Circadian rhythmicity in the GABAergic system in the suprachiasmatic nuclei of the rat

Neuroscienee Letters, 157 (1993 ) 199-202 © 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304°3940193l$ 06.00

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Circadian rhythmicity in the GABAergic system in the suprachiasmatic nuclei of the rat Ra61 Aguilar-Roblero, Luis Verduzco-Carbajal, Claudia Rodriguez, Jesus Mendez-Franco, Julio Mor~m and Miguel Perez de la M o r a Departamento de Neurociencias, Instituto de Fisiologia Celular, Universidad Nacional Autonoma de MOxico, Mkxico DF (Mexico)

(Received 5 April 1993; Revised version received 23 April 1993; Accepted 26 April 1993) Key words:

Neuroendocrine; Hypothalamus; Suprachiasmatic nucleus; Chronobiology; Glutamic acid decarboxylase; Biological rhythm

The participation of GABAergic mechanisms in the regulation of circadian rhythmicity by the suprachiasmatic nuclei (SCN) has been suggested from different lines of evidence. Little is known, however, whether GABA synthesis, release, uptake or content within the SCN may show a circadian pattern. The present results show that the activity of the GABAergic system within the SCN region of the rat exhibits circadian rhythmicity, which is manifested by correlative changes of the GABA content and the glutamic acid decarboxylase activity under the light/dark cycle, and by changes in the GABA content in animals kept under constant darkness.

The hypothalamic suprachiasmatic nuclei (SCN) have been shown in rodents to function as an oscillator involved in the regulation of several circadian rhythms [18]. Immunohistochemical studies have shown that more than 10 neuroactive substances are present within the neural elements in the SCN [3, 10, 28]. Most of these substances are distributed in either the dorsomedial (i.e. vasopressin) or the ventromedial (i.e. vasoactive intestinal polypeptide) regions of the nuclei, while only few of them (i.e. GABA) are found throughout the whole SCN [27]. Pharmacological manipulations of acetylcholine [16, 29], glutamate, aspartate [2] and neuropeptide Y [1] systems have shown that these neurotransmitters are able to modify the phase setting of circadian rhythms. The participation of GABAergic transmission in the regulation of circadian rhythmicity and SCN function has been suggested from immunohistochemical [8, 19, 21, 27], pharmacological [22-24] and electrophysiological [12, 15] studies. Little is known, however, whether GABA synthesis, release, uptake or content within the SCN may show a circadian pattern [2]. The present study is aimed towards establishing whether GABA synthesis and GABA content within the SCN show a circadian fluctuation. Correspondence." R. AguilaroRoblero, Departamento de Neurociencias, Instituto de Fisiologia Celular, UNAM, Apartado Postal 70-253, Mexico 04510 D.F., Mexico. Fax: (52-5) 622-5607 and 548-0387.

Male Wistar rats weighing between 200 and 300 g were housed in a sound-attenuated room kept at constant temperature (20 _+ I°C). The animals were maintained for at least 3 weeks under a 12:12 h light/dark cycle, lights were turned on at 08.00 h. The average light intensity in the room was 450 lux. After the habituation period to the light cycle, animals for the light-dark experiments (LD) were immediately processed as described below. Those animals used for experiments in constant darkness (DD) were separated from the colony and kept for 3 days under constant dim red light (50 lux) and subsequently processed. For each experiment, four or five animals were beheaded at 3 h intervals throughout the next 24 h starting at noon. This procedure was accomplished depending upon the type of illumination (bright white light or dim red light) in which the animal was at the time. For each animal the brain was quickly removed and collected in ice cold Krebs solution. A piece of 1.0 mm 3 from the anterior hypothalamus containing the SCN and the adjacent optic chiasm was dissected under a stereoscopic microscope. As a control a similar piece from the occipital cortex was also obtained. The blocks of tissue were immediately frozen with dry ice. In all cases the procedure was completed in less than 3 min. The tissue was stored individually in small plastic tubes kept at -70°C until further processing. The accuracy of the SCN dissection procedure was corroborated by histological examination of the remaining brain in 5 animals selected at random.

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In a first experiment we addressed whether there was a circadian rhythm in the GABA content of the SCN region; 72 animals were studied under either LD (n = 32) or D D (n = 40). In this design the number of animals per cell under LD and D D conditions was 4 and 5, respectively. Cellular contents of endogenous GABA were determined in ethanol extracts. The tissue was homogenized in 100/11 of 70% (v/v) ethanol and centrifuged at 5000 x g for 1 min. The GABA analysis was carried out after derivation with o-phtaldialdehyde by reverse phase H P L C according to Geddes and Wood [10] in a Beckman chromatograph equipped with an Ultrasphere column. In a second experiment we addressed whether there was a diurnal fluctuation in the G A D activity of the SCN region; 40 animals were studied under LD, n = 5 for each cell in the design. G A D activity was measured as described by Perez de la Mora et al. [19, 20]. The protein content was determined by the method of Lowry et al. [13]. The statistical analysis of the fluctuations in the content of GABA or in the activity of G A D as a function of the time of the day was performed by a one-way ANOVA. When necessary, the contrast of groups showing significant differences was accomplished by the Tukey test. The ~ level was set at 0.05. Examination of the histological sections showed in all cases complete ablation of the SCN, the dissected tissue also included the medial optic chiasm and the immediately surrounding hypothalamic areas. The GABA content within the suprachiasmatic region from animals kept under the light/dark cycle (LD) showed clear variations dependent upon the time of sacrifice (Fig. 1A, open circles). This fluctuation persisted even when the animals were kept under constant dim red light (Fig. 1A, filled circles). However, two main differences were evident between the animals kept in LD and those in DD: First, there was a shift in the time of the peak levels of GABA from 24 h in LD to 21 h in D D and second, there was a decrease in the daily average content of GABA from 17.5 + 6.7 (nmol/mg of protein; mean + S.D.) in animals from LD to 8.9 + 6.9 in animals from DD, In contrast to the SCN, the content of GABA within the occipital cortex showed non significant variations across the day in animals housed in LD (Fig. IA, triangles). G A D activity from the SCN region also showed variations depending on the time of the day (Fig. 1B, open circles). Although the statistical analysis detected only one significant peak at 24 h, visual inspection of the plotted data suggest that there may be a bimodal pattern with another peak of G A D activity between 15 and 18 h. In the occipital cortex, there is also a significant trend for a 24 h fluctuation in G A D activity, with a peak value at 24 h and a smaller peak at 15 h (Fig. 1B, filled circles). The relationship between G A D activity (circles) and

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Fig. 1. Temporal pattern of GABA content (top) and G A D activity (bottom) within the SCN and the occipital cortex. A: there was a clear circadian fluctuation in the content of GABA within the SCN from animals herd under LD cycles (open circles), which persisted in those animals held under constant darkness (filled circles), notice the shift in the time of the peak value of GABA in the SCN between both lighting conditions; while in the occipital cortex (triangles) the content of GABA remained constant in LD. B: there were diurnal fluctuations in the activity of the GAD both in the SCN (open circles) and the occipital cortex (filled circles) from animals held under LD. The asterisks indicate significant differences P < 0.05 (*LD, **DD).

GABA content (triangles) in the SCN is shown in Fig. 2. One can observe a clear correlation between the activity of the enzyme and the tissular content of the neurotransmitter. The present results show circadian rhythmicity on the activity of the GABAergic system within the SCN region of rats, which is manifested by correlative changes of the GABA content and the G A D activity along the 24 h. The fluctuation in the content of GABA persisted even under constant darkness, suggesting its endogenous nature. This notion is further supported by the shift in the time of the peak GABA values found in animals in D D with respect to those in ED (Fig. 1A). In regard to the activity of G A D within the SCN (Fig. IB), there is also a clear rhythmicity in LD which follows very closely with that of

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Fig. 2. Temporal correlation between the content of GABA (triangles, left scale) and the activity of the GAD (circles, right scale) within SCN. The horizontal bar indicates the light cycle, open bar lights-on, filled bar lights-off.

GABA levels (Fig. 2). This observation is suggestive that the rhythmicity in the activity of GAD could be endogenous as well, although that issue remains to be further studied. Furthermore, the regional specificity for the changes on the GABAergic system seems to be indicated by the lack of significant variations of GABA levels in the occipital cortex along the 24 h and for the lack of correlation between GABA levels and the GAD activity found in the same region. Diurnal variations in the content of GABA in the hypothalamus have been previously reported in rats [7] and hamsters [11], but the timing of the peak GABA levels were found during the daytime (lights on period). However, since GABA neurons are widely distributed through the hypothalamus, the timing of peak GABA levels in this region may reflect the phase of hypothalamic regions other than the SCN. It is well known that there is a postmortem increase in GABA levels [25]. However, since we were interested in the study of the temporal pattern of GABA content of the SCN along a 24 h period and we wanted to keep the animals undisturbed as much as possible, no attempt was made to prevent such an increase considering that relative GABA levels were satisfactory. The values for GABA levels and GAD activity reported in this work were similar to those reported by Tappaz et al. [26]. Previous studies have implicated GABAergic mechanisms in the regulation of circadian rhythmicity by light. Phase delays induced by brief pulses of light presented during early subjective night are specifically blocked by bicuculline [22], while phase advances induced during late subjective night are specifically blocked by diazepam [23]. Furthermore, it has been shown that these effects are mediated by the GABAA-benzodiazepine receptor. Administration of baclofen also blocks the phase shifting

effect of light, but in contrast to the previously mentioned drugs, baclofen affected both advances and delays [24]. This latter observation suggests that GABAa receptor is also involved in the modulation of circadian rhythmicity by light. The lower levels of GABA observed in the SCN of the rats maintained under DD conditions in comparison with those held under a LD regimen might be explained by a retinal control of GABA levels as has been suggested by Ralph and Menaker [24]. The present study provides evidence for a circadian behavior of the GABAergic system in the SCN and supports the involvement of GABAergic neurons in the regulation of circadian rhythms. However, since GABA containing neurons have also been reported in the retinal ganglion [6, 14] and the intergeniculate leaflet [17], two structures known to project to the SCN [4, 5], it is possible that the GABA levels and GAD-activity measured in the SCN would reflect not only what actually occurs in the SCN neurons and intrinsic axons, but rather the dynamics of the complete system. Further studies are needed to clarify this point. This study was partially supported by Grant IN200791 from DGAPA/UNAM. We thank Jacqueline Vazquez for proofreading the manuscript. 1 Albers, H.E. and Ferris, C.F., Neuropeptide Y role in light-dark cycle entrainment of hamster circadian rhythms, Neurosci. Lett., 50 (1984) 163-168. 2 Albers, H.E., Liou, S.Y., Ferris, C.F., Stopa, E.G. and Zoeller, R.T., Neurochemistry of circadian timing. In D.C. Klein, R.Y. Moore and S.M. Reppert (Eds.), Suprachiasmatic Nucleus. The Mind's Clock, Oxford University Press, New York, 1991, pp. 263 288. 3 Card, J.P. and Moore, R.Y., The suprachiasmatic nucleus of the golden hamster: Immunohistochemical analysis of cell and fiber distribution, Neuroscience, 13 (1984)415431. 4 Card, J.P. and Moore, R.Y., Organization of lateral geniculatehypotalamic connections in the rat, J. Comp. Neurol., 284 (1989) 135-147. 5 Card, J.P. and Moore, R.Y., The organization of visual circuits influencing the circadian activity of the suprachiasmatic nucleus. In D.C. Klein, R.Y. Moore and S.M. Reppert (Eds.), Suprachiasmatic Nucleus. The Mind's Clock, Oxford University Press, New York, 1991, pp. 51 76. 6 Caruso, D.M., Owczarzak, M.T. and Poucho, M.G., Colocalization of substance P and GABA in retinal ganglion cells: a computerassisted visualization, Vis. Neurosci., 5 (1990) 389 394. 7 Cattanebi, F., Maggi, A., Monduzzi, M., DeAngelis, L.D. and Racagni, G., GABA: circadian fluctuations in rat hypothalamus., J. Neurochem., 31 (1978) 565-567. 8 Francois-Bellan, A.M., Kachidian, P., Dusticier, G., Tonon, M.C., Vaudry, H. and Bosler, O., GABA neurons in the rat suprachiasmatic nucleus: involvement in chemospecific synaptic circuitry and evidence for GAD-peptide colocalization, J. Neurocytol., 19 (1990) 937 947. 9 Geddes, J.W. and Wood, J.D., Changes in the aminoacid content of nerve endings (synaptosomes) induced by drugs that alter the me-

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