The core mechanism of circadian timekeeping in arthropods and vertebrates consists of feedback loops involving several clock genes, including period (per) and timeless (tim). In the fruitfly Drosophila, circadian oscillations in per expression occur in chemosensory cells of the antennae, even when the antennae are excised and maintained in isolated organ culture. Here we demonstrate a robust circadian rhythm in Drosophila in electrophysiological responses to two classes of olfactory stimuli. These rhythms are observed in wild-type flies during light-dark cycles and in constant darkness, but are abolished in per or tim null-mutant flies (per01 and tim01) which lack rhythms in adult emergence and locomotor behaviour. Olfactory rhythms are also abolished in the per 7.2:2 transgenic line in which per expression is restricted to the lateral neurons of the optic lobe. Because per 7.2:2 flies do not express per in peripheral oscillators, our results provide evidence that peripheral circadian oscillators are necessary for circadian rhythms in olfactory responses. As olfaction is essential for food acquisition, social interactions and predator avoidance in many animals, circadian regulation of olfactory systems could have profound effects on the behaviour of organisms that rely on this sensory modality.

Photoreceptors are non-spiking neurons, and their synapses mediate the continuous release of neurotransmitters under the control of L-type voltage-gated calcium channels (VGCCs). Photoreceptors express endogenous circadian oscillators that play important roles in regulating photoreceptor physiology and function. Here, we report that the L-type VGCCs in chick cone photoreceptors are under circadian control. The L-type VGCC currents are greater when measured during the subjective night than during the subjective day. Using antibodies against the VGCCa1C and VGCCa1D subunits, we found that the immunofluorescence intensities of both VGCCa1C and VGCCa1D in photoreceptors are higher during the subjective night. However, the mRNA levels of VGCCa1D, but not VGCCa1C, are rhythmic. Nocturnal increases in L-type VGCCs are blocked by manumycin A, PD98059, and KN93, which suggest that the circadian output pathway includes Ras, Erk, and calcium-calmodulin dependent kinase II. In summary, four independent lines of evidence show that the L-VGCCs in cone photoreceptors are under circadian control. Keywords: avian, circadian, photoreceptor, retina, voltagegated calcium channel. Visual systems must anticipate daily changes in ambient illumination over 10-12 orders of magnitude. Circadian oscillators in the retina provide a mechanism for visual systems to initiate more sustained adaptive changes throughout the course of the day (Cahill and Besharse 1995;Green and Besharse 2004). The circadian oscillators in photoreceptors are endogenous and able to function independently in the absence of other retinal inputs (Cahill and Besharse 1993;Thomas et al. 1993;Ko et al. 2001). Photoreceptor circadian oscillators regulate retinomotor movement (Pierce and Besharse 1985;Burnside 2001), outer segment disc shedding and membrane renewal (LaVail 1980;Besharse and Dunis 1983), morphological changes at synaptic ribbons (Adly et al. 1999), gene expression (Korenbrot and Fernald 1989;Pierce et al. 1993;Haque et al. 2002), and the gating behavior of ion channels (Ko et al. 2001) among other photoreceptor activities. Importantly, photoreceptors are more sensitive to intense light damage at night than during the day, even in animals that have been maintained in constant darkness (DD) for several days after circadian lightdark (LD) cycle entrainment (Vaughan et al. 2002).Photoreceptors are non-spiking neurons, and they release glutamate continuously in the darkness as a result of depolarization-evoked activation of L-type voltage-gated calcium channels (VGCCs) (Barnes and Kelly 2002). The synthesis and release of the neurohormone melatonin in photoreceptors is also under circadian control (Cahill and Besharse 1993;Bernard et al. 1997;Ivanova and Iuvone 2003b), and melatonin synthesis and release can be blocked by dihydropyridine inhibitors of L-type VGCCs (Iuvone and Besharse 1986;Ivanova and Iuvone 2003a). In this regard, we previously showed that there is a circadian regulation of the apparent affinity of cGMP-gated ion channels (CNGCs) for cG...

Antennal neurons are both necessary and sufficient for olfaction rhythms, which demonstrates for the first time that a peripheral tissue can function as an autonomous pacemaker in Drosophila. These results reveal fundamental differences in the function and organization of circadian oscillators in Drosophila and mammals and suggest that components of the olfactory signal transduction cascade could be targets of circadian regulation.

Cryptochromes are flavin/pterin-containing proteins that are involved in circadian clock function in Drosophila and mice. In mice, the cryptochromes Cry1 and Cry2 are integral components of the circadian oscillator within the brain and contribute to circadian photoreception in the retina. In Drosophila, cryptochrome (CRY) acts as a photoreceptor that mediates light input to circadian oscillators in both brain and peripheral tissue. A Drosophila cry mutant, cryb, leaves circadian oscillator function intact in central circadian pacemaker neurons but renders peripheral circadian oscillators largely arrhythmic. Although this arrhythmicity could be caused by a loss of light entrainment, it is also consistent with a role for CRY in the oscillator. A peripheral oscillator drives circadian olfactory responses in Drosophila antennae. Here we show that CRY contributes to oscillator function and physiological output rhythms in the antenna during and after entrainment to light-dark cycles and after photic input is eliminated by entraining flies to temperature cycles. These results demonstrate a photoreceptor-independent role for CRY in the periphery and imply fundamental differences between central and peripheral oscillator mechanisms in Drosophila.

ARIA (for acetylcholine receptor-inducing activity), a protein purified on the basis of its ability to stimulate acetylcholine receptor (AChR) synthesis in cultured myotubes, is a member of the neuregulin family and is present at motor endplates. This suggests an important role for neuregulins in mediating the nerve-dependent accumulation of AChRs in the postsynaptic membrane. Nerve-muscle synapses have now been analyzed in neuregulin-deficient animals. Mice that are heterozygous for the deletion of neuregulin isoforms containing an immunoglobulin-like domain are myasthenic. Postsynaptic AChR density is significantly reduced, as judged by the decrease in the mean amplitude of spontaneous miniature endplate potentials and bungarotoxin binding. On the other hand, the mean amplitude of evoked endplate potentials was not decreased, due to an increase in the number of quanta released per impulse, a compensation that has been observed in other myasthenic states. Thus, the density of AChRs in the postsynaptic membrane depends on immunoglobulin-containing neuregulin isoforms throughout the life of the animal.