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Rat retinal benzodiazepine receptors are controlled by visual cortical mechanisms
Neurochem. Int. Vol. 10, No. 2, pp. 179-184, 1987 Printed in Great Britain. All rights reserved
0197-0186/87 $3.00+0.00 © 1987PergamonJournals Ltd
RAT RETINAL BENZODIAZEPINE RECEPTORS ARE C O N T R O L L E D BY VISUAL CORTICAL MECHANISMS REINHARD SCHLIEBS, THOMAS ROTHE and VOLKER BIGL Paul Flechsig Institute for Brain Research, Department of Neurochemistry, Karl Marx University, Leipzig, G.D.R. (Received 21 May 1986; accepted 29 August 1986)
A~traet--[3H]flunitrazepam binding was assayed in retinae of 25-day-old rats subjected either to unilateral enucleation at day 15, to intracranial unilateral cutting of the optic nerve at day 17, or to unilateral ablation of the visual cortex at day 17 postnatally. Unilateral enucleation resulted in an enhanced [3H]flunitrazepambinding in the retina of the remaining eye by 23% (P < 0.002, two-tailed Student t-test) as compared to unoperated controls. In rats with one optic nerve cut shortly before the optic chiasm, benzodiazepine binding in the retina of the lesioned side was significantlyhigher by 20.4 + 7.6% (P < 0.02, N = 10, paired test) in comparison to that in the retina with the intact optic nerve. Unilateral visual cortex ablation resulted in a 13% decrease (P < 0.02) in [3H]flunitrazepambinding in the retina contralateral to the brain lesion. In the lesioned rats of all three groups, the retinal benzodiazepine receptors were no longer capable of being modified by light/dark adaptation as is observed in normal rats. Our data suggest that (i) rat retinal benzodiazepine receptors are under a control from the visual cortex, and (ii) the benzodiazepine receptors of both eyes seem to be mutually tuned, presumably via a cortico-retinal feedback loop and an interhemispheric cortico-cortical information transfer. Recently the presence of high-affinity brain-specific benzodiazepine binding sites in preparations of rat retinae has been demonstrated (Howell et al., 1979; Paul et al., 1980; Rothe et al., 1985; Schliebs et al., 1986). Most of the specific binding of [3H]flunitrazepam is localized to the inner layers of the retina (Young and Kuhar, 1979). Lesion experiments with monosodium glutamate have emphasized the association of GABAergic mechanisms with benzodiazepine binding and suggested a predominant neuronal localization of the benzodiazepine binding sites in the inner plexiform layer (Regan et al., 1981). There is now some evidence that benzodiazepine receptors in the retina are involved in physiological events of light processing: Systemic administration of sedative dosages of diazepam reduced the lightevoked increase in retinal dopamine turnover, and intraocular application of flunitrazepam produced a suppression of light-enhanced dopamine synthesis in rat retina (Kamp and Morgan, 1982). Dark adaptation resulted in an increase in the affinity of [3H]diazepam (Biggio et al., 1981) and [3H]flunitrazepam (Rothe et al., 1985) for its specific binding site in the retina compared to that found in
light-adapted rats. In 25-day-old rats with one eyelid sutured at day l0 postnatally, the binding of [3H]flunitrazepam in the retina of the open eye was decreased (Rothe et al., 1985). However, dark adaptation of these monocularly deprived rats did not result in any change of benzodiazepine binding in the retina of either eye. This finding, that the retinal benzodiazepine receptors lose their ability to be modified by light/dark adaptation after monocular deprivation, suggested a physiological coupling between both retinae (Rothe et aL, 1985). Despite somewhat contradictionary results, there are both anatomical and physiological observations for centrifugal fibres to the retina in various species (Ebbesson and Meyer, 1981; Fergusson et aL, 1978; Halpern et aL, 1976; Hoogland and Welker, 1981; Itaya, 1980; Kawamura et al., 1974; Marchiavafa, 1976; Reprrant and Gallego, 1976; Rep~rant et al., 1980; Vanegas and Essayag-Millan, 1973; van Hasselt, 1972; Vesselkin et al., 1980; Weiler, 1985; for review of data until 1972, see van Hasseit, 1972/73). More recently, Molotchnikoff and Tremblay (1983) convincingly demonstrated that in rat responses of retinal ganglion cells might be modified and controlled by the visual cortex. In view of our recent finding that retinal benzo*Address correspondence to: Dr Reinhard Schliebs, Paul Flechsig Institute for Brain Research, Department of diazepine receptors might be tuned via retinopetal Neurochemistry, Karl Marx University, Karl-Marx- mechanisms (Rothe et al., 1985) the present study Stildter-StraBe 50, DDR-7039 Leipzig, G.D.R. was undertaken to investigate this centrifugal control 179
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o f retinal b e n z o d i a z e p i n e receptors in m o r e detail. T h e r e f o r e , [3H]flunitrazepam b i n d i n g was m e a s u r e d in rat retinae o n e week after the visual i n f o r m a t i o n t r a n s f e r was unilaterally i n t e r r u p t e d at different levels (enucleation o f the eyeball; c u t t i n g o f the o p t i c nerve intracranially) in o r d e r to see w h e t h e r there is a f u n c t i o n a l a s s s o c i a t i o n o f the activity o f rat retinal b e n z o d i a z e p i n e r e c e p t o r s b e t w e e n b o t h eyes. Rem o v a l o f the visual c o r t e x was p e r f o r m e d in o r d e r to study w h e t h e r rat retinal b e n z o d i a z e p i n e r e c e p t o r s are c o n t r o l l e d by visual cortical m e c h a n i s m s . EXPERIMENTAL PROCEDURES
Materials
[N-methyi-3H]flunitrazepam, specific activity 3056GBq/ mmol, was obtained from the Radiochemical Centre, Amersham, U.K., and used without further purification. Analytical grade chlordiazepoxide was a gift from VEB Arzneimittelwerk, Dresden, G.D.R. Treatment o f animals
Rats of either sex (strain BD III) reared under normal animal house conditions and under a 12 h light/dark cycle were used. Unilateral eyeball enucleation was performed under a light ether anaesthesia at the age of 15 days postnatally. Unoperated animals were used as controls. For lesion experiments, 17-day-old rats were anaesthetized with Nembutal (Nembutal sodium solution, Abbott Lab., Chicago, 40 mg/kg body weight) and placed in a K o p f stereotaxic apparatus. The skull was opened with a dental drill and unilateral optic nerve lesion and visual cortex ablation, respectively, were performed as follows: The right optic nerve was cut intracranially a few millimeters before reaching the optic chiasm by lowering a thin spatula 3~4 mm in front of the bregrna (i.e. the point where the midsagital suture crosses the suture between the frontal and parietal os of the skull) and 1 mm lateral to the midline to the base of the brain. Successful cutting of the optic nerve was indicated by a permanent enlargement of the pupil of the lesioned side. Lesions of the optic nerve at this level do not interfere with the retinal blood supply as the main retinal artery (arteria centralis retinae) meets the optic nerve but extracranially. Previous histological controls showed that after one week of survival light microscopically the retina appears normal. After a survival time long enough to allow for retrograde degeneration the ganglion cell layer has disappeared (see Wenk et al., 1981). Rats were killed by decapitation at the age of 25 days and the results of the lesion evaluated by visual inspection. Only those animals with a complete dissection of the optic nerve of one side leaving intact the other pathway were included in the biochemical analyses. Some control animals were sham operated by lowering the spatula only a few millimeters into the brain. No statistically significant differences in [3H]flunitrazepam binding between sham operated [126+9 (N = 6 ) fmol specifically bound [3H]flunitrazepam/mg protein] and untreated control animals (122 + 6 (4) fmol/mg protein) could be observed indicating that the alterations of other forebrain structures by the lesion method applied do not have any effect on retinal benzodiazepine binding.
The right visual cortex was removed by suction taking care to avoid any damage of the underlying white matter. The visual cortex was defined as visual areas 17, 18 and 18a. After killing the animals at the age of 25 days the localization and size of the ablation was checked macroscopically. Rats were only considered for biochemical analysis if their cortical lesion was properly placed. Unoperated rats were used as controls. Dark adaptation of rats was performed as described previously (Rothe et al., 1985). Briefly, 24-day-old rats adapted to a normal 12 h light/dark cycle from birth, were brought at the end of the light phase into a completely dark room and kept in the dark overnight. At the end of the fifth hour of the light phase of the following day the animals were killed under infrared light (filter UG7, VEB Schott Jena, lower transmission limit, 780 nm). Light adapted rats were killed at the age of 25 days at the end of the fifth hour of the light phase by decapitation. Tissue preparation
Retinae were rapidly isolated under a stereomicroscope and homogenized in 1 ml 50mM Tris-HC1, pH 7.4, and stored at - 2 0 ' C . Prior to assay, samples were thawed, diluted to a final volume of 3 ml with homogenization buffer, rehomogenized, and centrifuged at 75,000g for 10 min. The resulting pellet was washed once and the final pellet was resuspended in 0.7 ml 50 mM Tris-HC1 buffer (pH 7.4). This tissue preparation was immediately used for the binding assay. Benzodiazepine binding assay
[3H]flunitrazepam binding was assayed as described previously (Rothe et al., 1985). Briefly, glass tubes which contained 100/~1 of tissue suspension and 1.5nM [3H]flunitrazepam in a total volume of 500/~l 50 mM TrisHCI buffer, pH 7.4, were incubated for 30 rain at 0'C. The samples were rapidly filtered through glass fibre filters type NK VIII (VEB Feinpapierfabrik Neu-Kalil3, G.D.R., see also Schliebs et al., 1983). The filters were immediately washed three times with ice-cold incubation buffer, placed in scintillation vials and the retained radioactivity counted in a Packard TRICARB liquid scintillation spectrometer. Specific binding is defined as the difference between total [3H]flunitrazepam binding and the binding obtained in the presence of 100 # M chlordiazepoxide. Data are expressed as fmoles specifically bound [3H]flunitrazepam per mg of protein. Protein content was determined in aliquots of the tissue preparations by the method of Lowry et al. (1951). All samples were run in triplicates. As already shown previously (Rothe et al., 1985) specific [3H]flunitrazepam binding to rat retinal benzodiazepine receptors was saturable (dissociation constant K D = 2.4 _+ 0.3 nM; maximum receptor density: 338 + 22 fmol/mg protein) and was linearly dependent on protein content up to 0.2 mg protein/ml of incubation medium. Specific binding was shown to reach equilibrium during an incubation time of 30 min at 0c'C. There was no specific binding to the filter materials used (Schliebs et al., 1983). Statistical comparisons were made applying the twotailed Student t-test. RESULTS As s h o w n in Fig. 1 unilateral e n u c l e a t i o n resulted in an e n h a n c e m e n t o f [3H]flunitrazepam b i n d i n g in
Rat retinal benzodiazepine receptors
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Fig. i. [3H]flunitrazepam binding in the retina after light and dark adaptation of 25-day-old unilaterally enucleated and control rats. Data represent specifically bound amount of [3H]flunitrazepam assayed in triplicate at a ligand concentration of 1.5 nM and are the mean+SEM of seven (dark adaptation) and nine (light adaptation, control) separate determinations. After unilateral enucleation at postnatal day 15, rats were reared under a normal 12 h light/dark cycle until the age of 25 days and killed at the fifth hour of the light phase (light adapted rats, white columns). For dark adaptation, animals were brought into a completely dark room at the age of 24 days at the end of the light phase, kept in the dark overnight until the end of the fifth hour of light phase, and killed under infrared light (dark adapted animals, dotted columns). Animals which have not been subjected to any surgical operations were considered as controls. Contr., control rats; enucl., rats unilaterally enucleated. *P < 0.01, §P < 0.002, ~ P < 0.02, vs light-adapted controis, two-tailed Student t-test. the retina of the remaining eye by 23% as compared to that found in the retinae of unoperated control animals (P < 0.002, two-tailed Student t-test). The retinal benzodiazepine receptors of the remaining eye of enucleated rats were no longer capable of modification by dark adaptation (Fig. l). Unilateral optic nerve dissection was performed to allow for the assessment of benzodiazepine receptors also in the retina of the lesioned visual pathway. Using the paired test, [3H]flunitrazepam binding in the retina with the lesioned optic nerve was significantly higher by 20.4 + 7.6% (P < 0.02, N = 10) in comparison to that detected in the retina with the intact optic nerve. C o m p a r e d with untreated control animals a decreased benzodiazepine binding in the retina with intact optic nerve by 14% (P < 0.05) has been observed, whereas the binding level in the retina of the lesioned side was close to that assayed in the retina of unoperated control rats (Fig. 2). Unilateral visual cortex ablation affects [3H]flunitrazepam binding statistically significant
Fig. 2. Effect of unilateral cutting of the optic nerve on [3H]flunitrazepam binding in the retina of 25-day-old light adapted rats. Data represent specific binding assayed in triplicate at a ligand concentration of 1.5 nM and are the mean ___SEM of ten separate determinations. The right optic nerve was cut at postnatal day 17 by lowering a thin spatula to the base of the brain as indicated in Experimental Procedures. Sham operated animals (lowering the spatula only few millimeters into the brain) were considered as controls. Co., Control; c, retina contralateral to the lesion; i, retina ipsilateral to the lesion. Comparing binding data between both retinas of lesioned animals (paired test): i/c = 1.204 _ 0.076 (N = 10), P < 0.02, two-tailed Student t-test. §P < 0.05 vs control value.
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Fig. 3. Effect of unilateral visual cortex ablation on [3H]flunitrazepam binding in the retina of light (white columns) and dark adapted (dotted) 25-day-old rats. Data represent specific binding assayed in triplicate at a ligand concentration of 1.5 nM and are the mean+SEM of eight (light adapted) and five (dark adapted) separate determinations. Unilateral ablation of the visual cortex was performed at day 17 by aspiration. For light/dark adaptation and arrangement of data, see legend to Fig. 1. Contr., control retina; c, retina contralateral to the lesion; i, retina ipsilateral to the lesion. §P <0.02, ~ P <0.01 vs light adapted control, two-tailed Student t-test.
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only in the retina contralateral to the lesion. As shown in Fig. 3 a 13% decrease in binding (P < 0.02) in the retina contralateral to the brain lesion was detected in comparison to that observed in control animals. When the lesioned animals were dark adapted, no increase in [3H]flunitrazepam binding in the retina both contralateral and ipsilateral to the lesion could be observed as compared to light adapted controls. DISCUSSION
The centrifugal innervation of the mammalian retina is still an open question, although there are both anatomical and physiological observations for retinopetal fibres in various species (see e.g. van Hasselt, 1972/73; Molotchnikoff and Tremblay, 1983a, b). Histochemical, electron microscopical as well as electrophysiological studies have revealed the existence of an afferent innervation to the retina also in the rat (Itaya, 1980; ltaya and Itaya, 1985; Molotchnikoff and Tremblay, 1983a, b; Palkama and Sipponen, 1968). As recent findings indicated that retinal processes seem to be under a central nervous control (Molotchnikoff and Tremblay, 1983a; Rothe et al., 1985; Wenk et al., 1981), we were interested in whether rat retinal benzodiazepine receptors can be affected by lesioning selected structures of the primary visual system. Indeed, unilateral enucleation resulted in an increased level of benzodiazepine binding in the retina of the remaining eye indicating a retino-retinal coupling or a functional association of retinal benzodiazepine receptors between both eyes. After enucleation benzodiazepine receptors of the remaining eye were no longer modifiable by dark adaptation suggesting that the retinal receptors of both eyes are able to respond to changes in environmental lighting conditions only in accordance with each other which might be brought about either by a direct retino-retinal link or a biretinal central control. An alternative explanation of the loss of the darkinduced increase of binding in the enueleation experiments might be that dark adaptation normally leads to maximal binding which is also what is seen following enucleation, so a further increase in [3H]flunitrazepam binding cannot be obtained. Similarly, unilateral dissection of the optic nerve seems to interrupt retinopetal control and/or regulation mechanisms of retinal benzodiazepine receptors, thus again resulting in an uneven distribution of binding levels between both retinae. It is interesting to note that the binding level in the retina with intact
optic nerve is significantly lower as compared to untreated control animals, whereas enucleation results in elevated [3H]flunitrazepam binding in the retina of the remaining eye. The difference might be due to the more severe lesion produced by enucleation which includes also disruption of the afferent and efferent pathways of the eye muscles as well as the iris and the ciliary ganglion as compared to the dissection of the optic nerve. It has been proposed that the medial pretectal areas project to the retina and that the cortico-pretecto-retinal feedback loop is involved in reflex behavioural responses suggesting that cortico-retinal influences are associated with opto-motor reflexes (Itaya, 1980; Itaya and Itaya, 1985; Molotchnikoffand Tremblay, 1983a, b). There~ fore, in addition to the effects of interrupting the fibres travelling in the optic nerve, enucleation might interfere with brain structures involved in optomotoric reflex behaviour. Unilateral visual cortex ablation results in decreased binding in the retina of light adapted rats contralateral to the lesion, and the ability of benzodiazepine receptors to respond to environmental lighting is blocked in both retinas. Molotchnikoff and Tremblay (1983a) demonstrated by electrophysiological measurements that the activity of the efferent retinal impulses is at least partly under the control of the visual cortex, and the cortical influence seems to act mainly in the inner plexiform layer. As already demonstrated by Brooke et al. (1965) centrifugal fibres which seem to be involved in this visual cortical control of the retina terminate on retinal amacrine cells. Because [3H]flunitrazepam binding is also localized mainly in neurons in the inner plexiform layer (Regan et al., 1981; Young and Kuhar, 1979) rat retinal benzodiazepine receptors could be affected by such centrifugal fibres to the retina. Recently, it has been suggested that neuropeptides may be involved in this efferent retinal control (Stell et al., 1984; Weiler, 1985). Enkephalin was shown to be localized in different species almost exclusively in amacrine cells (Brecha et al., 1979; Eldred and Karten, 1983; Weiler, 1985). In the goldfish (Djamgoz et al., 1981) and chicken retina (Watt et al., 1984) it inhibits the release of [3H]GABA suggesting an interaction of enkephalin with retinal GABAergic transmission. Whether the changes in rat retinal benzodiazepine receptors following visual cortex ablation could be mediated through such neuropeptidergic mechanisms needs further clarification. The efferent control of rat retinal benzodiazepine receptors by the visual cortex as revealed from our lesion experiments seems to be mediated through
Rat retinal benzodiazepine receptors poly-synaptic rather than mono-synaptic pathways, presumably involving the medial pretectoretinal pathway recently described in the rat (Itaya and Itaya, 1985). Because the medial pretecto-retinal pathway is also crossing in the optic chiasm (Itaya, 1980; Itaya and Itaya, 1985), incoming information at the visual cortex from one eye can be used only to affect retinal mechanisms of the same eye. In order to explain the biretinal association of benzodiazepine receptors our data support the suggestion that retinopetal processes are mutually tuned at the level of the visual cortex by comparison of the information arrived in the visual cortex of each hemisphere. The functional role of the cortico-retinal feedback loop is still a question requiring clarification, but centrifugal influences may play a role in adjusting the retinal sensitivity as already had been proposed by Miles (1972) in birds. They also seem to play a role in adjusting and/or mutual tuning of retinal processes between both eyes as supported by our findings. Currently, evidence was presented that at least one-quarter of the photoreceptors in the monkey retina are immunoreactive with G A D or G A B A suggesting a putative role of G A B A as inhibitory transmitter in photoreceptor terminals (Nishimura et al., 1986). Until now there is, however, no indication that benzodiazepine receptors are involved in this G A B A e r g i c transmission of the outer retina. Acknowledgements--This work was supported by a grant of
the Ministry of Sciences and Technology of the G.D.R. The authors gratefully acknowledge the excellent technical assistance of Miss Renate Jendrek. The authors are indebted to Dr M. G. Stewart, The Open University, Milton Keynes, U.K., for his help with the English manuscript. REFERENCES Biggio G., Guarneri P. and Corda M. G. (1981) Benzodiazepine and GABA receptors in the rat retina: effect of light and dark adaptation. Brain Res. 216, 210-214. Brecha N., Karten H. J. and Laverack C. (1979) Enkephalin-containing amacrine cells in the avian retina: immunohistochemical localization. Proc. natn. Acad. Sei. U.S.A. 76, 3010-3014. Brooke R., Downer J. de C. and Powell T. P. S. (1965) Centrifugal fibres to the retina in the monkey and the cat. Nature, Lond. 207, 1365-1367. Djamgoz M. B. A., Stell W. K., Chin C. and Lam D. M. K. (1981) An opiate system in the goldfish retina. Nature, Lond. 292, 620-623. Ebbesson S. D. E. and Meyer D. L. (1981) Efferents to the retina have multiple sources in teleost fish. Science, N.Y. 214, 924-926. Eldred W. D. and Karten H. J. (1983) Characterization and quantification of peptidergic amacrine cells in the turtle NC.L 10/2--D
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0197-0186/87 $3.00+0.00 © 1987PergamonJournals Ltd
RAT RETINAL BENZODIAZEPINE RECEPTORS ARE C O N T R O L L E D BY VISUAL CORTICAL MECHANISMS REINHARD SCHLIEBS, THOMAS ROTHE and VOLKER BIGL Paul Flechsig Institute for Brain Research, Department of Neurochemistry, Karl Marx University, Leipzig, G.D.R. (Received 21 May 1986; accepted 29 August 1986)
A~traet--[3H]flunitrazepam binding was assayed in retinae of 25-day-old rats subjected either to unilateral enucleation at day 15, to intracranial unilateral cutting of the optic nerve at day 17, or to unilateral ablation of the visual cortex at day 17 postnatally. Unilateral enucleation resulted in an enhanced [3H]flunitrazepambinding in the retina of the remaining eye by 23% (P < 0.002, two-tailed Student t-test) as compared to unoperated controls. In rats with one optic nerve cut shortly before the optic chiasm, benzodiazepine binding in the retina of the lesioned side was significantlyhigher by 20.4 + 7.6% (P < 0.02, N = 10, paired test) in comparison to that in the retina with the intact optic nerve. Unilateral visual cortex ablation resulted in a 13% decrease (P < 0.02) in [3H]flunitrazepambinding in the retina contralateral to the brain lesion. In the lesioned rats of all three groups, the retinal benzodiazepine receptors were no longer capable of being modified by light/dark adaptation as is observed in normal rats. Our data suggest that (i) rat retinal benzodiazepine receptors are under a control from the visual cortex, and (ii) the benzodiazepine receptors of both eyes seem to be mutually tuned, presumably via a cortico-retinal feedback loop and an interhemispheric cortico-cortical information transfer. Recently the presence of high-affinity brain-specific benzodiazepine binding sites in preparations of rat retinae has been demonstrated (Howell et al., 1979; Paul et al., 1980; Rothe et al., 1985; Schliebs et al., 1986). Most of the specific binding of [3H]flunitrazepam is localized to the inner layers of the retina (Young and Kuhar, 1979). Lesion experiments with monosodium glutamate have emphasized the association of GABAergic mechanisms with benzodiazepine binding and suggested a predominant neuronal localization of the benzodiazepine binding sites in the inner plexiform layer (Regan et al., 1981). There is now some evidence that benzodiazepine receptors in the retina are involved in physiological events of light processing: Systemic administration of sedative dosages of diazepam reduced the lightevoked increase in retinal dopamine turnover, and intraocular application of flunitrazepam produced a suppression of light-enhanced dopamine synthesis in rat retina (Kamp and Morgan, 1982). Dark adaptation resulted in an increase in the affinity of [3H]diazepam (Biggio et al., 1981) and [3H]flunitrazepam (Rothe et al., 1985) for its specific binding site in the retina compared to that found in
light-adapted rats. In 25-day-old rats with one eyelid sutured at day l0 postnatally, the binding of [3H]flunitrazepam in the retina of the open eye was decreased (Rothe et al., 1985). However, dark adaptation of these monocularly deprived rats did not result in any change of benzodiazepine binding in the retina of either eye. This finding, that the retinal benzodiazepine receptors lose their ability to be modified by light/dark adaptation after monocular deprivation, suggested a physiological coupling between both retinae (Rothe et aL, 1985). Despite somewhat contradictionary results, there are both anatomical and physiological observations for centrifugal fibres to the retina in various species (Ebbesson and Meyer, 1981; Fergusson et aL, 1978; Halpern et aL, 1976; Hoogland and Welker, 1981; Itaya, 1980; Kawamura et al., 1974; Marchiavafa, 1976; Reprrant and Gallego, 1976; Rep~rant et al., 1980; Vanegas and Essayag-Millan, 1973; van Hasselt, 1972; Vesselkin et al., 1980; Weiler, 1985; for review of data until 1972, see van Hasseit, 1972/73). More recently, Molotchnikoff and Tremblay (1983) convincingly demonstrated that in rat responses of retinal ganglion cells might be modified and controlled by the visual cortex. In view of our recent finding that retinal benzo*Address correspondence to: Dr Reinhard Schliebs, Paul Flechsig Institute for Brain Research, Department of diazepine receptors might be tuned via retinopetal Neurochemistry, Karl Marx University, Karl-Marx- mechanisms (Rothe et al., 1985) the present study Stildter-StraBe 50, DDR-7039 Leipzig, G.D.R. was undertaken to investigate this centrifugal control 179
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o f retinal b e n z o d i a z e p i n e receptors in m o r e detail. T h e r e f o r e , [3H]flunitrazepam b i n d i n g was m e a s u r e d in rat retinae o n e week after the visual i n f o r m a t i o n t r a n s f e r was unilaterally i n t e r r u p t e d at different levels (enucleation o f the eyeball; c u t t i n g o f the o p t i c nerve intracranially) in o r d e r to see w h e t h e r there is a f u n c t i o n a l a s s s o c i a t i o n o f the activity o f rat retinal b e n z o d i a z e p i n e r e c e p t o r s b e t w e e n b o t h eyes. Rem o v a l o f the visual c o r t e x was p e r f o r m e d in o r d e r to study w h e t h e r rat retinal b e n z o d i a z e p i n e r e c e p t o r s are c o n t r o l l e d by visual cortical m e c h a n i s m s . EXPERIMENTAL PROCEDURES
Materials
[N-methyi-3H]flunitrazepam, specific activity 3056GBq/ mmol, was obtained from the Radiochemical Centre, Amersham, U.K., and used without further purification. Analytical grade chlordiazepoxide was a gift from VEB Arzneimittelwerk, Dresden, G.D.R. Treatment o f animals
Rats of either sex (strain BD III) reared under normal animal house conditions and under a 12 h light/dark cycle were used. Unilateral eyeball enucleation was performed under a light ether anaesthesia at the age of 15 days postnatally. Unoperated animals were used as controls. For lesion experiments, 17-day-old rats were anaesthetized with Nembutal (Nembutal sodium solution, Abbott Lab., Chicago, 40 mg/kg body weight) and placed in a K o p f stereotaxic apparatus. The skull was opened with a dental drill and unilateral optic nerve lesion and visual cortex ablation, respectively, were performed as follows: The right optic nerve was cut intracranially a few millimeters before reaching the optic chiasm by lowering a thin spatula 3~4 mm in front of the bregrna (i.e. the point where the midsagital suture crosses the suture between the frontal and parietal os of the skull) and 1 mm lateral to the midline to the base of the brain. Successful cutting of the optic nerve was indicated by a permanent enlargement of the pupil of the lesioned side. Lesions of the optic nerve at this level do not interfere with the retinal blood supply as the main retinal artery (arteria centralis retinae) meets the optic nerve but extracranially. Previous histological controls showed that after one week of survival light microscopically the retina appears normal. After a survival time long enough to allow for retrograde degeneration the ganglion cell layer has disappeared (see Wenk et al., 1981). Rats were killed by decapitation at the age of 25 days and the results of the lesion evaluated by visual inspection. Only those animals with a complete dissection of the optic nerve of one side leaving intact the other pathway were included in the biochemical analyses. Some control animals were sham operated by lowering the spatula only a few millimeters into the brain. No statistically significant differences in [3H]flunitrazepam binding between sham operated [126+9 (N = 6 ) fmol specifically bound [3H]flunitrazepam/mg protein] and untreated control animals (122 + 6 (4) fmol/mg protein) could be observed indicating that the alterations of other forebrain structures by the lesion method applied do not have any effect on retinal benzodiazepine binding.
The right visual cortex was removed by suction taking care to avoid any damage of the underlying white matter. The visual cortex was defined as visual areas 17, 18 and 18a. After killing the animals at the age of 25 days the localization and size of the ablation was checked macroscopically. Rats were only considered for biochemical analysis if their cortical lesion was properly placed. Unoperated rats were used as controls. Dark adaptation of rats was performed as described previously (Rothe et al., 1985). Briefly, 24-day-old rats adapted to a normal 12 h light/dark cycle from birth, were brought at the end of the light phase into a completely dark room and kept in the dark overnight. At the end of the fifth hour of the light phase of the following day the animals were killed under infrared light (filter UG7, VEB Schott Jena, lower transmission limit, 780 nm). Light adapted rats were killed at the age of 25 days at the end of the fifth hour of the light phase by decapitation. Tissue preparation
Retinae were rapidly isolated under a stereomicroscope and homogenized in 1 ml 50mM Tris-HC1, pH 7.4, and stored at - 2 0 ' C . Prior to assay, samples were thawed, diluted to a final volume of 3 ml with homogenization buffer, rehomogenized, and centrifuged at 75,000g for 10 min. The resulting pellet was washed once and the final pellet was resuspended in 0.7 ml 50 mM Tris-HC1 buffer (pH 7.4). This tissue preparation was immediately used for the binding assay. Benzodiazepine binding assay
[3H]flunitrazepam binding was assayed as described previously (Rothe et al., 1985). Briefly, glass tubes which contained 100/~1 of tissue suspension and 1.5nM [3H]flunitrazepam in a total volume of 500/~l 50 mM TrisHCI buffer, pH 7.4, were incubated for 30 rain at 0'C. The samples were rapidly filtered through glass fibre filters type NK VIII (VEB Feinpapierfabrik Neu-Kalil3, G.D.R., see also Schliebs et al., 1983). The filters were immediately washed three times with ice-cold incubation buffer, placed in scintillation vials and the retained radioactivity counted in a Packard TRICARB liquid scintillation spectrometer. Specific binding is defined as the difference between total [3H]flunitrazepam binding and the binding obtained in the presence of 100 # M chlordiazepoxide. Data are expressed as fmoles specifically bound [3H]flunitrazepam per mg of protein. Protein content was determined in aliquots of the tissue preparations by the method of Lowry et al. (1951). All samples were run in triplicates. As already shown previously (Rothe et al., 1985) specific [3H]flunitrazepam binding to rat retinal benzodiazepine receptors was saturable (dissociation constant K D = 2.4 _+ 0.3 nM; maximum receptor density: 338 + 22 fmol/mg protein) and was linearly dependent on protein content up to 0.2 mg protein/ml of incubation medium. Specific binding was shown to reach equilibrium during an incubation time of 30 min at 0c'C. There was no specific binding to the filter materials used (Schliebs et al., 1983). Statistical comparisons were made applying the twotailed Student t-test. RESULTS As s h o w n in Fig. 1 unilateral e n u c l e a t i o n resulted in an e n h a n c e m e n t o f [3H]flunitrazepam b i n d i n g in
Rat retinal benzodiazepine receptors
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Fig. i. [3H]flunitrazepam binding in the retina after light and dark adaptation of 25-day-old unilaterally enucleated and control rats. Data represent specifically bound amount of [3H]flunitrazepam assayed in triplicate at a ligand concentration of 1.5 nM and are the mean+SEM of seven (dark adaptation) and nine (light adaptation, control) separate determinations. After unilateral enucleation at postnatal day 15, rats were reared under a normal 12 h light/dark cycle until the age of 25 days and killed at the fifth hour of the light phase (light adapted rats, white columns). For dark adaptation, animals were brought into a completely dark room at the age of 24 days at the end of the light phase, kept in the dark overnight until the end of the fifth hour of light phase, and killed under infrared light (dark adapted animals, dotted columns). Animals which have not been subjected to any surgical operations were considered as controls. Contr., control rats; enucl., rats unilaterally enucleated. *P < 0.01, §P < 0.002, ~ P < 0.02, vs light-adapted controis, two-tailed Student t-test. the retina of the remaining eye by 23% as compared to that found in the retinae of unoperated control animals (P < 0.002, two-tailed Student t-test). The retinal benzodiazepine receptors of the remaining eye of enucleated rats were no longer capable of modification by dark adaptation (Fig. l). Unilateral optic nerve dissection was performed to allow for the assessment of benzodiazepine receptors also in the retina of the lesioned visual pathway. Using the paired test, [3H]flunitrazepam binding in the retina with the lesioned optic nerve was significantly higher by 20.4 + 7.6% (P < 0.02, N = 10) in comparison to that detected in the retina with the intact optic nerve. C o m p a r e d with untreated control animals a decreased benzodiazepine binding in the retina with intact optic nerve by 14% (P < 0.05) has been observed, whereas the binding level in the retina of the lesioned side was close to that assayed in the retina of unoperated control rats (Fig. 2). Unilateral visual cortex ablation affects [3H]flunitrazepam binding statistically significant
Fig. 2. Effect of unilateral cutting of the optic nerve on [3H]flunitrazepam binding in the retina of 25-day-old light adapted rats. Data represent specific binding assayed in triplicate at a ligand concentration of 1.5 nM and are the mean ___SEM of ten separate determinations. The right optic nerve was cut at postnatal day 17 by lowering a thin spatula to the base of the brain as indicated in Experimental Procedures. Sham operated animals (lowering the spatula only few millimeters into the brain) were considered as controls. Co., Control; c, retina contralateral to the lesion; i, retina ipsilateral to the lesion. Comparing binding data between both retinas of lesioned animals (paired test): i/c = 1.204 _ 0.076 (N = 10), P < 0.02, two-tailed Student t-test. §P < 0.05 vs control value.
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Fig. 3. Effect of unilateral visual cortex ablation on [3H]flunitrazepam binding in the retina of light (white columns) and dark adapted (dotted) 25-day-old rats. Data represent specific binding assayed in triplicate at a ligand concentration of 1.5 nM and are the mean+SEM of eight (light adapted) and five (dark adapted) separate determinations. Unilateral ablation of the visual cortex was performed at day 17 by aspiration. For light/dark adaptation and arrangement of data, see legend to Fig. 1. Contr., control retina; c, retina contralateral to the lesion; i, retina ipsilateral to the lesion. §P <0.02, ~ P <0.01 vs light adapted control, two-tailed Student t-test.
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only in the retina contralateral to the lesion. As shown in Fig. 3 a 13% decrease in binding (P < 0.02) in the retina contralateral to the brain lesion was detected in comparison to that observed in control animals. When the lesioned animals were dark adapted, no increase in [3H]flunitrazepam binding in the retina both contralateral and ipsilateral to the lesion could be observed as compared to light adapted controls. DISCUSSION
The centrifugal innervation of the mammalian retina is still an open question, although there are both anatomical and physiological observations for retinopetal fibres in various species (see e.g. van Hasselt, 1972/73; Molotchnikoff and Tremblay, 1983a, b). Histochemical, electron microscopical as well as electrophysiological studies have revealed the existence of an afferent innervation to the retina also in the rat (Itaya, 1980; ltaya and Itaya, 1985; Molotchnikoff and Tremblay, 1983a, b; Palkama and Sipponen, 1968). As recent findings indicated that retinal processes seem to be under a central nervous control (Molotchnikoff and Tremblay, 1983a; Rothe et al., 1985; Wenk et al., 1981), we were interested in whether rat retinal benzodiazepine receptors can be affected by lesioning selected structures of the primary visual system. Indeed, unilateral enucleation resulted in an increased level of benzodiazepine binding in the retina of the remaining eye indicating a retino-retinal coupling or a functional association of retinal benzodiazepine receptors between both eyes. After enucleation benzodiazepine receptors of the remaining eye were no longer modifiable by dark adaptation suggesting that the retinal receptors of both eyes are able to respond to changes in environmental lighting conditions only in accordance with each other which might be brought about either by a direct retino-retinal link or a biretinal central control. An alternative explanation of the loss of the darkinduced increase of binding in the enueleation experiments might be that dark adaptation normally leads to maximal binding which is also what is seen following enucleation, so a further increase in [3H]flunitrazepam binding cannot be obtained. Similarly, unilateral dissection of the optic nerve seems to interrupt retinopetal control and/or regulation mechanisms of retinal benzodiazepine receptors, thus again resulting in an uneven distribution of binding levels between both retinae. It is interesting to note that the binding level in the retina with intact
optic nerve is significantly lower as compared to untreated control animals, whereas enucleation results in elevated [3H]flunitrazepam binding in the retina of the remaining eye. The difference might be due to the more severe lesion produced by enucleation which includes also disruption of the afferent and efferent pathways of the eye muscles as well as the iris and the ciliary ganglion as compared to the dissection of the optic nerve. It has been proposed that the medial pretectal areas project to the retina and that the cortico-pretecto-retinal feedback loop is involved in reflex behavioural responses suggesting that cortico-retinal influences are associated with opto-motor reflexes (Itaya, 1980; Itaya and Itaya, 1985; Molotchnikoffand Tremblay, 1983a, b). There~ fore, in addition to the effects of interrupting the fibres travelling in the optic nerve, enucleation might interfere with brain structures involved in optomotoric reflex behaviour. Unilateral visual cortex ablation results in decreased binding in the retina of light adapted rats contralateral to the lesion, and the ability of benzodiazepine receptors to respond to environmental lighting is blocked in both retinas. Molotchnikoff and Tremblay (1983a) demonstrated by electrophysiological measurements that the activity of the efferent retinal impulses is at least partly under the control of the visual cortex, and the cortical influence seems to act mainly in the inner plexiform layer. As already demonstrated by Brooke et al. (1965) centrifugal fibres which seem to be involved in this visual cortical control of the retina terminate on retinal amacrine cells. Because [3H]flunitrazepam binding is also localized mainly in neurons in the inner plexiform layer (Regan et al., 1981; Young and Kuhar, 1979) rat retinal benzodiazepine receptors could be affected by such centrifugal fibres to the retina. Recently, it has been suggested that neuropeptides may be involved in this efferent retinal control (Stell et al., 1984; Weiler, 1985). Enkephalin was shown to be localized in different species almost exclusively in amacrine cells (Brecha et al., 1979; Eldred and Karten, 1983; Weiler, 1985). In the goldfish (Djamgoz et al., 1981) and chicken retina (Watt et al., 1984) it inhibits the release of [3H]GABA suggesting an interaction of enkephalin with retinal GABAergic transmission. Whether the changes in rat retinal benzodiazepine receptors following visual cortex ablation could be mediated through such neuropeptidergic mechanisms needs further clarification. The efferent control of rat retinal benzodiazepine receptors by the visual cortex as revealed from our lesion experiments seems to be mediated through
Rat retinal benzodiazepine receptors poly-synaptic rather than mono-synaptic pathways, presumably involving the medial pretectoretinal pathway recently described in the rat (Itaya and Itaya, 1985). Because the medial pretecto-retinal pathway is also crossing in the optic chiasm (Itaya, 1980; Itaya and Itaya, 1985), incoming information at the visual cortex from one eye can be used only to affect retinal mechanisms of the same eye. In order to explain the biretinal association of benzodiazepine receptors our data support the suggestion that retinopetal processes are mutually tuned at the level of the visual cortex by comparison of the information arrived in the visual cortex of each hemisphere. The functional role of the cortico-retinal feedback loop is still a question requiring clarification, but centrifugal influences may play a role in adjusting the retinal sensitivity as already had been proposed by Miles (1972) in birds. They also seem to play a role in adjusting and/or mutual tuning of retinal processes between both eyes as supported by our findings. Currently, evidence was presented that at least one-quarter of the photoreceptors in the monkey retina are immunoreactive with G A D or G A B A suggesting a putative role of G A B A as inhibitory transmitter in photoreceptor terminals (Nishimura et al., 1986). Until now there is, however, no indication that benzodiazepine receptors are involved in this G A B A e r g i c transmission of the outer retina. Acknowledgements--This work was supported by a grant of
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