Sustained activation of GABAA receptors in the suprachiasmatic nucleus mediates light-induced phase delays of the circadian clock: a novel function of ionotropic receptors

Abstract

The suprachiasmatic nucleus (SCN) contains a circadian clock that generates endogenous rhythmicity and entrains that rhythmicity with the day-night cycle. The neurochemical events that transduce photic input within the SCN and mediate entrainment by resetting the molecular clock have yet to be defined. Because GABA is contained in nearly all SCN neurons we tested the hypothesis that GABA serves as this signal in studies employing Syrian hamsters (Mesocricetus auratus). Activation of GABAA receptors was found to be necessary and sufficient for light to induce phase delays of the clock. Remarkably, the sustained activation of GABAA receptors for more than three consecutive hours was necessary to phase-delay the clock. The duration of GABAA receptor activation required to induce phase delays would not have been predicted by either the prevalent theory of circadian entrainment or by expectations regarding the duration of ionotropic receptor activation necessary to produce functional responses. Taken together, these data identify a novel neurochemical mechanism essential for phase-delaying the 'master' circadian clock within the SCN as well as identifying an unprecedented action of an amino acid neurotransmitter involving the sustained activation of ionotropic receptors.

Keywords: entrainment; hamster; neurotransmission; pacemaker; photic.

Figure 1

(A) Mean±SE of phase delays produced by 4 hourly injections of muscimol (21.9mM) into the SCN region between CT13.5 and CT16.5 (hit = animals receiving injections of muscimol within 500µm of the SCN; miss = animals receiving injections of muscimol more than 500µm from the SCN; *, p=0.021, vs. vehicle controls). (B) Mean±SE of phase delays produced by 4 hourly injections of muscimol (doses ranged from 6.6mM to 35.1mM) into the SCN region between CT13.5 and CT16.5. The magnitude of phase delays was dose-dependent (p=0.001). (C, D) Representative activity records demonstrating the effect of 4 hourly injections of vehicle (C) or muscimol (21.9mM) (D) into the SCN region between CT13.5 and CT16.5 on wheel running rhythms in DD. Bars depict the 4-hour injection period (white: saline; shaded: muscimol).

Figure 2

(A) Injection regimen used to determine the duration of GABA_A receptor activation necessary to induce a phase delay. All groups received a series of 4 hourly injections into the SCN region between CT13.5 and CT16.5. However, the number of consecutive injections containing muscimol (21.9mM) varied from 0 to 4 (VEH = vehicle; MUS = muscimol). (B) Mean±SE of phase delays produced by the 5 treatments outlined in A (* vs. 0 muscimol injections, p=0.002). (C) Injection regimen used to determine whether a single injection of muscimol at CT 16.5 might induce a phase delay or whether a single injection equal to the cumulative dose of 4 individual injections of muscimol might induce a phase delay. Animals in the 0.0 mM group received 4 hourly injections of vehicle into the SCN region between CT13.5 and CT16.5. Animals in the 21.9 and 87.6mM groups received 3 consecutive hourly injections of vehicle followed 1 hour later by a single injection of 21.9mM or 87.6mM muscimol into the SCN region (VEH = vehicle; MUS = muscimol). (D) Mean±SE of phase delays produced by the 3 treatments outlined in C.

Figure 3

(A) Mean±SE of phase delays produced by a light pulse (15 min, 150 lux) at CT13.5 followed by 8 hourly injections of vehicle or bicuculline (0.9mM) into the SCN region between CT14.5 and CT21.5 (*p=0.013). (B, C) Representative activity records demonstrating the effect of 8 hours of vehicle (B, white bar) or bicuculline (C, shaded bar) administration on light-induced phase delays in DD (sun blast = light pulse). (D) Light pulse and injection regimen used to determine whether the ability of light to induce phase delays requires the sustained activation of GABA_A receptors in the SCN. All animals received a light pulse (15 min, 150 lux) at CT13.5 followed by 8 hourly injections into the SCN region between CT14.5 and CT21.5. The number of consecutive injections containing bicuculline (0.9mM) varied from 0 to 8 (VEH = vehicle; BIC = bicuculline). (E) Mean±SE of phase delays produced by each of the treatment regimens outlined in D (*, p=0.035; **, p<0.001, vs. 0 inj controls). (F–I) Representative activity records demonstrating the effect of 0 (F, −96 min), 3 (G, −50 min), 6 (H, 19 min), and 8 hourly injections of bicuculline (I, −14 min) on light-induced phase delays in DD (sun blast = light pulse). White bars and shaded bars indicate times during which vehicle and bicuculline were administered, respectively.

Figure 4

(A) Light pulse and injection regimen used to determine whether the inhibition of GABA_A receptors during various phases between CT 14.5 and CT 21.5 would inhibit light-induced phase delays. All groups received a light pulse (15 min, 150 lux) at CT13.5 followed by 8 hourly intracranial injections containing either vehicle or bicuculline (0.9mM) into the SCN region between CT14.5 and CT21.5. Animals received one of 6 injection regimens (VEH = vehicle; BIC = bicuculline) (N’s: A=19, B=7, C=6, D=10, F=17. (B) Mean±SE of phase delays produced by the various injection regimens outlined in A.

Figure 5

Illustration of the minimal amount of damage produced by multiple injections into the SCN region as compared to the damage produced by single injections. (A) Photomicrograph from an animal that received a single injection into the SCN region. (B) Photomicrograph from a representative animal that received 8 hourly injections into the SCN region. In each case, a viscous ink was microinjected via the guide cannula to mark the drug injection track; it does not indicate the spread of drugs during experiments. Scale bars are 300µm.

Figure 6

Summary of the effects of over 1700 injections containing muscimol, bicuculline or vehicle into the SCN region on the phase of circadian rhythms. A. Solid red bar indicates the timing of SCN injections in which muscimol induces a significant phase delay in the locomotor rhythm. Open red bars indicate the timing of SCN injections of muscimol that did not produce significant phase delays. B. Solid blue bars indicate the timing of SCN injections in which bicuculline significantly inhibited light-induced phase delays. Open blue bars indicate the timing of SCN injections of bicuculline that did not significantly inhibit light-induced phase delays. Yellow bar indicates the timing of the 15-minute light pulse. C. Proposed sequence of neurochemical events within the SCN necessary for a light pulse to induce a phase delay. Light induces release of glutamate (GLU) that activates NMDA receptors within the SCN for seconds and possibly minutes (initial transient response). The transient responses to light induce activity in non-rhythmic SCN neurons that begins 30–60 minutes after the light pulse resulting in the sustained release of GABA for 6 or more hours. The sustained GABA release from non-rhythmic neurons results in the sustained activation of GABA_A receptors on rhythmic SCN neurons producing a phase delay in the molecular clock mechanisms.

Figure 7

Proposed regulation of the phase of the circadian clock and Period (Per) gene expression in the SCN by GABA_A receptor activation and inactivation. Left Panel: As described in Figure 6, light results in glutamate release from the retinohypothalamic tract (RHT). In response, there is a sustained release of GABA from, as well as a sustained induction of Per in, non-rhythmic neurons. In response, there is a sustained activation of GABA_A receptors and a sustained induction of Per in rhythmic neurons resulting in a phase delay of the circadian clock. Middle Panel: Acute activation of GABA_A receptors by injection of muscimol prior to a light pulse inhibits light induction of the sustained release of GABA from, as well as an inhibition of Per induction in, non-rhythmic neurons. Acute activation of GABA_A receptors inhibits NMDA-induced phase delays suggesting that activation of GABA_A receptors does not inhibit light-induced phase delays solely by inhibiting light-induced glutamate release (Mintz et al., 2002). Acute activation of GABA_A receptors ultimately blocks light-induced phase delays by preventing Per induction in rhythmic neurons. Right Panel: Sustained inhibition of GABA_A receptors by at least six hourly injections of bicuculline following a light pulse blocks light-induced phase delays by inhibiting Per induction in rhythmic neurons.