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Circadian activity of the GABAergic system in the golden hamster retina
Brain Research 912 (2001) 195–202 www.elsevier.com / locate / bres
Research report
Circadian activity of the GABAergic system in the golden hamster retina ´ I. Keller Sarmiento a , Carolina O. Jaliffa a , Daniel Saenz a , Ernesto Resnik b , Marıa a, Ruth E. Rosenstein * a
´ ´ Experimental, Departamento de Bioquımica ´ Humana, Facultad de Medicina, Laboratorio de Neuroquımica Retiniana y Oftalmologıa Universidad de Buenos Aires, Paraguay 2155, 5 to P, 1121, Buenos Aires, Argentina b Medical School, University of Minnesota, Minneapolis, MN 55455, USA Accepted 19 June 2001
Abstract Daily changes in g-aminobutyric acid (GABA) turnover rate were studied in the golden hamster retina. This parameter showed significant variations throughout the light–dark cycle, with minimal values during the day. Retinal glutamic acid decarboxylase (GAD) activity was higher at midnight than at noon. Moreover, [ 3 H]GABA binding significantly varied throughout the 24-h cycle, with maximal values during the day. Saturation studies performed at 12:00 and 24:00 h indicated that the maximal concentration of [ 3 H]GABA binding sites (Bmax ) was significantly higher at noon, whereas the dissociation constant (Kd ) remained unchanged. High K 1 -induced GABA release was significantly higher at midnight than at midday. Daily variations in retinal GABA turnover rate, GABA release, and in its specific binding persisted in golden hamsters exposed to constant darkness. In summary, these results support the idea of a circadian clock-controlled GABAergic activity in the hamster retina. 2001 Elsevier Science B.V. All rights reserved. Keywords: Hamster retina; GABA; Circadian rhythm
1. Introduction GABA is a major inhibitory neurotransmitter of the vertebrate retina, where it plays an important role in visual information processing [27,40]. Horizontal and amacrine cells were identified as the principal GABAergic neurons in the retina of several species [29,40]. In the rabbit retina, GABA immunostaining is sporadic in horizontal cells, while it is intense and reproducible in amacrine cells [29,32]. Although GABA-like immunoreactivity was observed in cat horizontal cell processes [11], the evidence for GABAergic horizontal cells in primate and rodent retina is still controversial. In the golden hamster retina, we have demonstrated GABA-like immunoreactivity in amacrine cells, in neurons localized in the ganglion cell layer, as well as in fibers and terminals at the inner Abbreviations: GABA, g-aminobutyric acid; GAD, glutamic acid decarboxylase; LD, light–dark cycle; DD, dark–dark cycle *Corresponding author. Tel.: 154-11-4508-3672, ext. 37; fax: 154-114508-3672, ext. 31. E-mail address: [email protected] (R.E. Rosenstein).
plexiform layer. In addition, a few horizontal cells also showed GABA-like immunolabeling [25]. Recently, we have shown that GABA increases melatonin content in the hamster retina, likely through a GABAA receptor-mediated mechanism [17]. The stimulatory effect of exogenous GABA was evident during the day but not during the night. The GABAA receptor antagonist, bicuculline, although having no effect per se under light conditions, significantly inhibited the darkness-induced stimulation of melatonin content, providing evidence for the involvement of endogenous GABA in the pathway of darkness input to the melatonin-generating system. Alternatively, since it has been shown that the hamster retina contains a circadian oscillator that regulates melatonin synthesis [38], these results permit the presumption that the retinal GABAergic system could mediate the action of a local pacemaker on melatonin biosynthesis. This hypothesis would have the pacemaker-induced regulation of melatonin levels by raising and lowering GABAergic activity at specific daytime points. A necessary (although not sufficient) condition to prove this idea would be to demonstrate daily variations and free running rhythms of retinal GABAergic activity.
0006-8993 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 01 )02736-6
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We have previously demonstrated that hamster retinal GABA turnover rate is significantly higher at 24:00 than at 12:00 h [17], but a detailed study on the temporal course of this and other GABAergic parameters was still lacking. Therefore, the aim of the present study was to examine GABA turnover rate, its specific binding, and GABA release in retinas of golden hamsters exposed to a light– dark (LD) or a constant dark cycle (DD).
2. Material and methods
2.1. Reagents and drugs GABA, L-glutamic acid, 3-mercaptopropionic acid, GABAse, and pyridoxal phosphate were obtained from Sigma (St Louis, MO, USA). [ 3 H]GABA, 1-[ 14 C]Lglutamic acid, and [ 3 H]muscimol were purchased from New England Nuclear Corp. (Boston, MA, USA).
2.2. Animals Male golden hamsters (average weight 120620 g), derived from a stock supplied by Charles River Breeding Laboratories (Wilmington, MA, USA), were purchased from a local dealer. Unless indicated, hamsters were kept under a photoperiod of 14 h of light–10 h of darkness (LD, lights on at 06:00 h), with free access to food and water. In some experiments, the animals were kept in constant darkness (DD) for 48 h. Since the average circadian period in our hamsters is of 24.1 h, after 48 h of constant darkness, circadian times would shift, maximally, 6 or 12 min. Thus, we assumed that the circadian times (CT, being CT512 defined as the time of locomotor activity onset), would approximately equal their previous zeitgeber times (ZT, with ZT512 defined as the time of lights off), and sacrificed the animals at six intervals through the 24 h-cycle. The animals were killed by decapitation, their eyes were immediately enucleated, and the retinas were excised and processed as described below. In the case of dark-exposed hamsters, sacrifice of animals and extraction of retinas were carried out under dim red light.
2.3. GABA turnover rate assessment GABA turnover rate was measured as previously described [35]. In order to prevent the postmortem increase in GABA content, the animals were injected with the GAD inhibitor, 3-mercaptopropionic acid (50 mg / kg i.p.) 2.5 min before sacrifice [39]. GABA turnover rate was assessed by the accumulation of GABA levels following inhibition of GABA transaminase [5]. The major assumption of the method used was that after the administration of g-vinyl GABA (a gift from Merrel Dow Research Institute, Strasbourg, France), the accumulation of GABA was linear for at least 1 h. g-Vinyl GABA was injected i.p. at a 1
g / kg dose, 62.5 min before sacrifice. Control animals, which received i.p. injections of vehicle (300 ml of saline), were used for the determination of the steady-state GABA concentrations. Retinas were excised, individually homogenized in distilled water and centrifuged at 12 0003 g for 15 min, the pellets being discarded. GABA concentrations were assessed by a radioreceptor assay as described by Bernasconi et al. [4], with detection range for GABA of 2.5–100 pmol. In order to ascertain whether the displacement of [ 3 H]muscimol from its binding sites was due to authentic GABA, tissue samples were incubated with the highly specific GABA-degrading enzyme system, GABAse, before the assay. Either standard or endogenous GABA was completely (i.e. more than 85%) degraded by GABAse.
2.4. GAD activity assessment GAD activity was determined as previously described [36]. Aliquots of homogenate (0.2–0.4 mg protein) corresponding to one retina / point were incubated in 1.5 mlEppendorf polypropylene tubes at 378C for 1 h. The incubation mixture (300 ml) contained 1-[ 14 C]L-glutamic acid (specific activity 49.6 Ci / mmol, 0.2 mCi / ml), 0.5–2 mM L-glutamic acid, 200 mM pyridoxal phosphate, and 10 mM b-mercaptoethanol. Incubations were carried out in triplicates at six different concentrations of L-glutamic acid (0.5–3 mM). The reaction was stopped by adding 10% trichloroacetic acid, and the 14 CO 2 released during an additional 2-h period was captured on filter paper embedded in hyamine hydroxide. Blanks included all reagents except that TCA was added before the homogenate. Double-reciprocal plots were constructed from the mean of triplicates, at each L-glutamic acid concentration.
2.5. [ 3 H] GABA release Retinas were incubated for 30 min at 378C with [ 3 H]GABA (0.5–1 mCi) in 500 ml of buffer containing 140 mM NaCl, 5 mM KCl, 2.5 mM CaCl 2 , 1 mM MgCl 2 , 10 mM Hepes, and 10 mM glucose, adjusted to pH 7.4 with Tris base. The tissues were washed several times in fresh buffer in order to remove the excess [ 3 H]GABA, and incubated for 10 min with gentle shaking in 500 ml of the same buffer or a high K 1 (50 mM) buffer in which osmolarity was conserved by equimolar reduction of Na 1 concentration. Tissues were digested with hyamine hydroxide, and radioactivity in the medium and that incorporated into the tissue were determined in a scintillation counter. Fractional release was calculated as the ratio of radioactivity released / total radioactivity uptaken by the tissue. Greater than 80% of the released radioactivity was identified as authentic GABA by thin layer chromatography.
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2.6. GABA binding assay [ 3 H]GABA binding was assessed by a standard procedure as previously described [1]. Briefly, retinal pools were homogenized in 4 ml of water, and centrifuged at 9003g for 20 min. The supernatant was centrifuged at 20 0003g for 20 min. The pellet was frozen at 2708C until the binding assay was performed (usually within 18 h). On the day of the experiment, the pellet was resuspended in 0.05 M Tris–HCl (pH 7.2) and Triton X-100 was added to achieve a final concentration of 0.01% (v / v). The mixture was incubated at 378C for 30 min in a shaking bath incubator. After centrifuging for 20 min at 20 0003g, the new pellet was washed five times with Tris–HCl and was finally resuspended in a fresh aliquot of Tris–HCl buffer. Aliquots of this homogenate (0.2–0.4 mg protein) were incubated for 10 min at room temperature in a total volume of 150 ml. The reaction was initiated by adding 10 nM of [ 3 H]GABA (80.8 Ci / mmol) together with varying concentrations of unlabeled GABA (6–200 nM), in the presence (nonspecific binding) or absence (total binding) of 200 mM unlabeled GABA. For single point binding assessment, unlabeled GABA was omitted. After incubation, the reaction was stopped by centrifuging the samples for 5 min at 20 0003g. The supernatant fraction was discarded and the pellet was gently washed twice with 1 ml of cold Tris–HCl buffer. Radioactivity was extracted by resuspending the pellet in 100 ml of hyamine hydroxide and leaving the samples overnight at room temperature. Under these conditions, [ 3 H]GABA binding was completely displaced by 20 mM muscimol. The dissociation constant (Kd ) and maximal concentration of binding sites were calculated by Scatchard analysis. Protein content was determined by the method of Lowry et al. [26], using bovine serum albumin as the standard. Statistical analysis of the results was made by Student’s t-test or by a two-way analysis of variance (ANOVA) followed by a Tukey’s test, as stated. All animal use procedures were in strict accordance with the NIH Guide for Care and Use of Laboratory Animals.
Fig. 1. Retinal GABA turnover rate throughout the 24-h cycle. Hamsters were injected (i.p.) with g-vinyl GABA (1 g / kg) 62.5 min before sacrifice, and received 50 mg / kg 3-mercaptopropionic acid 2.5 min before sacrifice. GABA levels were assessed by a radioreceptor assay. Data are the means6S.E.M. (n515 animals per group). This parameter showed significant daily variations (P,0.01, ANOVA); values at 08:00 and 12:00 h were significantly lower than at all the other intervals (**P,0.01, by Tukey’s test).
showed significant variations with a similar profile to that observed for GABA levels accumulated after the inhibition of its degradation (Fig. 2). Since both GABA content and GABA turnover rate showed higher values during the night, it seems likely that the synthesis of GABA in the retina varies during the 24-h cycle. In order to assess time-dependent variation in GABA synthesis, the activity of GAD was examined in the retina of animals killed at
3. Results Fig. 1 depicts the daily changes in GABA turnover rate of golden hamster retinas excised at six different intervals throughout the 24-h cycle. This parameter exhibited significant daily variations, showing minimal values during the day, at 08:00 and 12:00 h. GABA turnover rate was assessed following the administration of g-vinyl GABA. In these conditions, each point represents GABA levels accumulated after the inhibition of GABA transaminase activity. In the next experiment, GABA levels were measured similarly but without the inhibition of GABA degradation, in order to assess steady-state GABA concentrations along the 24-h cycle. This parameter also
Fig. 2. Daily variations in steady-state concentrations of GABA in the hamster retina. Animals received 50 mg / kg 3-mercaptopropionic acid 2.5 min before sacrifice. GABA levels were assessed by a radioreceptor assay. Data are means6S.E.M. (n515 animals per group). This parameter showed significant daily variations (P,0.01, ANOVA), the values during the day (except at 16:00 h) being significantly lower than the corresponding values during the night (**P,0.01, by Tukey’s test).
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12:00 or 24:00 h. Kinetics of GAD activity in retinas excised at midday and midnight are shown in Fig. 3. The Vmax of retinal GAD was significantly higher at 24:00 h (1.6260.07 nmol / min per mg protein) than at 12:00 h (1.1260.06 nmol / min per mg protein), while the Km remained unchanged (2.3560.2 and 2.1760.3 mM at 24:00 and 12:00 h, respectively). This result suggests an increase in the number of active GAD molecules during the night, in accordance with the daily variation of the retinal GABA turnover rate and steady-state GABA concentrations. Daily rhythmicity in retinal GABA binding is shown in Fig. 4. GABA binding was significantly higher at 08:00 and 12:00 h than at the rest of the daytime points. Saturation studies were performed in retinas of hamster killed at noon and midnight (Fig. 5). The calculated Bmax at 12:00 h (460625 fmol / mg protein) was significantly higher than at 24:00 h (205620 fmol / mg protein), whereas no significant changes were found for the binding affinity (Kd 54264 and 3763 nM at 12:00 and 24:00 h, respectively). This result indicate time-dependent variations in the density of retinal GABA receptors.
Fig. 4. [ 3 H]GABA binding in hamster retinas excised throughout the light–dark cycle. [ 3 H]GABA binding was assessed as described in Material and methods. Shown are means6S.E.M. (n512 animals / group). This parameter exhibited significant daily variations (P,0.01, ANOVA), the values at 08:00 and 12:00 h being significantly higher than at the rest of the daytime points (**P,0.01 by Tukey’s test).
In order to examine whether daily variation of GABA turnover and GABA binding are driven by a circadian clock, the same parameters were assessed in animals
Fig. 3. GAD activity in hamster retina was assessed by the CO 2 -trapping procedure using 1-[ 14 C]L-glutamate as substrate, as described in Material and methods. Slopes and Y-intercepts were calculated by the method of least squares. The points represent mean values from triplicates samples using one retina / point; differences among triplicates were less than 10%. In this experiment, representative of four, Vmax values were 1.02 and 1.58 nmol / min per mg protein at 12:00 and 24:00 h, respectively, and Km values were 2.01 mM (at 12:00 h) and 2.2 mM (at 24:00 h). Vmax was significantly higher (P,0.01, Student’s t-test) at 12:00 than at 24:00 h. Means6S.E.M. for Vmax and Km (n54) are given in the text.
Fig. 5. Scatchard regression plots of specific [ 3 H]GABA binding in the retina of hamster killed at 12:00 and 24:00 h. Slopes and Y-intercepts were calculated by the method of least squares. Each point represents mean values from triplicates using 10 animals per group, differences among triplicates were less than 10%. In this experiment, representative of four, Kd values were 40 and 35 nM at 12:00 and 24:00 h, respectively, and Bmax values were 450 and 185 fmol / mg protein at 12:00 and 24:00 h, respectively. Bmax was significantly higher (P,0.01, Student’s t-test) at 12:00 than at 24:00 h. Mean6S.E.M. for Bmax and Kd (n54) are given in the text.
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Daily variations of GABA release in the golden hamster retina were assessed. The basal and high K 1 -induced [ 3 H]GABA release was examined at midday and midnight (animals under a LD photoperiod) as well as at subjective midday and subjective midnight (animals kept under constant darkness). In agreement with the results obtained for GABA turnover rate and GABA synthesis, depolarization-induced GABA release was significantly higher at midnight (LD), and during the subjective midnight (DD) as shown in Fig. 8.
4. Discussion
Fig. 6. Circadian variations in retinal GABA turnover rate of hamsters kept under constant darkness for 2 days before sacrifice. Data are the means6S.E.M. (n515 animals per group). This parameter showed significant circadian variations (P,0.01, ANOVA); the values at clocktime 08:00 and 12:00 h were significantly lower than at the rest of the daytime points (**P,0.01, by Tukey’s test).
exposed to free running conditions. When animals were housed in constant darkness for 2 days before the experiment, and killed at equivalent daytime points, daily variations in retinal GABA turnover rate persisted (e.g. minimal values during the subjective day), but a significant increase in this parameter was already attained at clocktime 16:00 h (Fig. 6). Fig. 7 shows circadian variations in retinal [ 3 H]GABA binding in the retinas of hamsters kept under DD 2 days before sacrifice. A significant increase in this parameter was observed during the subjective day, indicating an endogenous control of its diurnal variation.
Fig. 7. Circadian variations of retinal [ 3 H]GABA binding of hamsters kept under constant darkness for 2 days before sacrifice. Shown are means6S.E.M. (n512 animals / group). This parameter showed significant circadian variations (P,0.01, ANOVA), the values at clocktime 08:00 and 12:00 h were significantly higher than at the rest of the daytime points (**P,0.01, by Tukey’s test).
Dopamine and GABA are the most studied retinal neurotransmitters regarding responsiveness to light and circadian rhythmicity [34]. Extensive evidence demonstrates that retinal dopamine transduces the light signal mimicking the acute inhibitory effect of light on melatonin biosynthesis. Accordingly, light increases the synthesis, release, and metabolism of dopamine in retinas of several species [6,13,16,41]. Moreover, it has been shown that rat retinal dopamine synthesis varies over the 24-h cycle with maximal values during the day [28]. In contrast to dopamine, no information is available about time-dependent variations of retinal GABAergic activity. Our results indicate that in the golden hamster retina, significant 24-h variations occur in both GABA steady-state concentrations and turnover rate. A time-dependent variation in GABA content of selected brain areas had been previously demonstrated in the rat tuberoinfundibular region and hypothalamus [9,10]. In the present work, besides GABA content, GABA turnover rate (an indicator that correlates with traffic neural signals in GABAergic neurons), was measured by its accumulation after inhibition of its degradation [14]. In a previous report, we observed the existence of daily variations in GABA turnover rate of cerebral cortex, cerebellum, hypothalamus, and pineal gland of golden hamsters, which also showed a tendency for maximal values towards the first half of the night [20]. Since in the present study GABA levels were measured by inhibiting its degradation, its synthesis is a presumptive target for time-dependent variations. Accordingly, a nocturnal increase in GAD Vmax was demonstrated with no change in the Km . In the rat brain, GABAA and benzodiazepine binding sites varied daily both in their number and affinity, with nocturnal and diurnal maxima, respectively [1,2], while a nocturnal peak in GABA postsynaptic effect was reported [21]. Since both GABA turnover rate and GABA release were higher during the night, it seems that an inverse relationship between these parameters and GABA binding occurs in the hamster retina, as an augmented value of this latter was observed during the day. From these results, it can be hypothesized that GABA binding sites are up- and down-regulated by their own ligand. As thoroughly
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Fig. 8. [ 3 H]GABA release in the golden hamster retina. Animals were kept under LD or DD cycles. Shown are means6S.E.M. (n512 animals / group). High K 1 (50 mM) induced a significant [ 3 H]GABA release at each time point (P,0.01, by Student’s t-test). This parameter was significantly higher at midnight than at midday (LD), and at subjective midnight than at subjective midday (DD) (**P,0.01, by Student’s t-test). No differences were found for the basal release of GABA.
washed membranes were used in all daytime points, variations in the occupancy of the receptor by endogenous GABA is unlikely. In fact, daily variations in GABA binding seem to be related to an increase in the density of GABA receptors, as indicated by the increase in Bmax at 12:00 relative to 24:00 h. The distribution of GABA receptors among the different cell types of the retina is not yet known in detail but the available results indicate that they are present in at least some members of each basic type of retinal cells [15]. Since GABA binding was assessed in the whole retina, we could not ascertain the locus of the observed phenomena. Therefore, the possibility that the measured changes reflect larger or lesser changes in specific retinal cell population(s) remains to be tested. A daily rhythm in the retina may be controlled by a circadian oscillator, by direct response to environmental lighting levels, or by a combination of these mechanisms. The observation that daily variations of GABA turnover rate, binding, and release persisted under constant conditions supports the idea of a clock-controlled function. Although the possibility that light may influence retinal GABAergic activity is not excluded, the light–dark cycle seems not to be necessary for the generation of its rhythm. In mammals, circadian oscillators have been identified in the suprachiasmatic nucleus (SCN) of the hypothalamus [23] and, more recently, in cultured neural retinas of the golden hamster [38]. GABA is detectable in nearly every neuron of the SCN [31], where a circadian rhythmicity of GABA content and GAD activity has been shown [3]. Besides the retina and the SCN, GABAergic neurons are also important in other components of the circadian
system, including the intergeniculate leaflet and the thalamic lateral geniculate complex [30,37]. Because of this key distribution, it was postulated that GABA is the principal neurotransmitter of the circadian timing system [8,31]. In view of this, and taken together with the rhythmical production of retinal melatonin and its regulation by GABA, it is tempting to speculate that the ocular circadian clock may act through the local GABAergic system to drive the melatonin rhythm. We have recently shown that dopamine significantly decreases hamster retinal melatonin levels [18]. Besides, we described day–night variations in hamster retinal dopamine turnover rate with maximal values at midday that persisted under DD conditions. Therefore, it is possible that a retinal circadian pacemaker may drive melatonin rhythm by raising or lowering the activity of GABAergic and dopaminergic neurons at specific circadian times in an independent way, being GABA and dopamine retinal paracrine signals for darkness and light, respectively. Alternatively, since a mutually inhibitory influence between dopaminergic and GABAergic system has been described in the retina of several species [19,22,33], it is possible that the retinal circadian clock, by influencing only one of those systems, could affect the other in a coordinate way. The possibility that the SCN pacemaker drives the retinal GABAergic and dopaminergic rhythms, is not presently excluded and is under current investigation. A close intimacy between retinal and SCN pacemakers must take place in order to finely tune circadian rhythmicity to environmental cycles. In fact, it has been shown that retinal GABAA receptors modulate the responsiveness of the circadian system to light [12]. Many aspects of retinal
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function, from gene transcription to complex intercellular interactions, are regulated by circadian oscillators [7,24,34]. Although much attention has been devoted to the study of retinal rhythm regulation, knowledge of the localization, and nature of the putative retinal clock is incomplete. Our results support the possibility that the retinal GABAergic system, as a mediating component of the ocular rhythm generator, may be involved in the responsiveness to light and circadian rhythmicity, being a part of the mechanisms underlying the temporal regulation of retinal physiology.
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Acknowledgements The authors wish to thank Dr Diego Golombek, Dr Carlos Mendez, and Dr Marcelo de las Heras for helpful discussion of this manuscript. This research was supported ´ Antorchas, Universidad de by grants from Fundacion ´ Buenos Aires, CONICET, Agencia Nacional de Promocion ´ ´ Cientıfica y Tecnica (ANPCyT), and the fellowship ˜ ´ Carrillo-Arturo Onativia’, ‘Ramon National Health Office, Buenos Aires, Argentina.
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[33] D.R. O’Brien, J.E. Dowling, Dopaminergic regulation of GABA release from the intact goldfish retina, Brain Res. 360 (1–2) (1985) 41–50. ´ A. Wirz-Justice, M. Terman, The visual input stage of [34] C.E. Reme, the mammalian pacemaking system. I. Is there a clock in the mammalian eye? J. Biol. Rhythm 6 (1991) 5–29. [35] R.E. Rosenstein, D.P. Cardinali, Melatonin increases in vivo GABA accumulation in rat hypothalamus, cerebellum, cerebral cortex and pineal gland, Brain Res. 398 (1986) 403–406. [36] R.E. Rosenstein, A.G. Estevez, D.P. Cardinali, Time-dependent effect of melatonin on glutamic acid decarboxylase activity and 36 Cl 2 uptake in rat hypothalamus, J. Neuroendocrinol. 1 (1989) 443–447. [37] K. Takatsuji, M. Tohyama, The organization of the rat geniculate
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Research report
Circadian activity of the GABAergic system in the golden hamster retina ´ I. Keller Sarmiento a , Carolina O. Jaliffa a , Daniel Saenz a , Ernesto Resnik b , Marıa a, Ruth E. Rosenstein * a
´ ´ Experimental, Departamento de Bioquımica ´ Humana, Facultad de Medicina, Laboratorio de Neuroquımica Retiniana y Oftalmologıa Universidad de Buenos Aires, Paraguay 2155, 5 to P, 1121, Buenos Aires, Argentina b Medical School, University of Minnesota, Minneapolis, MN 55455, USA Accepted 19 June 2001
Abstract Daily changes in g-aminobutyric acid (GABA) turnover rate were studied in the golden hamster retina. This parameter showed significant variations throughout the light–dark cycle, with minimal values during the day. Retinal glutamic acid decarboxylase (GAD) activity was higher at midnight than at noon. Moreover, [ 3 H]GABA binding significantly varied throughout the 24-h cycle, with maximal values during the day. Saturation studies performed at 12:00 and 24:00 h indicated that the maximal concentration of [ 3 H]GABA binding sites (Bmax ) was significantly higher at noon, whereas the dissociation constant (Kd ) remained unchanged. High K 1 -induced GABA release was significantly higher at midnight than at midday. Daily variations in retinal GABA turnover rate, GABA release, and in its specific binding persisted in golden hamsters exposed to constant darkness. In summary, these results support the idea of a circadian clock-controlled GABAergic activity in the hamster retina. 2001 Elsevier Science B.V. All rights reserved. Keywords: Hamster retina; GABA; Circadian rhythm
1. Introduction GABA is a major inhibitory neurotransmitter of the vertebrate retina, where it plays an important role in visual information processing [27,40]. Horizontal and amacrine cells were identified as the principal GABAergic neurons in the retina of several species [29,40]. In the rabbit retina, GABA immunostaining is sporadic in horizontal cells, while it is intense and reproducible in amacrine cells [29,32]. Although GABA-like immunoreactivity was observed in cat horizontal cell processes [11], the evidence for GABAergic horizontal cells in primate and rodent retina is still controversial. In the golden hamster retina, we have demonstrated GABA-like immunoreactivity in amacrine cells, in neurons localized in the ganglion cell layer, as well as in fibers and terminals at the inner Abbreviations: GABA, g-aminobutyric acid; GAD, glutamic acid decarboxylase; LD, light–dark cycle; DD, dark–dark cycle *Corresponding author. Tel.: 154-11-4508-3672, ext. 37; fax: 154-114508-3672, ext. 31. E-mail address: [email protected] (R.E. Rosenstein).
plexiform layer. In addition, a few horizontal cells also showed GABA-like immunolabeling [25]. Recently, we have shown that GABA increases melatonin content in the hamster retina, likely through a GABAA receptor-mediated mechanism [17]. The stimulatory effect of exogenous GABA was evident during the day but not during the night. The GABAA receptor antagonist, bicuculline, although having no effect per se under light conditions, significantly inhibited the darkness-induced stimulation of melatonin content, providing evidence for the involvement of endogenous GABA in the pathway of darkness input to the melatonin-generating system. Alternatively, since it has been shown that the hamster retina contains a circadian oscillator that regulates melatonin synthesis [38], these results permit the presumption that the retinal GABAergic system could mediate the action of a local pacemaker on melatonin biosynthesis. This hypothesis would have the pacemaker-induced regulation of melatonin levels by raising and lowering GABAergic activity at specific daytime points. A necessary (although not sufficient) condition to prove this idea would be to demonstrate daily variations and free running rhythms of retinal GABAergic activity.
0006-8993 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 01 )02736-6
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We have previously demonstrated that hamster retinal GABA turnover rate is significantly higher at 24:00 than at 12:00 h [17], but a detailed study on the temporal course of this and other GABAergic parameters was still lacking. Therefore, the aim of the present study was to examine GABA turnover rate, its specific binding, and GABA release in retinas of golden hamsters exposed to a light– dark (LD) or a constant dark cycle (DD).
2. Material and methods
2.1. Reagents and drugs GABA, L-glutamic acid, 3-mercaptopropionic acid, GABAse, and pyridoxal phosphate were obtained from Sigma (St Louis, MO, USA). [ 3 H]GABA, 1-[ 14 C]Lglutamic acid, and [ 3 H]muscimol were purchased from New England Nuclear Corp. (Boston, MA, USA).
2.2. Animals Male golden hamsters (average weight 120620 g), derived from a stock supplied by Charles River Breeding Laboratories (Wilmington, MA, USA), were purchased from a local dealer. Unless indicated, hamsters were kept under a photoperiod of 14 h of light–10 h of darkness (LD, lights on at 06:00 h), with free access to food and water. In some experiments, the animals were kept in constant darkness (DD) for 48 h. Since the average circadian period in our hamsters is of 24.1 h, after 48 h of constant darkness, circadian times would shift, maximally, 6 or 12 min. Thus, we assumed that the circadian times (CT, being CT512 defined as the time of locomotor activity onset), would approximately equal their previous zeitgeber times (ZT, with ZT512 defined as the time of lights off), and sacrificed the animals at six intervals through the 24 h-cycle. The animals were killed by decapitation, their eyes were immediately enucleated, and the retinas were excised and processed as described below. In the case of dark-exposed hamsters, sacrifice of animals and extraction of retinas were carried out under dim red light.
2.3. GABA turnover rate assessment GABA turnover rate was measured as previously described [35]. In order to prevent the postmortem increase in GABA content, the animals were injected with the GAD inhibitor, 3-mercaptopropionic acid (50 mg / kg i.p.) 2.5 min before sacrifice [39]. GABA turnover rate was assessed by the accumulation of GABA levels following inhibition of GABA transaminase [5]. The major assumption of the method used was that after the administration of g-vinyl GABA (a gift from Merrel Dow Research Institute, Strasbourg, France), the accumulation of GABA was linear for at least 1 h. g-Vinyl GABA was injected i.p. at a 1
g / kg dose, 62.5 min before sacrifice. Control animals, which received i.p. injections of vehicle (300 ml of saline), were used for the determination of the steady-state GABA concentrations. Retinas were excised, individually homogenized in distilled water and centrifuged at 12 0003 g for 15 min, the pellets being discarded. GABA concentrations were assessed by a radioreceptor assay as described by Bernasconi et al. [4], with detection range for GABA of 2.5–100 pmol. In order to ascertain whether the displacement of [ 3 H]muscimol from its binding sites was due to authentic GABA, tissue samples were incubated with the highly specific GABA-degrading enzyme system, GABAse, before the assay. Either standard or endogenous GABA was completely (i.e. more than 85%) degraded by GABAse.
2.4. GAD activity assessment GAD activity was determined as previously described [36]. Aliquots of homogenate (0.2–0.4 mg protein) corresponding to one retina / point were incubated in 1.5 mlEppendorf polypropylene tubes at 378C for 1 h. The incubation mixture (300 ml) contained 1-[ 14 C]L-glutamic acid (specific activity 49.6 Ci / mmol, 0.2 mCi / ml), 0.5–2 mM L-glutamic acid, 200 mM pyridoxal phosphate, and 10 mM b-mercaptoethanol. Incubations were carried out in triplicates at six different concentrations of L-glutamic acid (0.5–3 mM). The reaction was stopped by adding 10% trichloroacetic acid, and the 14 CO 2 released during an additional 2-h period was captured on filter paper embedded in hyamine hydroxide. Blanks included all reagents except that TCA was added before the homogenate. Double-reciprocal plots were constructed from the mean of triplicates, at each L-glutamic acid concentration.
2.5. [ 3 H] GABA release Retinas were incubated for 30 min at 378C with [ 3 H]GABA (0.5–1 mCi) in 500 ml of buffer containing 140 mM NaCl, 5 mM KCl, 2.5 mM CaCl 2 , 1 mM MgCl 2 , 10 mM Hepes, and 10 mM glucose, adjusted to pH 7.4 with Tris base. The tissues were washed several times in fresh buffer in order to remove the excess [ 3 H]GABA, and incubated for 10 min with gentle shaking in 500 ml of the same buffer or a high K 1 (50 mM) buffer in which osmolarity was conserved by equimolar reduction of Na 1 concentration. Tissues were digested with hyamine hydroxide, and radioactivity in the medium and that incorporated into the tissue were determined in a scintillation counter. Fractional release was calculated as the ratio of radioactivity released / total radioactivity uptaken by the tissue. Greater than 80% of the released radioactivity was identified as authentic GABA by thin layer chromatography.
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2.6. GABA binding assay [ 3 H]GABA binding was assessed by a standard procedure as previously described [1]. Briefly, retinal pools were homogenized in 4 ml of water, and centrifuged at 9003g for 20 min. The supernatant was centrifuged at 20 0003g for 20 min. The pellet was frozen at 2708C until the binding assay was performed (usually within 18 h). On the day of the experiment, the pellet was resuspended in 0.05 M Tris–HCl (pH 7.2) and Triton X-100 was added to achieve a final concentration of 0.01% (v / v). The mixture was incubated at 378C for 30 min in a shaking bath incubator. After centrifuging for 20 min at 20 0003g, the new pellet was washed five times with Tris–HCl and was finally resuspended in a fresh aliquot of Tris–HCl buffer. Aliquots of this homogenate (0.2–0.4 mg protein) were incubated for 10 min at room temperature in a total volume of 150 ml. The reaction was initiated by adding 10 nM of [ 3 H]GABA (80.8 Ci / mmol) together with varying concentrations of unlabeled GABA (6–200 nM), in the presence (nonspecific binding) or absence (total binding) of 200 mM unlabeled GABA. For single point binding assessment, unlabeled GABA was omitted. After incubation, the reaction was stopped by centrifuging the samples for 5 min at 20 0003g. The supernatant fraction was discarded and the pellet was gently washed twice with 1 ml of cold Tris–HCl buffer. Radioactivity was extracted by resuspending the pellet in 100 ml of hyamine hydroxide and leaving the samples overnight at room temperature. Under these conditions, [ 3 H]GABA binding was completely displaced by 20 mM muscimol. The dissociation constant (Kd ) and maximal concentration of binding sites were calculated by Scatchard analysis. Protein content was determined by the method of Lowry et al. [26], using bovine serum albumin as the standard. Statistical analysis of the results was made by Student’s t-test or by a two-way analysis of variance (ANOVA) followed by a Tukey’s test, as stated. All animal use procedures were in strict accordance with the NIH Guide for Care and Use of Laboratory Animals.
Fig. 1. Retinal GABA turnover rate throughout the 24-h cycle. Hamsters were injected (i.p.) with g-vinyl GABA (1 g / kg) 62.5 min before sacrifice, and received 50 mg / kg 3-mercaptopropionic acid 2.5 min before sacrifice. GABA levels were assessed by a radioreceptor assay. Data are the means6S.E.M. (n515 animals per group). This parameter showed significant daily variations (P,0.01, ANOVA); values at 08:00 and 12:00 h were significantly lower than at all the other intervals (**P,0.01, by Tukey’s test).
showed significant variations with a similar profile to that observed for GABA levels accumulated after the inhibition of its degradation (Fig. 2). Since both GABA content and GABA turnover rate showed higher values during the night, it seems likely that the synthesis of GABA in the retina varies during the 24-h cycle. In order to assess time-dependent variation in GABA synthesis, the activity of GAD was examined in the retina of animals killed at
3. Results Fig. 1 depicts the daily changes in GABA turnover rate of golden hamster retinas excised at six different intervals throughout the 24-h cycle. This parameter exhibited significant daily variations, showing minimal values during the day, at 08:00 and 12:00 h. GABA turnover rate was assessed following the administration of g-vinyl GABA. In these conditions, each point represents GABA levels accumulated after the inhibition of GABA transaminase activity. In the next experiment, GABA levels were measured similarly but without the inhibition of GABA degradation, in order to assess steady-state GABA concentrations along the 24-h cycle. This parameter also
Fig. 2. Daily variations in steady-state concentrations of GABA in the hamster retina. Animals received 50 mg / kg 3-mercaptopropionic acid 2.5 min before sacrifice. GABA levels were assessed by a radioreceptor assay. Data are means6S.E.M. (n515 animals per group). This parameter showed significant daily variations (P,0.01, ANOVA), the values during the day (except at 16:00 h) being significantly lower than the corresponding values during the night (**P,0.01, by Tukey’s test).
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12:00 or 24:00 h. Kinetics of GAD activity in retinas excised at midday and midnight are shown in Fig. 3. The Vmax of retinal GAD was significantly higher at 24:00 h (1.6260.07 nmol / min per mg protein) than at 12:00 h (1.1260.06 nmol / min per mg protein), while the Km remained unchanged (2.3560.2 and 2.1760.3 mM at 24:00 and 12:00 h, respectively). This result suggests an increase in the number of active GAD molecules during the night, in accordance with the daily variation of the retinal GABA turnover rate and steady-state GABA concentrations. Daily rhythmicity in retinal GABA binding is shown in Fig. 4. GABA binding was significantly higher at 08:00 and 12:00 h than at the rest of the daytime points. Saturation studies were performed in retinas of hamster killed at noon and midnight (Fig. 5). The calculated Bmax at 12:00 h (460625 fmol / mg protein) was significantly higher than at 24:00 h (205620 fmol / mg protein), whereas no significant changes were found for the binding affinity (Kd 54264 and 3763 nM at 12:00 and 24:00 h, respectively). This result indicate time-dependent variations in the density of retinal GABA receptors.
Fig. 4. [ 3 H]GABA binding in hamster retinas excised throughout the light–dark cycle. [ 3 H]GABA binding was assessed as described in Material and methods. Shown are means6S.E.M. (n512 animals / group). This parameter exhibited significant daily variations (P,0.01, ANOVA), the values at 08:00 and 12:00 h being significantly higher than at the rest of the daytime points (**P,0.01 by Tukey’s test).
In order to examine whether daily variation of GABA turnover and GABA binding are driven by a circadian clock, the same parameters were assessed in animals
Fig. 3. GAD activity in hamster retina was assessed by the CO 2 -trapping procedure using 1-[ 14 C]L-glutamate as substrate, as described in Material and methods. Slopes and Y-intercepts were calculated by the method of least squares. The points represent mean values from triplicates samples using one retina / point; differences among triplicates were less than 10%. In this experiment, representative of four, Vmax values were 1.02 and 1.58 nmol / min per mg protein at 12:00 and 24:00 h, respectively, and Km values were 2.01 mM (at 12:00 h) and 2.2 mM (at 24:00 h). Vmax was significantly higher (P,0.01, Student’s t-test) at 12:00 than at 24:00 h. Means6S.E.M. for Vmax and Km (n54) are given in the text.
Fig. 5. Scatchard regression plots of specific [ 3 H]GABA binding in the retina of hamster killed at 12:00 and 24:00 h. Slopes and Y-intercepts were calculated by the method of least squares. Each point represents mean values from triplicates using 10 animals per group, differences among triplicates were less than 10%. In this experiment, representative of four, Kd values were 40 and 35 nM at 12:00 and 24:00 h, respectively, and Bmax values were 450 and 185 fmol / mg protein at 12:00 and 24:00 h, respectively. Bmax was significantly higher (P,0.01, Student’s t-test) at 12:00 than at 24:00 h. Mean6S.E.M. for Bmax and Kd (n54) are given in the text.
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Daily variations of GABA release in the golden hamster retina were assessed. The basal and high K 1 -induced [ 3 H]GABA release was examined at midday and midnight (animals under a LD photoperiod) as well as at subjective midday and subjective midnight (animals kept under constant darkness). In agreement with the results obtained for GABA turnover rate and GABA synthesis, depolarization-induced GABA release was significantly higher at midnight (LD), and during the subjective midnight (DD) as shown in Fig. 8.
4. Discussion
Fig. 6. Circadian variations in retinal GABA turnover rate of hamsters kept under constant darkness for 2 days before sacrifice. Data are the means6S.E.M. (n515 animals per group). This parameter showed significant circadian variations (P,0.01, ANOVA); the values at clocktime 08:00 and 12:00 h were significantly lower than at the rest of the daytime points (**P,0.01, by Tukey’s test).
exposed to free running conditions. When animals were housed in constant darkness for 2 days before the experiment, and killed at equivalent daytime points, daily variations in retinal GABA turnover rate persisted (e.g. minimal values during the subjective day), but a significant increase in this parameter was already attained at clocktime 16:00 h (Fig. 6). Fig. 7 shows circadian variations in retinal [ 3 H]GABA binding in the retinas of hamsters kept under DD 2 days before sacrifice. A significant increase in this parameter was observed during the subjective day, indicating an endogenous control of its diurnal variation.
Fig. 7. Circadian variations of retinal [ 3 H]GABA binding of hamsters kept under constant darkness for 2 days before sacrifice. Shown are means6S.E.M. (n512 animals / group). This parameter showed significant circadian variations (P,0.01, ANOVA), the values at clocktime 08:00 and 12:00 h were significantly higher than at the rest of the daytime points (**P,0.01, by Tukey’s test).
Dopamine and GABA are the most studied retinal neurotransmitters regarding responsiveness to light and circadian rhythmicity [34]. Extensive evidence demonstrates that retinal dopamine transduces the light signal mimicking the acute inhibitory effect of light on melatonin biosynthesis. Accordingly, light increases the synthesis, release, and metabolism of dopamine in retinas of several species [6,13,16,41]. Moreover, it has been shown that rat retinal dopamine synthesis varies over the 24-h cycle with maximal values during the day [28]. In contrast to dopamine, no information is available about time-dependent variations of retinal GABAergic activity. Our results indicate that in the golden hamster retina, significant 24-h variations occur in both GABA steady-state concentrations and turnover rate. A time-dependent variation in GABA content of selected brain areas had been previously demonstrated in the rat tuberoinfundibular region and hypothalamus [9,10]. In the present work, besides GABA content, GABA turnover rate (an indicator that correlates with traffic neural signals in GABAergic neurons), was measured by its accumulation after inhibition of its degradation [14]. In a previous report, we observed the existence of daily variations in GABA turnover rate of cerebral cortex, cerebellum, hypothalamus, and pineal gland of golden hamsters, which also showed a tendency for maximal values towards the first half of the night [20]. Since in the present study GABA levels were measured by inhibiting its degradation, its synthesis is a presumptive target for time-dependent variations. Accordingly, a nocturnal increase in GAD Vmax was demonstrated with no change in the Km . In the rat brain, GABAA and benzodiazepine binding sites varied daily both in their number and affinity, with nocturnal and diurnal maxima, respectively [1,2], while a nocturnal peak in GABA postsynaptic effect was reported [21]. Since both GABA turnover rate and GABA release were higher during the night, it seems that an inverse relationship between these parameters and GABA binding occurs in the hamster retina, as an augmented value of this latter was observed during the day. From these results, it can be hypothesized that GABA binding sites are up- and down-regulated by their own ligand. As thoroughly
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Fig. 8. [ 3 H]GABA release in the golden hamster retina. Animals were kept under LD or DD cycles. Shown are means6S.E.M. (n512 animals / group). High K 1 (50 mM) induced a significant [ 3 H]GABA release at each time point (P,0.01, by Student’s t-test). This parameter was significantly higher at midnight than at midday (LD), and at subjective midnight than at subjective midday (DD) (**P,0.01, by Student’s t-test). No differences were found for the basal release of GABA.
washed membranes were used in all daytime points, variations in the occupancy of the receptor by endogenous GABA is unlikely. In fact, daily variations in GABA binding seem to be related to an increase in the density of GABA receptors, as indicated by the increase in Bmax at 12:00 relative to 24:00 h. The distribution of GABA receptors among the different cell types of the retina is not yet known in detail but the available results indicate that they are present in at least some members of each basic type of retinal cells [15]. Since GABA binding was assessed in the whole retina, we could not ascertain the locus of the observed phenomena. Therefore, the possibility that the measured changes reflect larger or lesser changes in specific retinal cell population(s) remains to be tested. A daily rhythm in the retina may be controlled by a circadian oscillator, by direct response to environmental lighting levels, or by a combination of these mechanisms. The observation that daily variations of GABA turnover rate, binding, and release persisted under constant conditions supports the idea of a clock-controlled function. Although the possibility that light may influence retinal GABAergic activity is not excluded, the light–dark cycle seems not to be necessary for the generation of its rhythm. In mammals, circadian oscillators have been identified in the suprachiasmatic nucleus (SCN) of the hypothalamus [23] and, more recently, in cultured neural retinas of the golden hamster [38]. GABA is detectable in nearly every neuron of the SCN [31], where a circadian rhythmicity of GABA content and GAD activity has been shown [3]. Besides the retina and the SCN, GABAergic neurons are also important in other components of the circadian
system, including the intergeniculate leaflet and the thalamic lateral geniculate complex [30,37]. Because of this key distribution, it was postulated that GABA is the principal neurotransmitter of the circadian timing system [8,31]. In view of this, and taken together with the rhythmical production of retinal melatonin and its regulation by GABA, it is tempting to speculate that the ocular circadian clock may act through the local GABAergic system to drive the melatonin rhythm. We have recently shown that dopamine significantly decreases hamster retinal melatonin levels [18]. Besides, we described day–night variations in hamster retinal dopamine turnover rate with maximal values at midday that persisted under DD conditions. Therefore, it is possible that a retinal circadian pacemaker may drive melatonin rhythm by raising or lowering the activity of GABAergic and dopaminergic neurons at specific circadian times in an independent way, being GABA and dopamine retinal paracrine signals for darkness and light, respectively. Alternatively, since a mutually inhibitory influence between dopaminergic and GABAergic system has been described in the retina of several species [19,22,33], it is possible that the retinal circadian clock, by influencing only one of those systems, could affect the other in a coordinate way. The possibility that the SCN pacemaker drives the retinal GABAergic and dopaminergic rhythms, is not presently excluded and is under current investigation. A close intimacy between retinal and SCN pacemakers must take place in order to finely tune circadian rhythmicity to environmental cycles. In fact, it has been shown that retinal GABAA receptors modulate the responsiveness of the circadian system to light [12]. Many aspects of retinal
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function, from gene transcription to complex intercellular interactions, are regulated by circadian oscillators [7,24,34]. Although much attention has been devoted to the study of retinal rhythm regulation, knowledge of the localization, and nature of the putative retinal clock is incomplete. Our results support the possibility that the retinal GABAergic system, as a mediating component of the ocular rhythm generator, may be involved in the responsiveness to light and circadian rhythmicity, being a part of the mechanisms underlying the temporal regulation of retinal physiology.
[12]
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Acknowledgements The authors wish to thank Dr Diego Golombek, Dr Carlos Mendez, and Dr Marcelo de las Heras for helpful discussion of this manuscript. This research was supported ´ Antorchas, Universidad de by grants from Fundacion ´ Buenos Aires, CONICET, Agencia Nacional de Promocion ´ ´ Cientıfica y Tecnica (ANPCyT), and the fellowship ˜ ´ Carrillo-Arturo Onativia’, ‘Ramon National Health Office, Buenos Aires, Argentina.
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