 36, 1400-l. Organisciak. D. T. and Noell, W. K. (1977). The rod outer segment phospholipid/opsin ratio of rats maintained in darkness or cyclic light. Invest. Ophthalmol. Vis. Sri. J6, 188-90. Penn, 1. S. and Anderson, K. E. ( 1987). Effect of light history on rod outer segment membrane composition in the rat. Exp. Eye Res. 44, 767-78. Penn. J. S.. Howard, A. G. and Williams. T. P. ( 1985). Light damage as a function of ’ light history ’ in the albino rat. In Retinal Degeneration : Experimental and Clinical Studies. Pp. 439-47. Alan R. Liss. Inc.: New York. Penn. J. S. and Williams, T. P. (1986). Photostasis: regulation of daily photon catch by rat retinas in response to various cyclic Iluminances. Exp. Ege Res. 43, 915-28. Pierce, M. E. and Besharse, J. C. (198 5). Circadian regulation of retinomotor movements. I. Interaction of melatonin and dopamine in the control of cone length. 1. Gen. Physiol. 86. 671-89. Plummer, C. J., Harris, J. P. and Phillipson, 0. T. (1986). Modification of retinal dopamine receptor binding by lesions of the tecto-retinal pathway in the rat. 1. PhHsiol. (Land.) 381. 52. Polyak, S. (1941). The Retina Pp. 328-42. The Ilniversity of Chicago Press : Chicago. Tamai, M., Teirstein. P.. Goldman, A., O’Brien, P. and Chader. G. (1978). The pineal gland does not control rod outer segment shedding and phagocytosis in the rat retina and pigment epithelium. Invest. Ophthalmol. Vis.
Sri. 17. 558-62. Teirstein, P.. Goldman, A. and O’Brien, P. (1980). Evidence for both local and central regulation of rat rod outer segment shedding. Invesr. Ophthalmol. Vis. Sci. 19. 1268-73. Tolman. B. L.. Koutz, C. A. and Kapp, L. M. (1989). Rod
PHOTORECEPTOR
PLASTICITY
outer segment diameter in rats increases with age. Invest. OphthaZmoZ. Vis. Sci. 30 (Suppl.), 294. Tran. V. T. and Zhu. N. L. (1991). Molecular characterization of dopamine D, receptors in the rat retina. Invest. Ophthalmol. Vis. Sci. 32 (Suppl.), 1260. Tsang, D.. Yew, 1). T. and Lam, S. T. L. (1985). Acute responses of rat retina after optic nerve ligation: a biochemical and histochemical study. Brain Res. 336. 289-95. Williams. T. P., Penn, J. S.. Bush. R. A. and Makino, C. L. (198X). Kenewal of rod outer segments and regulation
399
of daily photon-catch by the rat retina. In Proceedings of the Yamada Conference. Pp. 2 55-60. Yamada Science Foundation : Kyoto, Japan. Yamashita, S. and Teteda, H. (1981). FZferent neural control in the eyes of orb-weaving spiders. J. Comp. Physiol. 143.477-83. Zeytin. F. N., Rusk, S. F., Raymont, V. and Mandell, A. J. (1988). Fibroblast growth factor stabilizes ribonucleic acid and regulates differentiated functions in a multipeptide-secreting neuroendocrine cell line. Endocrinology 122, 1121-S.
Photoreceptor
Plasticity Unilateral
JERI-LYNN Institute
of Molecular
(Received
SCHREMSER
Biophysics,
Houston
in the Albino Optic Nerve
15 July
AND
Rat Retina Section
THEODORE
Florida State University, 1997 and accepted
Following
P. WILLIAMS
Tallahassee,
FL 32303-3015,
in revised form 14 November
U.S.A.
79?!)
Rhodopsin content of the retina increased when an albino rat was moved to a lower intensity cyclic light, and decreased when moved to a higher intensity. Unilateral optic nerve section was employed to test if intact optic nerves are necessary to maintain regulation of rhodopsin when albino rats are switched between two intensities, 3 lx and 600 lx (12 hr/12 hr. cyclic light). The ability to regulate rhodopsin content was altered but not lost in the eye which had the optic nerve transected. This alteration was in the direction of incomplete up-regulation when the intensity change was from high to low, and was in the direction of excessive down-regulation when the intensity change was from low to high. This indicates that efferent fibers from the brain to the retina may be involved in the regulation of rhodopsin content. Using histological techniques, cell number and rod outer segment (ROS) length were measured: surgery alone had no effect on ROS length or outer nuclear layer thickness and in all cases ROS lengths were inversely related to habitat illuminance. Key words: rhodopsin : rat : rod photoreceptors : optic nerve : plasticity.
1. Introduction
Optic Nerve and Retina
Regulation of Light-absorption
In addition to the well-characterized afferent fibers which transfer both visual and non-visual information to the brain, the optic nerve of a number of species has been shown to contain efferent fibers. There is evidence for centrifugal fibers to the retina in invertebrates (Chamberlain and Barlow, 1979), in rats (Itaya, 1980; Ohno, 1980; Drager et al., 1984: ltaya and Itaya, 1984; Hoogland et al., 1985; Muller and Hollander, 1988), and in other vertebrate species including primates (Polyak, 1941; Brooke et al., 1965) and humans (Honrubia and Elliot, 1968). It appears that these centrifugal fibers are not abundant, constituting less than 2% of the optic nerve fibers in the rat (Molotchnikoff and Tremblay, 1986) ; however, there is evidence indicating that these fibers have
Light history, the record of all light exposure an animal receives throughout its lifetime, is a contributing factor to the regulation of photon-catch. Photon-catching ability in the rat is adjusted by changing rod outer segment (ROS) length, rod cell number, rhodopsin packing per disc, rod cell diameter, disc packing density and regeneration rate (Organisciak and Noell, 1977: Battelle and LaVail, 1978 ; Penn and Williams, 19 8 6 ; Penn and Anderson, 19 8 7 : Tolman, Koutz and Rapp, 1989). Animals raised in different intensities, therefore, have different light histories. Rod photoreceptor outer
segment length and rhodopsin content are regulated according to the light intensity of the environment within constraints determined by the animal’s light history (Battelle and LaVail, 1978 ; Penn and Williams,
1986: Williams et al., 1988). Photostasis is the regulation of photon-catch (Penn and Williams, 1986). This phenomenon derives its name from the observation that groups of albino rats raised in a range of cyclic intensities, 3-400 lx, will
absorb statistically the same number of photons per rat per day. This phenomenon has also been demonstrated in Limulus polyphemus(Hoenig and Chamberlain, 1989). The fact that photostasis occurs in such diverse speciesindicates that it may play a significant
role at a fundamental yet to be determined.
level. What this role may be is
* For correspondence. 00144835/92/090393+07
508.00/0
extensive arborizations and are involved in modu-
lation of retinal function. To observe the influence of efferent fibers on the retina, studies have been done in which the optic nerve has been ligated (Tsang, Yew and Lam, 1985) or transected (Barlow, Chamberlain and Levinson, 1980; Teirstein, Goldman and O’Brien, 1980; Barlow et al., 1985; Bush and Williams, 1991). For example, Barlow et al. (1980) observed in Limulus that when the efferent input to the retina is cut the circadian rhythms in both the structure and physiology of the retina were abolished. Teirstein et al. (1980) investigated whether the process of disc shedding is affected by optic nerve section and concluded that intact optic nerves are not necessary for maintenance of the shedding rhythm, but may be necessary to synchronize and phase shift the disc shedding rhythm. Bush 0 1992 AcademicPressLimited
J.-L.
394
31x nat ws
SCHREMSER
,
,
I
I
e---
1
3 Ix
AND
T. P. WILLIAMS
600 Ix natlves
I
immigrants
Fro. 1. Light-history paradigm.Four groups,named accordingto their lighting regimes:3 lx natives; 3 lx immigrants: 600 lx natives: and 600 Ix immigrants.Nativeswere thoseanimalsthat remainedin their rearing intensity and immigrants (arrows) were thoseanimalsthat were moved to the new intensity.
and Williams ( 199 1) found that retinas with cut optic nerves suffered substantially less damage from light than did those with intact optic nerves. Williams et al. (1988) demonstrated that rats have the ability to up- and down-regulate rhodopsin content when they are moved to a new intensity. To study another possible influence of efferent fibers on the retina we asked the question: can the retina of an optic nerve sectioned eye still adjust rod outer segment (ROS) length and rhodopsin content when an animal is moved to a new lighting intensity? 2. Materials and Methods
Sprague-Dawley albino rats (breeder pairs from Charles River, Wilmington, MA) were born, raised and maintained in our laboratory, in controlled cyclic lighting, 12 hr/12 hr, light/dark. Two light intensities were used : a low-intensity condition of 3 lx; and a high-intensity condition of 600 lx. Special chambers were designed and built so that illumination could be set for 600 lx and delivered evenly to all rats. Illuminance was measured using both a Tektronix J16 digital photometer with a J6501 illuminance probe and a United Detector Technology Mode 350 Optometer. All intensities are reported in units of lx. One-hundred and eight rats were used in this experiment according to the NIH guidelines for the care and use of animals. All animals were 12 weeks of age at the time of unilateral optic nerve section (UONS) or sham surgery and 16 weeks when killed. Following surgery the animals were returned to their original intensities for a recovery period of 2 weeks and then some were switched to a ‘new intensity ’ for 2 weeks and others remained in their original intensity (Fig. 1). Natives were those animals that remained in their rearing intensity throughout their lifetime and TABLE I
Time of exposureto high intensity (600 Ix)
immigrants were those animals that were moved from their rearing intensity to the ‘new intensity ’ for 2 weeks. At 16 weeks of age, dark-adapted whole-eye rhodopsin levels, rod outer segment (ROS) length and outer nuclear layer (ONL) thickness were measured. There were four groups, named according to their lighting regimes : 3 lx natives ; 3 lx immigrants : 600 lx natives ; and 600 lx immigrants (Fig. 1). Each of the four groups was divided into three subgroups: sham eyes ; sectioned eyes (those eyes which have a severed optic nerve); and intact eyes. Table I condenses the following information but since this is an essential point to understand we will describe each group in detail. Six-hundred lux natives were born and raised in 600 lx for 16 weeks while 3 lx natives were born and raised in 3 lx for 16 weeks. A 600 lx immigrant was an animal that was raised in 3 lx until 14 weeks of age, then switched to 600 lx for 2 weeks ; therefore, a 600 lx immigrant had 2 weeks’ exposure to the high intensity. Finally, a 3 lx immi-
grant was raised in 600 lx for 14 to 3 lx for 2 weeks ; therefore, exposure to the high intensity. animals except 3 lx natives had
weeks then moved it had 14 weeks’ In summary, all at least 2 weeks’
exposure to the high intensity. UnilateraI Optic Nerve Section (UONS)
Rats were anesthetized with 0.40 cc per 100 g body weight of a mixture of 3.9 g chloral hydrate, 1.625 g pentobarbital and 3.9 g magnesium sulfate in a volume of 102.8 ml containing ethanol, propylene glycol and water. A ventral approach to the optic nerve was used because it results in IittIe or no trauma to neural tissue other than the optic nerve. For a detailed description of the surgery see Bush and Williams (199 1). Briefly, the rat was placed on its back on a surgical board and tissue covering both the hard and soft palates was removed. The exposed bone of the hard palate was cut away using a dental drill and a 1.5 mm round burr exposing the crania-pharyngeal canal as well as the presphenoid bone on the ventral
Exposure(weeks) 0
2 14 16
Group
__.---~
3 Ix natives 600 Ix immigrants 3 lx immigrants 600 lx natives
surface of the skull. A hole was drilled through the presphenoid bone exposing the optic nerves. If the dura was not penetrated by the drill, it was cut with a needle or sharp hook. One nerve was grasped with a
blunt hook (0.5 mm diameter) and was completely transected by the removal of a small section of the
PHOTORECEPTOR
PLASTICITY TABLE
395
II
Dark-adapted rhodopsin levels* for all groups Group
Sham
Intact
3 lx native 3 Ix immigrant 600 Ix native 600 lx immigrant
1.12fO.03 0~75+0~05 04 3 + 0.03 0.32 +0.04
l.Ol&-0.05 0.69+0.05 0.40 & 0.06 0.30+_0.03
*
arereportedasmolecules x 10 I’ perretina.
Mean
-f:S.F.M.
Vahcs
Sectioned 1.03+0.04 0.53$0.04 0.2 5 f 0.04 0.22+_0.01
was rinsed in buffer and dehydrated in a graded series of ethanol. Once dehydration was completed, the tissue was infiltrated with JB-4 embedding medium (Polysciences, Inc.) and embedded in JB-4/catalyst media. Four-micrometer thick sections were taken through the vertical meridian of each eye (centre of the optic disc) and were stained either with Sudan Black B (Allied Chemicals, Morristown, NJ) or hematoxylin and eosin (Polysciences, Inc.). Sudan Black B is an excellent lipophilic dye which stains ROS very well; it was used at a concentration of 0.3 g per 100 ml 70% EtOH for 40 set to 1 min and then cleared with xylene. Using a Zeiss standard microscope ( x 10 ocular) and a x 40 objective, the thickness of the ONL was measured with an ocular micrometer. The average thickness was used as an estimate of photoreceptor cell number. Beginning at the optic nerve, measurements were taken every 250 pm along both the inferior and superior halves of the retina. In a similar manner, ROS length measurements were taken using a x 100 objective with oil. The micrometer was positioned from baseto tip of the outer segmentsand followed the
angle of the outer segments to get an accurate length measurement. No oblique sectionswere usedfor either
measurement. 0.0
0.2
0.4
0.6 t?, fmolecules
0.8
I.0
I.2
I.4
x 10mi5)
Dark-adaptedRhodopsinLevels
FIG. 2. Dark-adapted whole-eye rhodopsin content of sectionedeye (K,) vs. that of intact eye(Ri) of sameanimal. Eachpoint on the graph representsone animal. If pointslie on the line, there are equal amountsof rhodopsinin each eye.Most points lie abovethe line indicating that the intact eye has more rhodopsinthan doesthe sectionedeye (P < 0.05). As expectedfrom earlier work, there is no significant difference between R, and R, of the 3 Ix native group. (A) 600 lx native: ( q ) 600 Ix immigrant: ( n ) 3 Ix native: (0) 3 Ix immigrant.
light. The animals were killed between 0800 and 1000 hr using CO, asphyxiation. Then rhodopsin was extracted as described elsewhere (Fulton et al., 1990).
nerve. The hole in the skull was closed using bone wax
was discarded and 0.7 ml of 1 “/” CTAB (cetyltri-
(Ethicon). To keep the air passageopen, an 8 mm to
methylammonium bromide, Sigma) was added to each pellet. The pellets were disrupted and mixed with the detergent so as to solubilize the ROS membranes and
Rats were dark-adapted for 16 hr prior to the assay and all subsequent procedures were done in dim red
Briefly, retinas were extruded through a slit in the
cornea and put into approximately
8 ml of distilled
water. Each retina was disrupted with a spatula and
centrifuged at 12 000 g for 15 min. The supernatant
1 cm length of Intramedic PE tubing (outer diameter 2.08 mm, inner diameter 1.57 mm) was inserted into the crania-pharyngeal canal. The palate was then repaired with bone wax. The completeness of the surgery was confirmed by observing the absence of the pupillary response and later by necropsy. Sham operations consisted of penetration of the palate and insertion of the PE tubing. Histology Histology was performed using the technique
reported by Penn, Howard and Williams (198 5). Rats were removed from their home cages and asphyxiated with CO,. Eyes were enucleated and fixed in a preparation of 1’2: paraformaldehyde and 2.5 % glutar-
aldehyde in 2 M Pipes at pH 7.2. After the eyes were fixed, both the lens and nasal portion of each eyecup were removed. The remaining portion of each eyecup
were centrifuged again for 15 min. Extraction volumes were measured and each supernatant was scanned
from 350 to 700 nm using an HP-8452A Diode Array Spectrophotometer to generate an absorbance spectrum before and after complete bleaching. Once a difference spectrum was obtained, rhodopsin concentration was calculated using the Beer-Lambert equation, assuming an absorbance coefficient of 42 000 K’ cm-’ at h max. This concentration was then converted to units of molecules per eye. Statistical Analysis Both Student’s t-tests and paired t-tests were performed using the data from the rhodopsin. ONL and ROS length measurements. Average rhodopsin levels shown in Table II were not used in calculating
J.-L.
396
significance for intact vs. sectioned eyes. Kather, the rhodopsin data in Fig. 2 were used for this comparison using paired t-tests. The level of significance was set at 0.05 for rhodopsin measurements and 0.01 for ONI, and ROS measurements.
3. Results There was no significant difference in rhodopsin content between sham and intact eyes of all groups (Table II); therefore, within each animal the eye with the optic nerve intact is used as a control for the eye with the optic nerve sectioned. For all three subgroups. sham, sectioned and intact, rhodopsin content of the retina in the immigrant groups is up-regulated when the animal is moved from a high-intensity light (600 lx) to a low-intensity light (3 lx) and is downregulated when the animal is moved from a lowintensity light to a high-intensity one. Dark-adapted rhodopsin levels are also significantly reduced (P < 0.01) in the high light intensity natives compared to those of the low intensity natives (Table II). The results given in Fig. 2 show that the retina’s response to optic nerve section depends upon the lighting environment to which the retina had been exposed. The sectioned eye had significantly less(P < 0.05) rhodopsin than did the intact eye in the same animal in the following lighting regimes: 3 lx immigrants, 600 lx immigrants and 600 lx natives (Fig. 2). Only in the 3 lx native case were the rhodopsin values the same [intact 1.02 (+ 0.04) x lOI and sectioned 1.03 * (0.04) x 101” (mean? S.E.M.)]. in agreement with Bush, Young and Williams (198 7). No ROS length differences were found within a given lighting environment among sham, sectioned or intact eyes. However, if lighting was changed, ROS
(B)
(A)
2.5:fold 1.7:fold
decrease
increase
600
Ix
native 13.4
+ I.2 ,unl
i
FIG. 3. Average rod outer segmentlengthsin micronsfor all groups (meank~.~.~.). A, Rod outer segment lengths decrease when the animal is moved to i higher intensity of
light, from 3 to 600 lx. B, Rod outer segment lengths increase when the animal light, from 600 to 3 lx.
is moved to a lower intensity
of
SCHREMSER
AND
T. P. WILLIAMS
lengths changed inversely with intensity. r.,gardless of whether the optic nerve was intact or :*\c +-el (Fig. 3). When animals were switched from :X ii, : ? lx. ROS length increased 1.7-fold. When animals were switched from 3 to 600 lx, ROS length decreased2.5fold. Average ROS length in /r.m for each lighting environment is shown in Fig. 3. Cell number, as measured by ONL thickness, was not significantly different among sham, sectioned or intact eyes for each lighting regime. Differences did arise between 3 lx natives and all other lighting regimes. Three lx immigrants, 600 lx immigrants and 600 Ix natives, all of which had at least 2 weeks’ exposure to 600 lx (Table I), had statistically the same number of cells. Average ONL thickness (mm) for these three groups was 0.024 kO.003 mm (mean + S.D.), which was significantly less(P < 0.01) than that found in the 3 Ix native group. 0.034 _+0.001 mm (mean * s.n.). 4. Discussion In the 3 lx native group (low intensity for their entire lifetime) the optic nerve sectioned retinas and the optic nerve intact retinas have equal rhodopsin content. In all other groups, the retinas with the optic nerve sectioned maintained lower rhodopsin concentrations than did those with intact optic nerves (Fig. 2). One explanation for why all retinas with a sectioned optic nerve, except 3 lx natives, have lessrhodopsin is that only those eyes that had been exposed to highintensty light, 600 lx, for at least 2 weeks (Table I) were challenged by that intensity and responded by partial regulation of rhodopsin. The 3 lx native animals were never required to readjust and perhaps this is how they are able to maintain their high levels in both eyes. That the regulation of rhodopsin levels was incomplete clearly shows that the regulatory mechanism is altered by ONS. Some ideas about how it may be altered will be discussedbelow. Note that even though the sectioned eye has less rhodopsin it still follows the same general pattern of regulation that a sham or non-surgery eye does (Williams et al., 1988). The sectioned eye is able to upregulate its rhodopsin content when moved from a high-intensity light to a low-intensity light (seeTable II and Fig. 2, 600 lx native vs. 3 lx immigrant); however. not to the same extent as occurs in a nonsurgical control animal (Williams et al., 1988). The sectioned eye is also able to down-regulate its rhodopsin content when moved from a low- to a highintensity light (see Table II and Fig. 2, 3 Ix native vs. 600 lx immigrant). In this case, however, the sectioned eye down-regulates to a level lower than anticipated, based on measurements for a 600 lx native. Given these results, we speculate that efferent input is involved in making fine adjustments in adaptation
PHOTORECEPTOR
PLASTICITY
of rhodopsin content to a new lighting environment but coarse adjustments to a new environment are controlled locally. Another possibility is that cutting the efferent input to the retina may upset the balance between up- and down-regulation of rhodopsin and deregulate the system so that it would resort to a default mode of down-regulation when challenged by light. A third possible explanation is that all regulation is local and that the efferent pathway is not involved. If this were the case, optic nerve section may disrupt normal local regulation not through interruption of efferent pathways but possibly through intra-retinal release of growth factors (Faktorovich et al., 1990). This last possibility is problematic since one effect of basic iibroblast growth factor (bFGF), the growth factor used in the Faktorovich et al. (1990) study, is to stabilize RNA and therefore contribute to an increase in translational activity and ultimately increased protein synthesis (Zeytin et al., 1988). Since it may be expected that growth factors would be released in the retina with the optic nerve sectioned, one may hypothesize that protein synthesis would increase. However, as regards rhodopsin, we find the opposite result, the retina with the optic nerve sectioned has less of the pigment. A role for growth factors cannot be fully excluded, however, given their diverse actions in other systems. The sectioned eye was able to regulate ROS length in the same way as did the intact eye, indicating that control of length changes may be a local retinal event. ROS disc shedding, which is one factor involved in modifying ROS length, has been shown to be both locally and centrally controlled (Teirstein et al., 1980). IaVail ( 19 76) demonstrated that ROS disc shedding follows a circadian rhythm. The regulatory mechanism of the disc shedding rhythm was thought to involve the pineal gland, humoral factors or be endogenous to the eye itself. LaVail (19 76) showed that the pineal gland may be involved by making reserpine injections into rats and finding that the burst of disc shedding was blocked. Reserpine abolishes some circadian rhythms by depletion of noradrenaline in the afferent nerve terminals in the pineal gland (Axelrod and Zatz, 1977). Later studies (Tamai et al., 1978: LaVail and Ward, 1978) showed that pinealectomy had no effect on disc shedding rhythms in rat. thereby eliminating the possible role of the pineal gland in the regulatory mechanism. Teirstein et al. (I 980) demonstrated, using monocularly occluded rats, that humoral factors, which would be expected to affect both eyes, cannot be responsible for the direct regulation of ROS disc shedding. The regulatory mechanism may therefore be endogenous to the eye. The data of the present study support the hypothesis that the disc shedding regulatory mechanism is endogenous to the eye since the ability to change ROS length was not affected by IJONS. This study as well as others (Battelle and LaVail, 1978 : Penn and Williams. 1986 ; Penn and Anderson,
397
1987; Williams et al., 1988) clearly shows that ROS Iength is not a constant in the rat eye if the animal is exposed to an intensity different from that to which it is accustomed. To summarize, ROS length is inversely related to habitat illuminance. This dynamic quality of the photoreceptor rod outer segments allows it to be a vehicle for adaptation to a new lighting environment (Williams et al., 1988). We tend to favor the hypothesis that fine adjustments in adaptation are controlled through efferent input. There is growing evidence that the loss of the efferent pathway may change the neurochemistry of the retina (for review see Bush and Williams, 1991). These efferent fibers which are thought to be GABAergic (Ohno, 1980) tonically inhibit dopaminergic (DA) neurons of the retina (Kamp and Morgan 1981: Marshburn and Iuvone, 1981). There is a category of amacrine cells located in the inner nuclear layer of the retina which is dopaminergic. Cutting the optic nerve which contains these efferent fibers may disinhibit the dopaminergic neurons and increase dopamine release, For example, a reduction in DA receptor binding in the rat retina has been shown to occur following a lesion of the pretectum, a potential site of centrifugal fiber origin (Itaya and Itaya, 1984 ; Plummer, Harris and Phillipson, 1986). This reduction in receptor binding may reflect an increase in dopamine release. Dopamine receptors have been found on photoreceptor cells (Brann and jelsema, 1985; Tran and Zhu, 1991). Changes in levels of dopamine may therefore influence second-messenger systems in the photoreceptors via dopamine receptors. We speculate that these second-messenger systems could affect gene expression and thereby change opsin content of the retina. As dopamine functions as a neuromodulator in the retina, many changes in related receptor systems and other transmitters may occur in relation to the alterations in retinal dopamine levels. These changes in dopamine and other transmitters have been shown to affect the physiology of the retinal cells. specifically photoreceptor cells (Pierce and Besharse. 198 5 ; Dearry and Burnside, 1986; Eder, Drake and Williams, 1991). The alteration in the physiology of the photoreceptors could be the factor linking optic nerve section to altered rhodopsin regulation. In invertebrates, both structure and physiology of the photoreceptors are affected following ONS (Fleissner and Fleissner, 1978; Barlow et al., 1980; Yamashita and Teteda, 1981). IJsing the albino rat as a model we have found that UONS may influence the physiology of vertebrate photoreceptors. In conclusion, the ability of &heoptic nerve sectioned eye to regulate rhodopsin levels is altered but not lost. The retina with the optic nerve sectioned is not able to up-regulate rhodopsin levels fully compared to the companion retina with an intact optic nerve. The ONS retina down-regulates rhodopsin more than does the intact optic nerve retina. It is possible that both local
J.-L.
and central regulatory mechanisms may play roles in the overall regulation of rhodopsin levels: the former regulating coarse adaptations and the latter regulating tine adaptations.
Acknowledgement This work was supported
by NIH grant ROI EY07753
References Axelrod, J. and Zatz, M. (1977). The p-adrenergic receptor and the regulation of circadian rhythms in the pineal gland. In Biochemicul Actions of Hormones. Vol. IV [Ed. Litwack, G.). Academic Press: New York, U.S.A. Barlow, R. B. Jr (1983). Circadian rhythms in the Limulus visual system. 1. Neurosci. 3, 856-70. Barlow. R. B. Jr. Chamberlain, S. C. and Levinson. J. 2. (1980). Limulus brain modulates the structure and function of the lateral eyes. Science 210, 1037-g. Barlow, R. B. Jr, Kaplan. E., Renninger. G. H. and Saito. T. ( 198 5). Efferent control of circadian rhythms in the Limulus lateral eye. Neurosci. Res. 2 (Suppl.), S65-S78. Battelle. B.-A. and LaVail, M. M. (19 78). Rhodopsin content and rod outer segment length in albino rat eyes: modification by dark adaptation. Exp. Eye Res. 26. 487-97. Brann. M. R. and Jelsema, C. L. (198 5 ). Dopamine receptors on photoreceptor membranes couple to a GTP-binding protein which is sensitive to both pertussis and cholera toxin. Biochem. Biophys. Res. Commun. 133. 222-7. Brooke, R. N. L., deDowner. D. J. and Powell, T. P. S. (1965). Centrifugal fibers to the retina in the monkey and cat. Nature 207. 1365-7. Bush, R. A. and Williams, T. P. (1991). The effect of unilateral optic nerve section on retinal light damage in rats. Exp. Eye Res. 52, 139-53. Bush. R. A., Young, C. T. and Williams, T. P. (1987). Rhodopsin decreases in rat retina following optic nerve section. Invest. Ophthalmol. Vis. Sci. 28 (Suppl.), 142. Chamberlain, S. C. and Barlow, R. B. (1979). Light and efferent activity control rhabdom turnover in Limulus photoreceptors. Science 206, 361-3. Dearry, A. and Burnside, B. (1986). Dopaminergic regulation of cone retinomotor movement. I. Induction of cone contraction is mediated by D2 receptors. 1. Neurochem. 46, 1022-3 I. Drager. U. C.. Edwards, D. I,. and Barnstable. C. J. (1984). Antibodies against filamentous components in discrete cell types of the mouse retina. 1. Neurosci. 4, 202 542. Eder, D. J., Drake, M. A. and Williams, T. P. (1991). Compression of the photoreceptor cell responsivity in the isolated rat retina by physiological concentrations of dopamine. Invest. Ophthalmol. Vis. Sci. 32 (Suppl.). 1260. Faktorovich, E. G., Steinberg, R. H., Yasumura. D., Matthes. M. T. and LaVail, M. M. (1990). Photoreceptor degeneration in inherited retinal dystrophy delayed by basic fibroblast growth factor. Nature 347. 83-6. Fleissner. G. and Fleissner. G. (1978). The optic nerve mediates the circadian pigment migration in the median eyes of the scorpion. Camp. Biochem. Physiol. 61A. 69-71. F&on, A. B., Dodge, J., Schremser, J.-L.. Armstrong, A.. Lanier, F., Dawson, W. W. and Williams. T. P. (1990). The quantity of rhodopsin in human eyes. Curr. Eye Res. 9, 1211-6.
SCHREMSER
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