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GABAergic modulation of optic nerve-evoked field potentials in the rat suprachiasmatic nucleus
BRAIN RESEARCH ELSEVIER
Brain Research 694 (1995) 264-27(/
Research report
GABAergic modulation of optic nerve-evoked field potentials in the rat suprachiasmatic nucleus Robert L. Gannon a, Matthew J. Cato a, Kathryn Hart Kelley ~, Deborah L. Armstrong b, Michael A. Rea a,* Biological Rhythms and lntegratit~e Neurosciences Research Institute, Armstrong Laboratory / CFTO, Brooks AFB, TX 78235, USA h Division of Life Sciences, University of Texas at San Antonio, San Antonio, TX, USA Accepted 21 June 1995
Abstract
The suprachiasmatic nuclei (SCN) at the base of the hypothalamus are known to be the site of the endogenous circadian pacemaker in mammals. The SCN are innervated by the retinohypothalamic tract, which conveys photic information to the SCN. GABA is one of the most abundant neurotransmitters in the SCN, and has been implicated in the modulation of photic responses of the SCN circadian pacemaker. This study sought to examine the effect of GABAergic compounds on optic nerve-evoked SCN field potentials recorded in rat horizontal hypothalamic slices. The GABA A agonist muscimol (10 #M) potentiated SCN field potentials by 23%, while application of the GABA A antagonist bicuculline (10 /xM) inhibited SCN field potentials by a similar amount, (22%). Conversely, the GABA n agonist baclofen (1.0 /.LM) inhibited SCN field potentials by 48%, while the GABA B antagonist phaclofen (0.5 mM) augmented SCN field potentials by 62%. Recordings performed at both day and night times indicate that there were no qualitative day-night differences in GABAergic activity on SCN field potentials. This study concludes that, in general, GABA A activity tends to increase, and GABA n activity tends to decrease the response of SCN neurons to optic nerve stimulation. Keywords: Hypothalamus; Brain slice; y-Aminobutyric acid; Circadian rhythm
1. Introduction
Circadian rhythms in mammals arise through the action of an endogenous oscillator located in the suprachiasmatic nuclei (SCN) at the base of the hypothalamus [16,17,20]. Under normal conditions, the circadian 'clock' is synchronized (entrained) to the environmental light:dark cycle through photic signals communicated from the retina to the SCN by the retinohypothalamic tract (RHT) [9,18]. Photic entrainment of the SCN circadian clock is achieved by daily adjustments of the phase of the circadian oscillation in response to retinal illumination [5,28]. Light exposure during the night (or dark period) causes either phase delays or phase advances of the circadian oscillation depending on the time of exposure, while light during the day (or light period) does not alter the phase of the oscillation. The photic input to the SCN is known to be modulated
* Corresponding author. Fax: (1) (2101 536-3513. 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved
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by at least three major neurotransmitters, glutamate, serotonin, and y-aminobutyric acid (GABA). Glutamatergic antagonists block light induced phase shifts of hamster circadian wheel running activity, as well as optic nerve evoked SCN field potentials [23,24], and glutamate is believed to be the primary excitatory neurotransmitter released from RHT terminals. In addition, previous work from this laboratory found that serotonergic agonists also inhibit light-induced phase shifts of hamster circadian wheel running and optic-nerve evoked field potentials [24], suggesting that serotonin may function as a modulator of photic responsiveness in the SCN. However, the most abundant neurotransmitter type in the SCN is G A B A [19,31]. GABAergic interneurons comprise a dense network within the SCN [19,31], including a reciprocal innervation to each side of the nucleus [3]. This dense innervation by GABA in the SCN suggests a role of these neurons in regulating pacemaker activity. In addition, systemic administration of various GABAergic agents has been reported to attenuate light-induced phase shifts and c-fos expression in the SCN [4,21], suggesting that, like sero-
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tonin, GABA may modulate photic responsiveness in the SCN. In order to further elucidate the role of GABA as a modulator of photic responses in the SCN, we investigated the effects of selective agonists and antagonists of GABA A and GABA B receptors on optic nerve evoked synaptic activity in the rat SCN. The results indicate that GABA B receptors mediate tonic inhibition of optic nerve-evoked responses in the SCN, while GABA A receptors appear to facilitate responses of SCN neurons to optic nerve activity.
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Male Sprague-Dawley rats (Charles River Laboratories; 150-200 g) were maintained under a 12:12 h light/dark cycle for several weeks prior to use. Food and water were provided ad libitum. Four hs after light on (day slices), rats were anesthetized with halothane and decapitated, and the brains were rapidly removed and cooled with ice-cold artificial cerebrospinal fluid (ACSF) consisting of (in mM): NaCI (122), KCI (3.8), MgSO 4 (1.2), KH2PO 4 (1.2), NaHCO 3 (25), CaCI 2 (2.5) and glucose (10). The brains were further freehand dissected so that only a tissue block containing the hypothalamus and optic nerves remained. The tissue block was affixed to a vibratome pedestal using cyanoacrylic glue and a 400-500 /xm thick horizontal hypothalamic slice was cut which contained the suprachiasmatic nuclei and both attached optic nerves. Night slices were prepared in the same manner, only rats were sacrificed between 1 h before and 1 h after lights out. Hypothalamic slices were incubated in ACSF for 4 h at room temperature under an atmosphere of 95%:5% O2/CO 2. After incubation, slices were transferred to a temperature controlled Hatton-type recording chamber and super/used with ACSF at 0.9 ml/min using a peristaltic pump at a temperature of 35°C. Slices were completely submerged in the recording chamber, which had a volume of 0.25 ml. Drugs were prepared in ACSF and applied to the slice by means of a 3 way valve in the line delivering ACSF to the slice. Bipolar suction electrodes were used to deliver a square wave pulse to the optic nerve (1.0 Hz, 0.3 ms) at a maximal stimulation intensity of 0.7 mA. The resultant extracellular field potentials were recorded in the contralateral suprachiasmatic nucleus using 3.0 M NaCl-filled glass microelectrodes with a resistance of approximately 1 M/-2. Field potentials were amplified with a DAM 80 preamplifier (World Precision Instruments) and responses were recorded as the average of 16 trials using either a Gould Model 1604 digital oscilloscope or a Macintosh Ilci running Labview software (version 2.4, National Instruments, Austin, TX). In some experiments, a glass microelectrode (1.0 M O ) was inserted into the optic nerve between the stimulating electrode and the slice in order to measure the evoked volley of compound action potentials traveling along the optic nerve. Field potentials were quantified as a
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Fig. 1. Amplitude variation of SCN field potentials. A: stimulus response curve for SCN field potentials. Square-wave pulses (0.3 ms, 1 Hz) of varying intensities were delivered to the optic nerve and the resulting field potential amplitudes were recorded in the contralateral SCN (filled squares). The optic nerve volley evoked by the stimulus was concurrently recorded (filled circles). B: scatter plot for all the control SCN field potential amplitudes recorded in the present report are grouped and illustrated. Field potentials were evoked with identical stimuli (square wave pulses of 0.7 mA, 0.3 ms, 1 Hz). Values had a mean of 327_+13 /.LV (mean_S.E.M., n = 47). Inset: the field potentials were typically characterized by a single large negative wave of less than 10 ms duration. Peak amplitudes were determined as the difference (in p.V) between the two points represented by the dotted lines illustrated on the field potential.
measure of the peak amplitude in /.tV of the large negative wave (Fig. 1), [26]. The following drugs were purchased from Research Biochemicals International (Natick, MA): (-)-bicuculline methiodide, phaclofen, (_+)-baclofen, muscimol hydrobromide. All other reagents were obtained from Sigma (St. Louis, MO).
3. Results
A total of 60 hypothalamic slices were used in the present study. Field potential recordings were taken from hypothalamic slices for only 2-3 h in order to maintain comparable circadian timing schedules between studies.
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Fig. 2. Muscimol potentiates SCN field potentials. 10-min applications of muscimol to hypothalamic slices enhanced the optic nerve evoked SCN field potential amplitudes by 23%. The amplitude of control SCN field potential responses was 346 ± 30 p.V (mean ± S.E.M., n = 9). Representative field potentials for each condition are illustrated. Calibration bar = 10 ms × 0.2 mV. * P < 0.05, statistical difference from control.
Stimulus response curves were generated for SCN field potentials and optic nerve volleys recorded in hypothalamic slices (Fig. 1). SCN field potentials were evoked after a minimum stimulus of 0.4 mA was delivered to the optic nerve, and reached a plateau amplitude at 0.6-0.7 mA (Fig. 1). Control values of SCN field potential responses from hypothalamic slices for all 47 preparations reported here are illustrated in Fig. lB. The average response evoked by a 0.7 mA stimulus was 327 + 13 /xV (mean + S.E.M.). A ten min perfusion with the GABA A agonist, muscimol, potentiated the optic nerve-evoked SCN field potential by 23 + 2% (mean + S.E.M., P < 0.005 when tested as percent control, Student's t-test; Fig. 2). The effect was reversible after a 10 min washout of the drug. Preliminary experiments with muscimol showed that the hypothalamic slice preparations were sensitive to repeated applications
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[Bicuculline] (/aM) Fig. 3. Bicuculline inhibits SCN field potentials. A: the amplitudes of SCN field potentials were reduced by 22% from control levels after 10-rain applications of the GABA A antagonist bicuculline to hypothalamic slices. In this group of experiments, the amplitudes of control SCN field potential responses averaged 296 5:24/.tV ( + S.E.M., n = 6). Representative field potentials for each condition are illustrated. Calibration = 10 m s × 0 . 2 mV. * P < 0.05 from control, Student's t-test. B: concentration-response for bicuculline inhibition of SCN field potential responses. SCN field potential responses were recorded after 10-min applications of each concentration, and expressed as the percentage of the control (pretreatment) response. Concentrations higher than 10 ~M were not tested. Control amplitudes were 290 ± 31 p.V, (mean 5: S.E.M., n = 5). * indicates statistical significance of P <0.05 from control, Student's t-test.
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[Baclofen] (pM) Fig. 4. Baclofen inhibits SCN field potentials. A: the GABA a agonist baclofen strongly inhibited SCN field potentials by 48% after 10-min applications of the agonist to hypothalamic slices. Control amplitude levels of SCN field potentials were 346 5:17 ~V, (mean 5: S.E.M., n = 4). * * denotes significant inhibition from control, P < 0.01, Student's t-test. Representative field potentials for each condition are shown. Calibration = 10 m s × 0 . 2 mV. B: concentration-response curve for baclofen inhibition of SCN field potentials. SCN field potential responses were recorded after 10-min applications of baclofen to hypothalamic slices. Data are expressed as the percentage of the control (pretreatment) response recorded before drug application. Baclofen (10 p,M) completely inhibited the SCN field potential. Control response amplitudes averaged 390 _+1.0 /.tV, (mean + S.E.M., n = 3). Statistically significant inhibition from control levels are indicated: * P < 0.05, * * P < 0.01, Student's t-test.
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of muscimol, manifested by persistent signal inhibition after the second application, even with extensive washing. Therefore, only one application of muscimol to each slice preparation could be performed, and a proper concentration-response curve could not be generated for this agonist. Nevertheless, 1 0 / x M was the lowest concentration of muscimol that consistently potentiated field potentials in this preparation. At a concentration of 10 /zM the G A B A A antagonist, bicuculline, inhibited optic nerve-evoked SCN field potentials by 22 __+2% (mean + S.E.M., Fig. 3). The effect of bicuculline was readily reversible, with responses returning to control levels after only a ten min washout of the drug. Exposure of slices to varying concentrations of bicuculline revealed a shallow concentration-response curve; with a
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[Phaclofen] (mM) Fig. 5. Phaclofen potentiates SCN field potentials. A: 10-min application of the GABAB antagonist, phaclofen (0.5 raM), potentiated SCN field potential amplitudes by 62 + 30%. Control levels for SCN field potentials averaged 346 -1-47/xV, (mean + S.E.M., n = 6). Representative field potentials for each condition are illustrated. Calibration = 10 ms x 0.2 mV. * indicates a statistically significant difference (P < 0.05) relative to control responses as determined by the Student's t-test. B: concentration-response curve for the facilitory effects of phaclofen on SCN field potentials. SCN field potentials were recorded before and after 10-min applications of phaclofen to hypothalamic slices. Data are expressed as the percentage of control responses. The mean amplitude for control responses was 370+20 ~V, (+S.E.M., n = 4). * indicates a statistically significant enhancement over control values, P < 0.05, Student's t-test.
Table 1 Comparison of GABA agonist and antagonist activity on optic nerveevoked SCN field potentials recorded from hypothalamic slices during day and night times Percent change in amplitude from control
Dayslices Nightslices
Muscimoi (10/zM)
Bicuculline (10/zM)
Baclofen (1 /zM)
Phaclofen (0.5 mM)
(+)23±2 n=9 (+)46±2 n=3
(-)22±2 n=6 (-)23±3 n=3
(-)48±4 n=4 (-)48±3 n=4
(+)62±30 n=6 (+)52±14 n=3
Values from day slices are reprinted here from Figs. 2-5 for comparison. Night slice values were recorded 3-8 h after lights off. Mean amplitude of control night slice field potentials was 451 ± 20 /~V ( + S.E.M.).
maximal inhibitory effect of only 29 + 7% (mean + S.E.M.) with 10 /xM bicuculline, the highest dose tested (Fig. 3). In contrast to the slight inhibitory effects of bicuculline reported above, the G A B A B agonist, baclofen, completely blocked optic nerve-evoked SCN field potentials when applied to slices at a concentration of 10 /.tM (Fig. 4). A near 50% inhibition (48 + 4%, mean + S.E.M.) of SCN field potentials was achieved after bath application of 1.0 /zM baclofen (Fig. 4) and this concentration was used in subsequent experiments. The inhibitory effects of 1.0 /.~M baclofen on SCN field potentials were readily reversible with significant recovery occurring during the first ten min (Fig. 4). The G A B A a antagonist, phaclofen, potentiated optic nerve-evoked SCN field potentials. A concentration response curve for phaclofen effects on SCN field potentials was generated, and phaclofen significantly enhanced evoked potentials by 39 + 12% (mean + S.E.M., P < 0.05 from control, Student's t-test) at a concentration of 0.5 mM (Fig. 5). In separate experiments, a ten min application of 0.5 mM phaclofen to hypothalamic slices augmented the SCN field potentials by 62 + 30%, (mean + S.E.M., Fig. 5). The effects of phaclofen were readily reversible, as demonstrated by recovery to near control values within 10 min after washout was initiated (Fig. 5). The same concentrations of G A B A agonists and antagonists as used in Figs. 2 - 5 were applied to hypothalamic slices recorded from during the mid to late portion of the donor animals' night period. The effects of G A B A drugs on optic nerve-evoked SCN field potentials in night slices were qualitatively similar to those observed in slices recorded from during the day (Table 1).
4. D i s c u s s i o n
The role of the SCN in the generation and entrainment of circadian rhythms in mammals is well established [17,20], and extensive neurochemical characterization of
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the SCN has been reported [32]. However, relatively little is known concerning how these neurochemical systems regulate circadian clock oscillations within the SCN. Most intrinsic SCN neurons appear to be GABAergic, and it has been suggested that GABA is the principal neurotransmitter in the SCN circadian system [19,31]. GABA content in the rat SCN has been reported to increase at night [1], suggesting that GABAergic neurons are under circadian regulation. However, several published studies have failed to demonstrate circadian variability in the sensitivity of spontaneously firing SCN neurons to exogenously applied GABA [11,14,15]. Exogenously applied GABA has been shown to inhibit the response of SCN neurons to optic nerve stimulation [13]. Furthermore, systemic administration of a GABA B agonist or a GABA A antagonist inhibits light-induced phase shifts of the SCN-regulated hamster activity rhythms [21,22]. These observations suggest that GABA may indeed modulate photic responsiveness in the SCN. Therefore, in the current study, we further examined the role of GABA agonists and antagonists on an index of SCN photic responsiveness, optic nerve-evoked field potentials in the rat hypothalamic slice. In the present study, the GABA A agonist, muscimol, consistently potentiated optic nerve evoked field potentials in the hamster SCN (Fig. 2). This observation is consistent with a role for GABA in the modulation of photic responsiveness in the SCN, and demonstrates that GABA A receptor mediated activity may facilitate photic responses in the SCN. However, two groups have reported that bath applied muscimol inhibited the spontaneous firing of SCN neurons [12,26,29], and systemic injections of muscimol at night failed to potentiate light-induced phase advances or delays of the free-running activity rhythm in hamsters [22]. Therefore, it is at present unclear whether or not GABA A agonists will potentiate light-induced responses of the SCN pacemaker. An investigation of the effects of local administration of GABA A agonists, into the region of the SCN, on light-induced phase shifts of the circadian pacemaker is needed to help define the action of GABA A agonists in the SCN. Intracellular and extracellular recordings of SCN neurons in the hypothalamic slice preparation have demonstrated that the GABA g antagonist, bicuculline, blocks fast IPSPs in rat and guinea pig SCN neurons in response to hypothalamic stimulation [10], and prevents the GABA-induced reduction in spontaneous firing rate of neurons in the rat and hamster SCN [12,25]. In the present study, however, only a slight inhibition of rat SCN field potentials was observed after application of 10 /xM bicuculline (Fig. 3), indicating that there may be only a minimal tonic inhibitory regulation by GABA A receptors of RHT-mediated responses in SCN neurons. To our knowledge, there is only a single report which examined the effects of GABA A antagonists on light-induced phase shifts in rodent circadian activity rhythms. Systemic injections of bicuculline into hamsters attenuated
light-induced phase delays, but not phase advances, of circadian wheel running [21]. However, systemic injections of bicuculline done by the same laboratory a few years later failed to block light-induced c-los expression in the SCN, leading the authors to conclude that the locus of action for the bicuculline blockade of phase delays was probably not at the R H T / S C N synpases [4]. Therefore, as for GABA g agonists, the effects of local, SCN-directed injections of GABA A antagonists on light-induced phase shifts need to be determined before a role of GABA g receptors in SCN function can fully be ascertained. Nevertheless, the present results indicate that muscimol potentiates and bicuculline inhibits optic nerve stimulation-induced field potentials in the SCN in vitro, supporting a role for GABA A receptors in the regulation of light-induced SCN cellular activity. The present study finds that the GABA B agonist, baclofen, strongly attenuates optic nerve evoked SCN field potentials, confirming a previous observation [25]. Systemically administered baclofen has been reported to attenuate both light-induced phase advances and delays of the running activity rhythm in hamsters [22], as well as light-induced expression of c-los within the SCN [4]. These observations, together with the results of the present study, indicate that GABA B receptors may regulate the response of SCN neurons and, indeed, the SCN circadian oscillator, to photic stimulation. The application of the GABA B antagonist, phaclofen, to the rat SCN in vitro resulted in a concentration-dependent increase in SCN field potential amplitude (Fig. 5). The effects of GABA B antagonists on evoked responses in the SCN have not been previously reported. The potentiating effect of phaclofen on evoked responses in the SCN is consistent with the possibility that there is a tonic GABA 8-receptor mediated inhibition of optic nerve evoked responses in the SCN in vitro, suggesting that GABA may dynamically regulate photic responsiveness in the nucleus. If tonic GABAergic activity in the SCN, mediated through GABA a receptors, serves to limit the response of the SCN oscillator to photic influence, then it would be predicted that administration of GABAa antagonists into the SCN region might potentiate photic phase shifts of SCN-driven circadian rhythms. This issue was partially addressed by Ralph and Menaker [22] who showed that local administration of 6-aminovaleric acid, a somewhat selective GABA B antagonist [2], which blocked the inhibition of light-induced phase advances of the free running activity rhythm in hamsters caused by systemic baclofen, did not potentiate light-induced phase shifts when administered alone. However, a more thorough examination of the effects of local administration of GABA B antagonists is required in order to assess a possible modulatory role for tonic GABA B receptor activity in the photic regulation of the SCN circadian oscillator. The effects of baclofen on photic and optic nerve-evoked responses in the SCN are very similar to those of sero-
R.L. Gannon et al. / Brain Research 694 (1995) 264-270
tonin. Both baclofen and serotonin attenuate optic nerve evoked SCN field potentials in a concentration-dependent manner (Fig. 4), [13,24]. In addition, both baclofen and the serotonin agonist, 8-OH-DPAT, (1) cause phase advances of the free running activity rhythm in hamsters when administered during the subjective day [27,30], and (2) attenuate light-induced phase shifts of the free running activity rhythm in hamsters [22,24]. Furthermore, baclofen application to the rat SCN in vitro has been reported to increase the release of newly synthesized serotonin in the SCN area [7], and systemic administration of baclofen increases the synthesis and release of serotonin in the rat SCN [8]. Since (1) the effects elicited by baclofen and serotonin agonists on SCN neuronal activity and SCNdriven circadian rhythms are qualitatively similar, (2) baclofen increases serotonin synthesis and release in the SCN, and (3) serotonergic and GABAergic neurons have convergent innervation in the SCN [6], it is quite possible that the effects of baclofen in the SCN are produced through an increase in serotonin release in the SCN. The possibility that GABAergic compounds act at pre- or postsynaptic GABA receptors to modulate serotonin release in the SCN and, thereby, influence RHT transmission and photic stimulation remains to be investigated. The responses of SCN field potentials after application of GABAergic compounds were qualitatively similar for day and night slices in the present study. The lack of day-night differences in response to GABAergic drug application on SCN neuronal activity is in agreement with previous findings utilizing exogenously applied GABA. No day-night differences were observed in the responsiveness of SCN neurons to either iontophoresed [14,15] or bath applied [11] GABA in rat and hamster preparations. Therefore, these results suggest that circadian changes in GABA activity within the SCN probably do not account for the responsiveness of the SCN to photic stimuli being limited to only the dark phase of the circadian cycle. However, it is clear that GABAergic compounds can influence both photic responses to, and cellular activity within, the SCN; and GABA is, therefore, likely to have a critical, as yet undetermined, role in the function of the SCN pacemaker. In conclusion, the field potentials evoked in rat hypothalamic slices are sensitive to drugs that act at both GABA A and GABA B receptors. The GABA A agonist, muscimol, potentiates, and the GABA A antagonist, bicuculline, attenuates optic nerve evoked field potentials in the SCN. In contrast, the GABA B antagonist, phaclofen, potentiates, and the GABA B agonist, baclofen, attenuates optic nerve evoked field potentials in the SCN. Similar effects were seen in hypothalamic slices recorded from at either day or night times, indicating a lack of circadian sensitivity in these responses. These results suggest that the responses of SCN neurons to optic nerve stimulation are under tonic, submaximal inhibition by GABAergic elements in the hypothalamic slice.
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Acknowledgements The animals involved in this study were procured, maintained and used in accordance with the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources -National Research Council. Supported by AFOSR 2312W6 (M.A.R.)
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[30]
[31] [32]
hypothatamic slice preparations, dap. J. Pharm., 33 (1983) 12251231. Shibata, S., Oomura, Y., Hattori, K. and Kita, H., Responses of suprachiasmatic nucleus neurons to optic nerve stimulation in rat hypothalamic slice preparation, Brain Res., 302 (1984) 83-89. Smith, R.D., Turek, F.W. and Slater, N.T., Bicuculline and picrotoxin block phase advances induced by GABA agonists in the circadian rhythm of locomotor activity in the golden hamster by a phaclofen-insensitive mechanism, Brain Res., 530 (1990) 275-282. Takahashi, J.S., DeCoursey, L., Bauman, L. and Menaker, M., Spectral sensitivity of a novel photoreceptive system mediating entrainment of mammalian circadian rhythms, Nature, 308 (1983) 186-188. Tominaga, K., Shibata, S., Hamada, T. and Watanabe, S., GABA A receptor agonist muscimol can reset the phase of neuronal activity rhythm in the rat suprachiasmatic nucleus in vitro, Neurosci. Lett., 166 (1994) 81-84. Tominaga, K., Shibata, S., Ueki, S. and Watanabe, S., Effects of 5-HTIA receptor agonists on the circadian rhythm of wheel-running activity in hamsters, Eur. J. PharmacoL, 214 (1992) 79-84. van den Pol, A.N., Glutamate and GABA presence and action in the suprachiasmatic nucleus, J. Biol. Rhythms, 8 (1993) S l l - S 1 5 . van den Pol, A.N. and Tsujimoto, K.L., Neurotransmitters of the hypothalamic suprachiasmatic nucleus: immunocytochemical analysis of 25 neuronal antigens, Neuroscience, 15 (1985) 1049-1086.
Brain Research 694 (1995) 264-27(/
Research report
GABAergic modulation of optic nerve-evoked field potentials in the rat suprachiasmatic nucleus Robert L. Gannon a, Matthew J. Cato a, Kathryn Hart Kelley ~, Deborah L. Armstrong b, Michael A. Rea a,* Biological Rhythms and lntegratit~e Neurosciences Research Institute, Armstrong Laboratory / CFTO, Brooks AFB, TX 78235, USA h Division of Life Sciences, University of Texas at San Antonio, San Antonio, TX, USA Accepted 21 June 1995
Abstract
The suprachiasmatic nuclei (SCN) at the base of the hypothalamus are known to be the site of the endogenous circadian pacemaker in mammals. The SCN are innervated by the retinohypothalamic tract, which conveys photic information to the SCN. GABA is one of the most abundant neurotransmitters in the SCN, and has been implicated in the modulation of photic responses of the SCN circadian pacemaker. This study sought to examine the effect of GABAergic compounds on optic nerve-evoked SCN field potentials recorded in rat horizontal hypothalamic slices. The GABA A agonist muscimol (10 #M) potentiated SCN field potentials by 23%, while application of the GABA A antagonist bicuculline (10 /xM) inhibited SCN field potentials by a similar amount, (22%). Conversely, the GABA n agonist baclofen (1.0 /.LM) inhibited SCN field potentials by 48%, while the GABA B antagonist phaclofen (0.5 mM) augmented SCN field potentials by 62%. Recordings performed at both day and night times indicate that there were no qualitative day-night differences in GABAergic activity on SCN field potentials. This study concludes that, in general, GABA A activity tends to increase, and GABA n activity tends to decrease the response of SCN neurons to optic nerve stimulation. Keywords: Hypothalamus; Brain slice; y-Aminobutyric acid; Circadian rhythm
1. Introduction
Circadian rhythms in mammals arise through the action of an endogenous oscillator located in the suprachiasmatic nuclei (SCN) at the base of the hypothalamus [16,17,20]. Under normal conditions, the circadian 'clock' is synchronized (entrained) to the environmental light:dark cycle through photic signals communicated from the retina to the SCN by the retinohypothalamic tract (RHT) [9,18]. Photic entrainment of the SCN circadian clock is achieved by daily adjustments of the phase of the circadian oscillation in response to retinal illumination [5,28]. Light exposure during the night (or dark period) causes either phase delays or phase advances of the circadian oscillation depending on the time of exposure, while light during the day (or light period) does not alter the phase of the oscillation. The photic input to the SCN is known to be modulated
* Corresponding author. Fax: (1) (2101 536-3513. 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved
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by at least three major neurotransmitters, glutamate, serotonin, and y-aminobutyric acid (GABA). Glutamatergic antagonists block light induced phase shifts of hamster circadian wheel running activity, as well as optic nerve evoked SCN field potentials [23,24], and glutamate is believed to be the primary excitatory neurotransmitter released from RHT terminals. In addition, previous work from this laboratory found that serotonergic agonists also inhibit light-induced phase shifts of hamster circadian wheel running and optic-nerve evoked field potentials [24], suggesting that serotonin may function as a modulator of photic responsiveness in the SCN. However, the most abundant neurotransmitter type in the SCN is G A B A [19,31]. GABAergic interneurons comprise a dense network within the SCN [19,31], including a reciprocal innervation to each side of the nucleus [3]. This dense innervation by GABA in the SCN suggests a role of these neurons in regulating pacemaker activity. In addition, systemic administration of various GABAergic agents has been reported to attenuate light-induced phase shifts and c-fos expression in the SCN [4,21], suggesting that, like sero-
R.L. Gannon et al. / B r a i n Research 694 (1995) 264-270
tonin, GABA may modulate photic responsiveness in the SCN. In order to further elucidate the role of GABA as a modulator of photic responses in the SCN, we investigated the effects of selective agonists and antagonists of GABA A and GABA B receptors on optic nerve evoked synaptic activity in the rat SCN. The results indicate that GABA B receptors mediate tonic inhibition of optic nerve-evoked responses in the SCN, while GABA A receptors appear to facilitate responses of SCN neurons to optic nerve activity.
265
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Male Sprague-Dawley rats (Charles River Laboratories; 150-200 g) were maintained under a 12:12 h light/dark cycle for several weeks prior to use. Food and water were provided ad libitum. Four hs after light on (day slices), rats were anesthetized with halothane and decapitated, and the brains were rapidly removed and cooled with ice-cold artificial cerebrospinal fluid (ACSF) consisting of (in mM): NaCI (122), KCI (3.8), MgSO 4 (1.2), KH2PO 4 (1.2), NaHCO 3 (25), CaCI 2 (2.5) and glucose (10). The brains were further freehand dissected so that only a tissue block containing the hypothalamus and optic nerves remained. The tissue block was affixed to a vibratome pedestal using cyanoacrylic glue and a 400-500 /xm thick horizontal hypothalamic slice was cut which contained the suprachiasmatic nuclei and both attached optic nerves. Night slices were prepared in the same manner, only rats were sacrificed between 1 h before and 1 h after lights out. Hypothalamic slices were incubated in ACSF for 4 h at room temperature under an atmosphere of 95%:5% O2/CO 2. After incubation, slices were transferred to a temperature controlled Hatton-type recording chamber and super/used with ACSF at 0.9 ml/min using a peristaltic pump at a temperature of 35°C. Slices were completely submerged in the recording chamber, which had a volume of 0.25 ml. Drugs were prepared in ACSF and applied to the slice by means of a 3 way valve in the line delivering ACSF to the slice. Bipolar suction electrodes were used to deliver a square wave pulse to the optic nerve (1.0 Hz, 0.3 ms) at a maximal stimulation intensity of 0.7 mA. The resultant extracellular field potentials were recorded in the contralateral suprachiasmatic nucleus using 3.0 M NaCl-filled glass microelectrodes with a resistance of approximately 1 M/-2. Field potentials were amplified with a DAM 80 preamplifier (World Precision Instruments) and responses were recorded as the average of 16 trials using either a Gould Model 1604 digital oscilloscope or a Macintosh Ilci running Labview software (version 2.4, National Instruments, Austin, TX). In some experiments, a glass microelectrode (1.0 M O ) was inserted into the optic nerve between the stimulating electrode and the slice in order to measure the evoked volley of compound action potentials traveling along the optic nerve. Field potentials were quantified as a
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Fig. 1. Amplitude variation of SCN field potentials. A: stimulus response curve for SCN field potentials. Square-wave pulses (0.3 ms, 1 Hz) of varying intensities were delivered to the optic nerve and the resulting field potential amplitudes were recorded in the contralateral SCN (filled squares). The optic nerve volley evoked by the stimulus was concurrently recorded (filled circles). B: scatter plot for all the control SCN field potential amplitudes recorded in the present report are grouped and illustrated. Field potentials were evoked with identical stimuli (square wave pulses of 0.7 mA, 0.3 ms, 1 Hz). Values had a mean of 327_+13 /.LV (mean_S.E.M., n = 47). Inset: the field potentials were typically characterized by a single large negative wave of less than 10 ms duration. Peak amplitudes were determined as the difference (in p.V) between the two points represented by the dotted lines illustrated on the field potential.
measure of the peak amplitude in /.tV of the large negative wave (Fig. 1), [26]. The following drugs were purchased from Research Biochemicals International (Natick, MA): (-)-bicuculline methiodide, phaclofen, (_+)-baclofen, muscimol hydrobromide. All other reagents were obtained from Sigma (St. Louis, MO).
3. Results
A total of 60 hypothalamic slices were used in the present study. Field potential recordings were taken from hypothalamic slices for only 2-3 h in order to maintain comparable circadian timing schedules between studies.
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Fig. 2. Muscimol potentiates SCN field potentials. 10-min applications of muscimol to hypothalamic slices enhanced the optic nerve evoked SCN field potential amplitudes by 23%. The amplitude of control SCN field potential responses was 346 ± 30 p.V (mean ± S.E.M., n = 9). Representative field potentials for each condition are illustrated. Calibration bar = 10 ms × 0.2 mV. * P < 0.05, statistical difference from control.
Stimulus response curves were generated for SCN field potentials and optic nerve volleys recorded in hypothalamic slices (Fig. 1). SCN field potentials were evoked after a minimum stimulus of 0.4 mA was delivered to the optic nerve, and reached a plateau amplitude at 0.6-0.7 mA (Fig. 1). Control values of SCN field potential responses from hypothalamic slices for all 47 preparations reported here are illustrated in Fig. lB. The average response evoked by a 0.7 mA stimulus was 327 + 13 /xV (mean + S.E.M.). A ten min perfusion with the GABA A agonist, muscimol, potentiated the optic nerve-evoked SCN field potential by 23 + 2% (mean + S.E.M., P < 0.005 when tested as percent control, Student's t-test; Fig. 2). The effect was reversible after a 10 min washout of the drug. Preliminary experiments with muscimol showed that the hypothalamic slice preparations were sensitive to repeated applications
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[Bicuculline] (/aM) Fig. 3. Bicuculline inhibits SCN field potentials. A: the amplitudes of SCN field potentials were reduced by 22% from control levels after 10-rain applications of the GABA A antagonist bicuculline to hypothalamic slices. In this group of experiments, the amplitudes of control SCN field potential responses averaged 296 5:24/.tV ( + S.E.M., n = 6). Representative field potentials for each condition are illustrated. Calibration = 10 m s × 0 . 2 mV. * P < 0.05 from control, Student's t-test. B: concentration-response for bicuculline inhibition of SCN field potential responses. SCN field potential responses were recorded after 10-min applications of each concentration, and expressed as the percentage of the control (pretreatment) response. Concentrations higher than 10 ~M were not tested. Control amplitudes were 290 ± 31 p.V, (mean 5: S.E.M., n = 5). * indicates statistical significance of P <0.05 from control, Student's t-test.
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[Baclofen] (pM) Fig. 4. Baclofen inhibits SCN field potentials. A: the GABA a agonist baclofen strongly inhibited SCN field potentials by 48% after 10-min applications of the agonist to hypothalamic slices. Control amplitude levels of SCN field potentials were 346 5:17 ~V, (mean 5: S.E.M., n = 4). * * denotes significant inhibition from control, P < 0.01, Student's t-test. Representative field potentials for each condition are shown. Calibration = 10 m s × 0 . 2 mV. B: concentration-response curve for baclofen inhibition of SCN field potentials. SCN field potential responses were recorded after 10-min applications of baclofen to hypothalamic slices. Data are expressed as the percentage of the control (pretreatment) response recorded before drug application. Baclofen (10 p,M) completely inhibited the SCN field potential. Control response amplitudes averaged 390 _+1.0 /.tV, (mean + S.E.M., n = 3). Statistically significant inhibition from control levels are indicated: * P < 0.05, * * P < 0.01, Student's t-test.
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of muscimol, manifested by persistent signal inhibition after the second application, even with extensive washing. Therefore, only one application of muscimol to each slice preparation could be performed, and a proper concentration-response curve could not be generated for this agonist. Nevertheless, 1 0 / x M was the lowest concentration of muscimol that consistently potentiated field potentials in this preparation. At a concentration of 10 /zM the G A B A A antagonist, bicuculline, inhibited optic nerve-evoked SCN field potentials by 22 __+2% (mean + S.E.M., Fig. 3). The effect of bicuculline was readily reversible, with responses returning to control levels after only a ten min washout of the drug. Exposure of slices to varying concentrations of bicuculline revealed a shallow concentration-response curve; with a
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[Phaclofen] (mM) Fig. 5. Phaclofen potentiates SCN field potentials. A: 10-min application of the GABAB antagonist, phaclofen (0.5 raM), potentiated SCN field potential amplitudes by 62 + 30%. Control levels for SCN field potentials averaged 346 -1-47/xV, (mean + S.E.M., n = 6). Representative field potentials for each condition are illustrated. Calibration = 10 ms x 0.2 mV. * indicates a statistically significant difference (P < 0.05) relative to control responses as determined by the Student's t-test. B: concentration-response curve for the facilitory effects of phaclofen on SCN field potentials. SCN field potentials were recorded before and after 10-min applications of phaclofen to hypothalamic slices. Data are expressed as the percentage of control responses. The mean amplitude for control responses was 370+20 ~V, (+S.E.M., n = 4). * indicates a statistically significant enhancement over control values, P < 0.05, Student's t-test.
Table 1 Comparison of GABA agonist and antagonist activity on optic nerveevoked SCN field potentials recorded from hypothalamic slices during day and night times Percent change in amplitude from control
Dayslices Nightslices
Muscimoi (10/zM)
Bicuculline (10/zM)
Baclofen (1 /zM)
Phaclofen (0.5 mM)
(+)23±2 n=9 (+)46±2 n=3
(-)22±2 n=6 (-)23±3 n=3
(-)48±4 n=4 (-)48±3 n=4
(+)62±30 n=6 (+)52±14 n=3
Values from day slices are reprinted here from Figs. 2-5 for comparison. Night slice values were recorded 3-8 h after lights off. Mean amplitude of control night slice field potentials was 451 ± 20 /~V ( + S.E.M.).
maximal inhibitory effect of only 29 + 7% (mean + S.E.M.) with 10 /xM bicuculline, the highest dose tested (Fig. 3). In contrast to the slight inhibitory effects of bicuculline reported above, the G A B A B agonist, baclofen, completely blocked optic nerve-evoked SCN field potentials when applied to slices at a concentration of 10 /.tM (Fig. 4). A near 50% inhibition (48 + 4%, mean + S.E.M.) of SCN field potentials was achieved after bath application of 1.0 /zM baclofen (Fig. 4) and this concentration was used in subsequent experiments. The inhibitory effects of 1.0 /.~M baclofen on SCN field potentials were readily reversible with significant recovery occurring during the first ten min (Fig. 4). The G A B A a antagonist, phaclofen, potentiated optic nerve-evoked SCN field potentials. A concentration response curve for phaclofen effects on SCN field potentials was generated, and phaclofen significantly enhanced evoked potentials by 39 + 12% (mean + S.E.M., P < 0.05 from control, Student's t-test) at a concentration of 0.5 mM (Fig. 5). In separate experiments, a ten min application of 0.5 mM phaclofen to hypothalamic slices augmented the SCN field potentials by 62 + 30%, (mean + S.E.M., Fig. 5). The effects of phaclofen were readily reversible, as demonstrated by recovery to near control values within 10 min after washout was initiated (Fig. 5). The same concentrations of G A B A agonists and antagonists as used in Figs. 2 - 5 were applied to hypothalamic slices recorded from during the mid to late portion of the donor animals' night period. The effects of G A B A drugs on optic nerve-evoked SCN field potentials in night slices were qualitatively similar to those observed in slices recorded from during the day (Table 1).
4. D i s c u s s i o n
The role of the SCN in the generation and entrainment of circadian rhythms in mammals is well established [17,20], and extensive neurochemical characterization of
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the SCN has been reported [32]. However, relatively little is known concerning how these neurochemical systems regulate circadian clock oscillations within the SCN. Most intrinsic SCN neurons appear to be GABAergic, and it has been suggested that GABA is the principal neurotransmitter in the SCN circadian system [19,31]. GABA content in the rat SCN has been reported to increase at night [1], suggesting that GABAergic neurons are under circadian regulation. However, several published studies have failed to demonstrate circadian variability in the sensitivity of spontaneously firing SCN neurons to exogenously applied GABA [11,14,15]. Exogenously applied GABA has been shown to inhibit the response of SCN neurons to optic nerve stimulation [13]. Furthermore, systemic administration of a GABA B agonist or a GABA A antagonist inhibits light-induced phase shifts of the SCN-regulated hamster activity rhythms [21,22]. These observations suggest that GABA may indeed modulate photic responsiveness in the SCN. Therefore, in the current study, we further examined the role of GABA agonists and antagonists on an index of SCN photic responsiveness, optic nerve-evoked field potentials in the rat hypothalamic slice. In the present study, the GABA A agonist, muscimol, consistently potentiated optic nerve evoked field potentials in the hamster SCN (Fig. 2). This observation is consistent with a role for GABA in the modulation of photic responsiveness in the SCN, and demonstrates that GABA A receptor mediated activity may facilitate photic responses in the SCN. However, two groups have reported that bath applied muscimol inhibited the spontaneous firing of SCN neurons [12,26,29], and systemic injections of muscimol at night failed to potentiate light-induced phase advances or delays of the free-running activity rhythm in hamsters [22]. Therefore, it is at present unclear whether or not GABA A agonists will potentiate light-induced responses of the SCN pacemaker. An investigation of the effects of local administration of GABA A agonists, into the region of the SCN, on light-induced phase shifts of the circadian pacemaker is needed to help define the action of GABA A agonists in the SCN. Intracellular and extracellular recordings of SCN neurons in the hypothalamic slice preparation have demonstrated that the GABA g antagonist, bicuculline, blocks fast IPSPs in rat and guinea pig SCN neurons in response to hypothalamic stimulation [10], and prevents the GABA-induced reduction in spontaneous firing rate of neurons in the rat and hamster SCN [12,25]. In the present study, however, only a slight inhibition of rat SCN field potentials was observed after application of 10 /xM bicuculline (Fig. 3), indicating that there may be only a minimal tonic inhibitory regulation by GABA A receptors of RHT-mediated responses in SCN neurons. To our knowledge, there is only a single report which examined the effects of GABA A antagonists on light-induced phase shifts in rodent circadian activity rhythms. Systemic injections of bicuculline into hamsters attenuated
light-induced phase delays, but not phase advances, of circadian wheel running [21]. However, systemic injections of bicuculline done by the same laboratory a few years later failed to block light-induced c-los expression in the SCN, leading the authors to conclude that the locus of action for the bicuculline blockade of phase delays was probably not at the R H T / S C N synpases [4]. Therefore, as for GABA g agonists, the effects of local, SCN-directed injections of GABA A antagonists on light-induced phase shifts need to be determined before a role of GABA g receptors in SCN function can fully be ascertained. Nevertheless, the present results indicate that muscimol potentiates and bicuculline inhibits optic nerve stimulation-induced field potentials in the SCN in vitro, supporting a role for GABA A receptors in the regulation of light-induced SCN cellular activity. The present study finds that the GABA B agonist, baclofen, strongly attenuates optic nerve evoked SCN field potentials, confirming a previous observation [25]. Systemically administered baclofen has been reported to attenuate both light-induced phase advances and delays of the running activity rhythm in hamsters [22], as well as light-induced expression of c-los within the SCN [4]. These observations, together with the results of the present study, indicate that GABA B receptors may regulate the response of SCN neurons and, indeed, the SCN circadian oscillator, to photic stimulation. The application of the GABA B antagonist, phaclofen, to the rat SCN in vitro resulted in a concentration-dependent increase in SCN field potential amplitude (Fig. 5). The effects of GABA B antagonists on evoked responses in the SCN have not been previously reported. The potentiating effect of phaclofen on evoked responses in the SCN is consistent with the possibility that there is a tonic GABA 8-receptor mediated inhibition of optic nerve evoked responses in the SCN in vitro, suggesting that GABA may dynamically regulate photic responsiveness in the nucleus. If tonic GABAergic activity in the SCN, mediated through GABA a receptors, serves to limit the response of the SCN oscillator to photic influence, then it would be predicted that administration of GABAa antagonists into the SCN region might potentiate photic phase shifts of SCN-driven circadian rhythms. This issue was partially addressed by Ralph and Menaker [22] who showed that local administration of 6-aminovaleric acid, a somewhat selective GABA B antagonist [2], which blocked the inhibition of light-induced phase advances of the free running activity rhythm in hamsters caused by systemic baclofen, did not potentiate light-induced phase shifts when administered alone. However, a more thorough examination of the effects of local administration of GABA B antagonists is required in order to assess a possible modulatory role for tonic GABA B receptor activity in the photic regulation of the SCN circadian oscillator. The effects of baclofen on photic and optic nerve-evoked responses in the SCN are very similar to those of sero-
R.L. Gannon et al. / Brain Research 694 (1995) 264-270
tonin. Both baclofen and serotonin attenuate optic nerve evoked SCN field potentials in a concentration-dependent manner (Fig. 4), [13,24]. In addition, both baclofen and the serotonin agonist, 8-OH-DPAT, (1) cause phase advances of the free running activity rhythm in hamsters when administered during the subjective day [27,30], and (2) attenuate light-induced phase shifts of the free running activity rhythm in hamsters [22,24]. Furthermore, baclofen application to the rat SCN in vitro has been reported to increase the release of newly synthesized serotonin in the SCN area [7], and systemic administration of baclofen increases the synthesis and release of serotonin in the rat SCN [8]. Since (1) the effects elicited by baclofen and serotonin agonists on SCN neuronal activity and SCNdriven circadian rhythms are qualitatively similar, (2) baclofen increases serotonin synthesis and release in the SCN, and (3) serotonergic and GABAergic neurons have convergent innervation in the SCN [6], it is quite possible that the effects of baclofen in the SCN are produced through an increase in serotonin release in the SCN. The possibility that GABAergic compounds act at pre- or postsynaptic GABA receptors to modulate serotonin release in the SCN and, thereby, influence RHT transmission and photic stimulation remains to be investigated. The responses of SCN field potentials after application of GABAergic compounds were qualitatively similar for day and night slices in the present study. The lack of day-night differences in response to GABAergic drug application on SCN neuronal activity is in agreement with previous findings utilizing exogenously applied GABA. No day-night differences were observed in the responsiveness of SCN neurons to either iontophoresed [14,15] or bath applied [11] GABA in rat and hamster preparations. Therefore, these results suggest that circadian changes in GABA activity within the SCN probably do not account for the responsiveness of the SCN to photic stimuli being limited to only the dark phase of the circadian cycle. However, it is clear that GABAergic compounds can influence both photic responses to, and cellular activity within, the SCN; and GABA is, therefore, likely to have a critical, as yet undetermined, role in the function of the SCN pacemaker. In conclusion, the field potentials evoked in rat hypothalamic slices are sensitive to drugs that act at both GABA A and GABA B receptors. The GABA A agonist, muscimol, potentiates, and the GABA A antagonist, bicuculline, attenuates optic nerve evoked field potentials in the SCN. In contrast, the GABA B antagonist, phaclofen, potentiates, and the GABA B agonist, baclofen, attenuates optic nerve evoked field potentials in the SCN. Similar effects were seen in hypothalamic slices recorded from at either day or night times, indicating a lack of circadian sensitivity in these responses. These results suggest that the responses of SCN neurons to optic nerve stimulation are under tonic, submaximal inhibition by GABAergic elements in the hypothalamic slice.
269
Acknowledgements The animals involved in this study were procured, maintained and used in accordance with the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources -National Research Council. Supported by AFOSR 2312W6 (M.A.R.)
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