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Novel phase-shifting effects of GABAA receptor activation in thesuprachiasmatic nucleus of a diurnal rodent

C. M. Novak and H. E. AlbersDepartments of Biology and Psychology, Center for Behavioral Neuroscience,Georgia State University, Atlanta, Georgia 30302-4010

Submitted 2 October 2003; accepted in final form 28 November 2003

Novak, C. M., and H. E. Albers. Novel phase-shifting effects ofGABAA receptor activation in the suprachiasmatic nucleus of a diurnalrodent. Am J Physiol Regul Integr Comp Physiol 286: R820–R825, 2004.First published December 4, 2003; 10.1152/ajpregu.00575.2003.—Thevast majority of neurons in the suprachiasmatic nucleus (SCN), theprimary circadian pacemaker in mammals, contain the inhibitoryneurotransmitter GABA. Most studies investigating the role of GABAin the SCN have been performed using nocturnal rodents. Activationof GABAA receptors by microinjection of muscimol into the SCNphase advances the circadian activity rhythm of nocturnal rodents, butonly during the subjective day. Nonphotic stimuli that reset thecircadian pacemaker of nocturnal rodents also produce phase ad-vances during the subjective day. The role of GABA in the SCN ofdiurnal animals and how it may differ from nocturnal animals is notknown. In the studies described here, the GABAA agonist muscimolwas microinjected directly into the SCN region of diurnal unstripedNile grass rats (Arvicanthis niloticus) at various times in their circa-dian cycle. The results demonstrate that GABAA receptor activationproduces large phase delays during the subjective day in grass rats.Treatment with TTX did not affect the ability of muscimol to inducephase delays, suggesting that muscimol acts directly on pacemakercells within the SCN. These data suggest that the circadian pacemak-ers of nocturnal and diurnal animals respond to the most abundantneurochemical signal found in SCN neurons in opposite ways. Thesefindings are the first to demonstrate a fundamental difference in thefunctioning of circadian pacemaker cells in diurnal and nocturnalanimals.

circadian rhythm; muscimol; grass rat; Arvicanthis niloticus; phase-response curve

THE SUPRACHIASMATIC NUCLEUS (SCN) of the hypothalamus is theprimary circadian pacemaker in mammals. Neurons within theSCN contain both GABA and its synthetic enzyme (glutamicacid decarboxylase; Ref. 23) as well as GABAA receptors (15,39). The SCN shows a daily rhythm in GABA content, and ithas been estimated that all or nearly all neurons within the SCNcontain GABA (1, 39, 53). Thus far, GABA in the SCNappears to mediate the phase-shifting effects of at least somenonphotic stimuli as well as modulate the phase-shifting effectsof light (16, 37). GABAA receptor activation in the SCN bymuscimol during the subjective day induces phase advances inhamsters, and small phase delays are observed during thesubjective night (50). In fact, the phase-shifting effects ofmuscimol are very similar to the phase-shifting effects ofneuropeptide Y (NPY; 2) as well as other nonphotic stimulisuch as triazolam treatment (52) and forced activity (41).Because arousal-induced phase shifts are inhibited by NPY

antisera within the SCN (4) and NPY-induced phase shifts areinhibited by GABAA receptor antagonists (22), it is likely thatactivation of GABAA receptors within the SCN is necessaryfor nonphotic stimuli to induce phase shifts of circadianrhythms.

The great majority of studies on the neuronal mechanismscontrolling circadian rhythms have been conducted using noc-turnal animals. The few studies that have examined circadiancontrol in diurnal species have found that the patterns ofneuronal metabolic activity in the SCN, as well as the re-sponses to light, are very similar to nocturnal animals (26, 30,33, 45, 46). There is reason to believe, however, that the roleof GABA in the SCN may differ between diurnal and nocturnalspecies. As described above, GABA appears to mediate theeffects of nonphotic stimuli on rhythms (20, 22). In nocturnalanimals, arousal during the subjective day, when nocturnalanimals are normally quiescent, is crucial for the induction ofnonphotic phase shifts (41). It is therefore possible that thephase-shifting effects of nonphotic stimuli, as well as theneural mechanisms that mediate these effects, may differ indiurnal animals. The present study determined if the phase-shifting actions of GABA in the SCN of a diurnal rodentspecies, Arvicanthis niloticus (unstriped Nile grass rat), differfrom those reported in nocturnal rodents.

EXPERIMENTAL PROCEDURES

Male and female A. niloticus (n � 94) were used in the followingstudies. All animals used were raised from a colony at Georgia StateUniversity, descended from the colony at Michigan State University(29). Animals had access to food and water ad libitum. Animals werehoused in Plexiglas cages (20 � 40 � 20 cm) in a 12:12-h light/darkcycle and after surgery were individually housed in cages equippedwith a running wheel.

Each grass rat underwent surgical implantation of a guide cannula(26 gauge; Plastics One). The animals were deeply anesthetized(pentobarbital sodium, 50 mg/kg dose, 1 ml/kg volume ip) and placedin a stereotaxic frame. The guide cannula was aimed at the SCNregion using the following coordinates: 9.5° angle toward the midline,�1.5 mm anterior to bregma, �1.4 mm lateral to bregma, and �3.0to �3.1 mm ventral to dura mater; the final position of the 32-gauge,14-mm microinjection needle was 6.0 to 6.1 mm below dura. Cranio-plastic cement (Plastics One) was used to secure the cannula to theskull.

Running wheels were outfitted with magnetic switches that re-corded each wheel revolution. For each wheel, the number of wheelrevolutions per 5-min period was recorded using Mini-mitter equip-ment (dataports and QA-4s) and VitalView software (Mini-mitter;Bend, OR). After animals were entrained to a 12:12-h light/dark cycle,

Address for reprint requests and other correspondence: C. M. Novak, Centerfor Behavioral Neuroscience, Georgia State Univ., PO Box 4010, BiologyDept., Atlanta, GA 30302-4010 (E-mail: biocmn@langate.gsu.edu).

The costs of publication of this article were defrayed in part by the paymentof page charges. The article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Am J Physiol Regul Integr Comp Physiol 286: R820–R825, 2004.First published December 4, 2003; 10.1152/ajpregu.00575.2003.

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they were released into constant darkness (DD). After 7–10 days inDD, animals were given their first of up to three microinjections. Atleast 10 days separated each set of injections. During microinjections,animals were removed from their cage and gently restrained while themicroinjection needle was inserted into the guide cannula. Muscimol(Sigma; 1–10 mM) or saline vehicle (200 nl) were microinjected intothe SCN area using a 1-�l Hamilton syringe. Muscimol induced anextended behavioral stupor in grass rats, which usually started within15 min after microinjection. The change in behavior occurred regard-less of what time of day the animal received muscimol, and animalsfully recovered within a few hours. Similar effects have been seen inhamsters under the same conditions. Also, microinjections of baclofen(GABAB agonist) produce a similar behavioral change with minimalphase alterations (C. M. Novak, unpublished observation). It is there-fore unlikely that any phase shifts seen after microinjection of mus-cimol are due to nonspecific effects (such as changes in body tem-perature) secondary to the behavioral change.

Linear regression was used to 1) predict the onset of wheel runningactivity and 2) calculate the phase shift value after each injection (9).A regression line was fitted to the activity onsets (defined as the first5-min bin with 10 or more revolutions, followed by 3 similar bins) forthe 7 days preceding the injection. The resulting predicted onsetvalues were used to calculate the injection time for each animal. Asecond regression line was fitted to the 10 onsets after the injectionday (the first 2–3 onsets were not included because of transient onsetsafter phase shifts). This yielded two regression values, both predictingthe onset time of the day after injection. The postinjection regressionvalue was subtracted from the preinjection value to calculate the phaseshift value. Phase advances were therefore positive numbers, andphase delays were negative numbers. Phase shift values were notincluded if 1) the injection of muscimol did not induce the typicalbehavioral stupor (5 data points excluded), 2) the standard error ofeither regression line was over 20 min (variability in onset times wasnot related to treatment or dose), 3) the differences between the slopesof the pre- and postinjection regression lines was �0.01 (14.4 min, toaccount for any possible changes in circadian period due to thetreatment; 23 total data points excluded), or 4) the microinjection sitewas not within 300 �m of the SCN (see below).

Although A. niloticus are strictly diurnal in the field (5), animals inthe laboratory show a variety of patterns of wheel running activity.Access to a running wheel induces varying degrees of crepuscular(twilight active) activity in some grass rats, which is likely due tomasking. Although this wheel-induced masking presents a limitationto the use of A. niloticus as a diurnal model, there is no better speciesto use for an experiment of this nature. Because of the crepusculartendencies exhibited by many grass rats, the morning activity onsetcould not always be used to calculate a regression line. Depending onwhich onset (morning associated or evening associated) had leastday-to-day variability (i.e., standard error of onset values), that onsetwas used to calculate injection time and phase shift values (see Ref.42). At the conclusion of each experiment, an ANOVA was used todetermine if either the sex of the animal or the onset used to calculatephase shift or injection time affected the phase shift values; nosignificant effects were found (P � 0.05).

The phase-shifting effects of muscimol were determined for eachhour over the course of the subjective day to describe the effects ofmuscimol using a phase-response curve (PRC). In a PRC, the mag-nitude of the phase shift is plotted on the vertical axis, with phasedelays below the vertical axis and phase advances above it. Time ofday is plotted as circadian time (CT; hours after morning activityonset) on the horizontal axis, starting with CT 0, the start of subjectiveday, with CT 12 defining the start of subjective night. In this way, theamount and direction of the phase shifts induced by a stimulus can bedescribed over the course of the day. To determine the phase-responsecurve for muscimol, the GABAA agonist (Sigma; 10 mM in salinevehicle) was microinjected at each hour of the cycle (the morningactivity onset was defined as CT 0 if this onset was used to determine

phase; if the evening activity onset was used, this was considered CT12; Ref. 42). The resulting phase shift for each injection was plottedacross the circadian cycle. The data were analyzed in CT groups (datapoints were divided into 8 groups according to injection time: CT 0–2,3–5, 6–8, 9–11, 12–14, 15–17, 18–20, and 21–23). A one-wayANOVA was used to determine if muscimol-induced phase shiftsdiffered over time of day, and post hoc (least significant difference)tests were used to probe differences between CT groups.

After the time (i.e., CT) of maximal response to muscimol wasascertained, a dose-response curve for muscimol was determined.Grass rats were microinjected with muscimol at CT 4 using thefollowing doses: 0 mM (vehicle microinjection), 1 mM, 5 mM, and 10mM. A one-way ANOVA was used to determine if muscimol induceda significant phase shift at CT 4, and the least significant differencetest was used to reveal significant differences between doses ofmuscimol.

Lastly, the ability of muscimol to induce phase delays in thepresence of TTX (reversibly blocks sodium-dependent action poten-tials) was assessed. At CT 4, grass rats were microinjected with 200nl of muscimol (10 mM), either alone or in a cocktail with TTX(Sigma; 5 �M in saline vehicle), TTX alone, or vehicle. Administra-tion of TTX in a cocktail successfully blocks the effects of a GABAreceptor agonist (baclofen, but not muscimol) in vitro (3); TTX hasalso been microinjected in a cocktail with muscimol previously in thislab (14). The resulting phase shifts were calculated, and a two-wayANOVA was used to determine if muscimol-induced phase shiftswere affected by the addition of TTX (using muscimol and TTX as theindependent variables, phase shift values as the dependent variable).Moreover, in a separate experiment, the effect of TTX on light-induced phase shifts was assessed as a positive control for TTX. Inhamsters, SCN microinjections of TTX attenuate the phase-shiftingeffects of light (38) but not the phase-shifting effects of muscimol(24). Grass rats were exposed to a light pulse (500 lx, 15 min) at CT14–16, when light induces phase delays; animals were microinjectedwith either TTX or vehicle immediately (usually within 1–2 min)before light exposure. An independent-samples t-test was used todetermine the effects of TTX on light-induced phase delays in grassrats.

After all activity data were collected, brains were extracted toascertain cannula placement. Each animal was injected with a lethaldose of pentobarbital sodium (0.5 ml/animal ip; 50 mg/ml) thenmicroinjected with 200 nl india ink through each guide cannula. Thebrains were removed and placed into 10% buffered formalin for atleast 24 h. Brains were sectioned into 100-�m sections using avibratome, and the SCN sections were evaluated using a microscope.Only animals with injection sites placed within 300 �m of the borderof the SCN that did not penetrate the third ventricle were included inthe analysis (see Fig. 1).

RESULTS

Muscimol induces phase delays during the subjective day.As shown in Fig. 2, microinjections of muscimol inducedphase shifts in wheel running activity when microinjected intothe SCN region but not at all times of the circadian cycle.During the subjective day, especially between CT 3 and 5,muscimol induced phase delays in the rhythm of wheel runningactivity. No consistent phase-shifting effect of muscimol wasfound during the subjective night. An ANOVA revealed asignificant effect of time of injection on muscimol-inducedphase shifts (P � 0.01). Muscimol induced significantlygreater phase delays at CT 3–5 compared with all other CTgroups.

Before the slope criterion was applied, a correlation test wasrun to determine if the magnitude of the phase shift wascorrelated to a change on slope (circadian period). No signif-

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icant correlation was found (r � �0.035, P � 0.759), indicat-ing that muscimol-induced phase shifts were not associatedwith a change in circadian period compared with microinjec-tions of muscimol that did not induce phase shifts.

Muscimol-induced phase delays are dose dependent. Mus-cimol induced significant phase delays at CT 4 (P � 0.01). Asseen in Fig. 3, a post hoc test revealed that 10 mM muscimolinduced significantly larger phase delays than the 5 mM, 1mM, or vehicle groups (P � 0.01).

Muscimol-induced phase delays are not blocked by TTX.The two-way ANOVA showed no significant interaction be-tween muscimol and TTX on phase shifts; there was a signif-icant main effect of muscimol (P � 0.001) but not TTX. Asillustrated in Fig. 4, muscimol induced phase delays, and TTXhad no significant effect on muscimol-induced phase delays. Inthe control experiment, light-induced phase delays in grass ratsat CT 14–16 and TTX significantly attenuated these phaseshifts (P � 0.05). The mean phase delay after light�vehiclewas �72.29 min � 6.98 SE, whereas the mean phase delayafter treatment with light�TTX was significantly lower,�48.73 min � 5.00 SE (33% decrease). These results demon-strate that TTX reduced light-induced but not muscimol-in-duced phase delays in A. niloticus.

DISCUSSION

The studies described here demonstrate that the GABAA

receptor activation in the SCN of a diurnal rodent, A. niloticus,induces large phase delays of the circadian wheel runningrhythm during the subjective day, with 10 mM as the criticaldose to achieve phase delays. These results differ dramaticallyfrom the phase-shifting effects of muscimol in nocturnal ani-mals. In hamsters, muscimol produces phase advances duringthe subjective day (50). The phase-shifting effects of muscimolin grass rats and hamsters may also differ during the subjectivenight. In hamsters, muscimol induces small phase delays dur-

Fig. 1. Photomicrograph of a microinjection site in the region of the supra-chiasmatic nucleus (SCN; anterior SCN pictured here). Arrow points to the inkinjection, delineating final placement of microinjection needle. If the tip of theneedle was placed within 300 �m of the SCN border without penetrating thewall of the 3rd ventricle (3V), data from that animal were included in theanalysis. oc, optic chiasm.

Fig. 2. Phase shifts produced by microinjection of the GABAA agonistmuscimol (200 nl, 10 mM) into the SCN region, plotted as a phase-responsecurve (A). Large phase delays were seen in the early- to mid-subjective day.Significantly larger phase delays were found after muscimol microinjection atcircadian time (CT) 3–5 than at any other time point (*P � 0.05). Samplesizes: CT 0–2 (6), CT 3–5 (8), CT 6–8 (8), CT 9–11 (7), CT 12–14 (6), CT15–17 (9), CT 18–20 (10), CT 21–23 (5). A representative single-plottedactogram of wheel-running activity after muscimol microinjection (B) showsphase delays after microinjection of muscimol at CT 3 (large delay) and CT 1(small delay), but not CT 10. Arrows indicate the first onset after injection,with injection time marked by asterisks. Crepuscular pattern of wheel runningcan also be seen, with the CT 3 microinjection indicated on the morningactivity component, and the CT 10 and CT 1 microinjections marked on theevening activity component.

Fig. 3. Significantly larger phase delays were seen after microinjection of 10mM muscimol (200 nl) into the SCN region compared with 5 mM or vehicle(*P � 0.01; A). A single-plotted actogram (B) shows phase shifts induced by10 mM muscimol but not 5 mM, 1 mM, or vehicle. Again, the wheel-inducedcrepuscular activity components can be seen, with arrows pointing to theevening activity component.

Fig. 4. Microinjections of muscimol (musc) into the SCN of A. niloticus at CT4 induced significantly larger phase delays than vehicle (veh; *P � 0.05).Addition of TTX (5 �M, Sigma) to reversibly block sodium-dependent actionpotentials did not have any significant effect on muscimol-induced phasedelays. Microinjection of TTX did, however, significantly reduce light-inducedphase delays at CT 14–16.

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ing the subjective night (50), whereas no consistent phaseadvances or delays were seen after muscimol microinjectionduring the subjective night in grass rats. As seen in hamsters,inhibition of sodium-dependent action potentials in the SCNwith TTX had no discernable effect on muscimol-inducedphase delays. Taken together, these data suggest that muscimolacts directly on SCN pacemaker cells or cells that communi-cate with pacemaker cells by mechanisms other than sodium-dependent action potentials in both hamsters and grass rats.These data further suggest that the circadian pacemakers ofdiurnal and nocturnal animals respond differently to GABA.These findings are the first to demonstrate a significant differ-ence in a fundamental characteristic of the circadian clockmechanism between diurnal and nocturnal animals. Moreover,it is the first indication that there may be diurnal/nocturnaldifferences within cells housing circadian oscillatory mecha-nism.

GABA-induced phase resetting in the SCN can be demon-strated at the cellular level. Activation of GABAA receptorsduring the subjective day phase advances the circadian rhythmof rat SCN neuronal activity in vitro in a manner similar to itseffect on behavioral rhythms (51). Indeed, GABA-inducedphase shifts can be found at the level of a single neuron (32).Also, single-unit discharge of most SCN neurons is inhibitedby GABA and GABAergic agents (6, 12, 31, 47), as is thedaytime peak of metabolic activity within the SCN. GABAmay also act as a synchronizing agent within the SCN, entrain-ing each cellular oscillator to the same phase (32). It has alsobeen proposed that GABA is involved in optic nerve stimula-tion-induced suppressions of neuronal activity in both rats anddiurnal Octodon degus (27). Lastly, GABA suppresses regularintracellular Ca2� oscillations in SCN cells (54); this may beone mechanism through which GABA might alter the molec-ular oscillatory mechanism within SCN neurons. Taken to-gether with previous studies, the data presented here suggestthe possibility that the different responses to GABAA receptoractivation in diurnal and nocturnal rodents might originate atthe level of individual SCN neurons.

In addition to the opposite effects of muscimol in producingphase shifts during the day, other effects of muscimol aresimilar between species. Muscimol induces a behavioral stuporin both grass rats and hamsters. The magnitudes of the phaseshifts produced by muscimol are not strikingly different be-tween species (50). In addition, in both grass rats and hamsters,muscimol-induced phase shifts are not significantly affected byinhibition of sodium-dependent action potentials by TTX (3,24). These data strongly suggest that muscimol activatesGABAA receptors on SCN pacemaker cells rather than onneurons that communicate with SCN pacemaker cells viaconventional synaptic transmission. Other explanations arepossible, however. For example, a nonsynaptic chemical mes-senger such as nitric oxide (NO) may be involved in mediatingphase shifts (10). Phase resetting by NO is restricted to thesubjective night, however, making it unlikely that NO mediatesmuscimol-induced phase shifting during the subjective day(11). Another possibility is that muscimol may act on cells thatcommunicate with pacemaker neurons via gap junctions orother mechanisms not involving sodium-dependent action po-tentials. In support of this hypothesis is the finding that mus-cimol has been shown to inhibit dye transfer between SCNneurons, indicating that GABAA receptor activation may in-

fluence the strength of gap junction communication and theresulting spread of depolarization (7, 48). The possible role ofgap junctions in GABA-related phase shifting during the dayremains unclear. If gap junctions or NO are necessary formuscimol-induced phase shifting to occur, it seems likely thateither mechanism affects only local neuronal networks.

One role of GABA within the SCN is thought to be themediation of nonphotic phase shifts. Specifically, in nocturnalanimals, GABA receptor activation within the SCN produces asimilar pattern of phase shifting as forced activity or arousal(41), NPY (21), and activation of the serotonin system (36, 43).Both NPY from the intergeniculate leaflet and 5-HT from thedorsal and median raphe nuclei relay information about non-photic events to the SCN and related brain structures (4, 13,34). Antagonists to GABAA receptors microinjected within theSCN also block the phase-shifting effects of NPY (22). GABAreceptor activation within the SCN may be the final commonpathway for nonphotic stimuli to act on pacemaker mecha-nisms in nocturnal animals. Whether it serves the same purposein diurnal animals remains to be determined. In fact, very littleis known about the roles of 5-HT or NPY in the rhythms ofdiurnal animals (18, 49). Taken together with previous results,the data described here demonstrate that the SCN is sensitive tothe phase-shifting effects of GABAA receptor activation duringthe subjective day, regardless of whether the animal is diurnalor nocturnal. However, phase shifting effects of activation ofGABAA receptors is opposite in nocturnal animals and diurnalgrass rats.

It might be assumed that the same stimulus that causesnonphotic phase shifts in nocturnal animals (i.e., arousal oractivity) would induce nonphotic phase shifts in diurnal ani-mals. If this were the case, it follows that the circadian systemof diurnal animals would be sensitive to these nonphoticstimuli at a different time of day (during the night) thannocturnal animals (during the day). Evidence suggests thatscheduled activity has, at most, a modest influence on phase indiurnal rodents (17, 25, 28). From these studies combined withthe data presented here, it seems that the phase of the circadianpacemaker, not the phase of the animal’s activity cycle (i.e.,diurnal or nocturnal), determines when an animal is sensitive toa nonphotic stimulus (17, 25, 28). On the other hand, thedirection of the phase shift induced by GABAA receptoractivation (i.e., phase delays in grass rats but phase advances inhamsters) may be coupled to the daily activity pattern of thespecies. The environmental stimulus or behavioral change thatmay ultimately cause an alteration of SCN GABAA receptoractivation in grass rats is unknown. Other possible entrainingagents should be considered in addition to induced activity. Forexample, social contact has been shown to promote rhythmsynchronization in other diurnal (44) and nocturnal (8, 35, 40)animals. Olfactory cues can entrain and accelerate reentrain-ment in diurnal Octodon degus (19). The ability of arousal oractivity to act as an entraining agent may not be a generalproperty of the circadian system of mammals.

The data presented here suggest that the circadian pace-maker of both nocturnal and diurnal animals is sensitive tononphotic stimuli during the daytime but that the direction ofthe phase shift, as well as the stimulus needed to induce thephase shift, may differ according to species. These data implythat information on nonphotic entrainment and the neuralmechanisms mediating nonphotic entrainment in nocturnal

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animals may not generalize directly to diurnal animals, includ-ing humans.

ACKNOWLEDGMENTS

We thank D. Smith and especially C. Ehlen, who helped with theseexperiments, as well as the GSU animal care staff. Dr. L. Smale providedhelpful comments on the manuscript.

GRANTS

This work was supported by National Institutes of Mental Health Grant58789 (to H. E. Albers) and National Research Service Award 12956 (to C. M.Novak).

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