The Journal of Neuroscience, August 1989, g(8): 2858-2885 GABA Regulation of Circadian Responses to Light. I. Involvement of GABA,-Benzodiazepine and GABA, Receptors Martin R. Ralph and Michael Menaker” Institute of Neuroscience, University of Oregon, Eugene, Oregon 97403 Light-induced phase shifts of the circadian locomotor rhythm neurotransmission.In the golden hamster, Mesocricetus aura- of hamsters can be blocked by agents that alter GABA neu- tus, light-induced phase delays are blocked by the GABA an- rotransmission. The GABA antagonist bicuculline blocks tagonist, bicuculline, but responsesare unaffected by this drug phase delays induced by light and the benzodiazepine di- at times when light induces phase advances (Ralph and Men- azepam, which can potentiate GABA activity, blocks light- aker, 1985). Conversely, diazepam, a benzodiazepine (BZ) that induced phase advances. In the experiments reported here, potentiatesGABA neurotransmissionthrough action at GABA,- we found that the bicuculline blockade of phase delays was BZ receptors, blocks light-induced phaseadvances but not de- reduced by agents that mimic or potentiate GABA activity. lays (Ralph and Menaker, 1986). Taken together, these results Conversely, the diazepam blockade of phase advances was suggestthat GABA may be involved in the regulation of cir- reduced by both competitive and noncompetitive antago- cadian responsesto light. However, while bicuculline is thought nists of GABA. This indicates that the GABA-benzodiazepine to be a relatively selective antagonist of GABA, diazepam has receptor-ionophore complex is the most likely site of action numerous other effects in the CNS that may contribute to the for the effects of these drugs on circadian rhythms. However, blockade of the responseto light. Such mechanismsinclude competitive GABA agonists did not mimic the blocking ef- inhibition of adenosineuptake (Phillis et al., 1980, 1981; Wu fects of benzodiazepines, nor did the antagonist picrotoxin et al., 198l), inhibition of phosphodiesteraseactivity (Beer et mimic the blocking effect of bicuculline. Therefore, the clas- al., 1972) and blockade of calcium channels (Taft and De- sic action of GABA, increased chloride conductance, may Lorenzo, 1984). not be the effector mechanism in this case. We also found The experiments reported here were designedto determine that the GABA, agonist baclofen blocked both phase ad- whether, and in what specific ways, GABA is involved in the vances and delays and that the blockade of advances was light responsivenessof the circadian system in mammals. If reversed by the antagonist delta-aminovaleric acid. Taken GABA neurotransmissionis an important component of mam- together, these results indicate that GABA is involved in the malian circadian systems,then agentsthat affect it should affect regulation of circadian responses to light and that the reg- circadian rhythms in ways that reflect their known pharmaco- ulation is mediated by both GABA, and GABA, receptors. logical actions and interactions. We have tested the specific hypothesis that the effects of both The entrainment of circadian rhythms by light cycles is thought bicuculline and diazepam on light-induced phaseshifts are me- to occur primarily through daily, light-induced phaseadvances diated by GABA,-BZ receptors. The GABA, receptor is part and delays of endogenouscircadian oscillations (Pittendrigh, of a supramolecularreceptor complex that includes,along with 1960, 1965; DeCoursey, 1964). Single light pulses, given to the GABA site, binding sites for BZs, barbiturates, and picro- animals otherwise held in constant dark, induce phaseshifts in toxin. The effects of the variety of drugs that are active at these free-running circadian rhythms. Pulsesgiven in the early sub- sites have been reviewed extensively (seeOlsen, 1982; Ticku, jective night induce phase delays, whereas pulsesgiven in the 1983; Haefely et al., 1985). The cellular effectsof drugs at these late subjective night induce phaseadvances. For nocturnal ro- sites are most often interpreted as being mediated through dents, subjective night is considered to be the 12 hr interval changesin the conductance of a chloride ionophore associated following the onset of running wheel activity in constant lighting with the complex; however, recent reports have suggestedthat, conditions. in addition, the effects of drugs acting at the BZ site may be Recently, we have reported phase-dependentdifferences in mediated through the inhibition of adenylate cyclase activity the sensitivity ofthese light responsesto agentsthat affect GABA (Fung and Fillenz, 1984; Gray et al., 1984). We have tested the involvement of GABA,-BZ receptors by examining numerous Received Aug. 29, 1988; revised Jan. 25, 1989; accepted Jan. 30, 1989. agents that affect GABA neurotransmission.If the hypothesis We wish to thank Dr. M. E. Lickey, Dr. G. M. Cahill, and M. Max for their is correct, then theseshould alter the circadian responsesto light critical discussion of the manuscript. This work is part of a dissertation submitted by M. R. R. in partial fulfillment of the requirements for the degree of Ph.D. at in ways that reflect the known interactions of these drugs with the University of Oregon and was supported by National Institutes of Health GABA and the receptor complex. grants MH 17148 to M. R. R. and HD 13162 and a grant from Upjohn Co., Attempts to understand the putative actions of GABA in Kalamazoo, Michigan, to M. M. Correspondence should be addressed to Dr. Martin R. Ralph at his present regulating the effects of light on circadian responsesare com- address: Department ofPsychology, University ofToronto, Toronto, Canada M5S plicated by the fact that the effects of GABA in the CNS are 1Al. a Present address: Department of Biology, University of Virginia, Charlottes- known to be mediated by at least 2 receptor types. The GABA, ville, Virginia, 2290 1. receptor is distinct from GABA, and does not appear to be Copyright 0 I989 Society for Neuroscience 0270-6474/89/082858-08$02.00/O associated with a BZ receptor site nor a chloride ionophore The Journal of Neuroscience, August 1989, 9(E) 2859 (Bowery et al., 1983). Moreover, it is relatively insensitive to 1.0 GABA, agonists and to bicuculline (Bowery et al., 198 1, 1983). q vehicle This receptor, therefore, cannot account for the effects of di- q THIP azepam and bicuculline on circadian rhythms. On the other .a Ei diazepam hand, because the GABA, agonist baclofen decreases synaptic transmission in the suprachiasmatic nucleus (Shibata et al., 1986), the only identified site ofrhythm generation in mammals (Rusak .E and Zucker, 1979; Turek, 1985) it is possible that GABA may influence circadian rhythms or their responses to light via GABA, receptors, as well as via GABA,-BZ receptors. To test this hy- .4 pothesis, we have examined the effects of baclofen and a GABA, antagonist, delta-aminovaleric acid (DAVA). i Materials and Methods Male golden hamsters 8-12 weeks of age (Lakeview, Charles River), C were housed individually, and their locomotor rhythms (wheel-running injection light/ light/ activity) were recorded on Esterline-Angus event recorders as previously control. agonist bic;;cuIIi:e/ described (see Pittendrigh, 1965; Takahashi et al., 1984). Animals were initially exposed to a light cycle of 14 hr of light at 100 lux and 10 hr of dark (LD 14: 10) to which the animals were allowed to entrain before Figure I. Reduction of the bicuculline blockade of light-induced phase being released into constant dark (DD). After free-running for 7 d in delays by GABA,-BZ agonists. Injection control, animals given either DD, each animal was given either an intraperitoneal injection of a drug vehicle only (0.2 ml), vehicle + THIP, or vehicle + diazepam, respec- or combination of drugs specific for GABA,-BZ or GABA, sites, or a tively, at CT 13.5. Light group, animals given the same injections as drug injection followed by a 15 min pulse of 5 15 nm monochromatic controls prior to a 15 min phase-delaying light pulse; Light/bicuculline light atan intensity of 0.23 MW cmm2 srl). Injections were made in the group, animals given the same injections as controls along with 3.5 mgl dark with the aid of an infrared viewer (FJW Industries). Experiments kg bicuculline prior to a 15 min phase-delaying light pulse. The open were conducted at 2 circadian time (CT)points: at CT 13.5 (115 hr after histogram in the last set shows the bicuculline blockade of the light- activity onset) when light normally induces phase delays, and at CT 18 induced phase shift. * p < 0.05, ** p < 0.01 compared with bicuculline (6 hr after activity onset) when light normally induces phase advances. plus light; and *** p < 0.01 compared with vehicle control. n = 6 per The total fluence delivered during a light pulse (3 x lOI pho- group. Error bars represent SEM. tons cm -2 srml) was calculated to produce approximately half-maximal phase shifts in control animals at either time point (see Takahashi et al., 1984). 30 min prior to the light pulse, and DAVA was administered 5 min Involvement of GABA,-BZ receptors. Experiments conducted at CT prior to the light pulse via bilateral intracranial cannulae aimed at a 13.5 were designed to test whether the effects of bicuculline could be point 0.5 mm dorsal to the SCN. The placement of cannula guides, mimicked by other GABA,-BZ antagonists or reduced by GABA,-BZ implanted 2-3 weeks prior to the experiment, was later verified histo- agonists. Experiments conducted at CT 18 were designed to test whether logically. the effects ofdiazepam could be mimicked by other GABA,-BZagonists or reduced by GABA,-BZ antagonists. All drugs were administered in Results 100% DMSO (0.2 ml). Experimental animals were given an i.p. injection Interactions of GABA,-BZ drugs at CT 13.5 of a drug or drug combination prior to a light pulse (at 1 of the 2 time points). Control animals (at both time points) were given either an Bicuculline, 4.0 mgkg, significantly blocked the phase-delaying injection of the vehicle prior to a light pulse or an injection of one of effect of the light pulse at CT 13.5.