The period clock gene is expressed in central nervous system

The period protein (PER) is an essential component of the circadian clock in Drosophila melanogaster. Although PER-containing pacemaker cells have been previously identified in the brain, the neuronal network that comprises the circadian clock remained unknown. Here it is shown that some PER neurons are also immunostained with an antiserum against the crustacean pigment-dispersing hormone (PDH). This antiserum reveals the entire arborization pattern of these pacemaker cells. The arborizations of these neurons are appropriate for modulation of the activity of many neurons and they might interact with PER-containing glial cells. A putative physiological role of PDH in the circadian system is discussed. The importance of the period gene (per) for circadian rhythmicity in the fruitfly Drosophila melanogaster is well documented (for review, see ref. 1). Mutations at the period locus lengthen, shorten, or apparently abolish the circadian rhythm of eclosion and locomotor activity of adult flies (1, 2). The period gene product (PER), a predominantly nuclear protein (3), appears to be involved in circadian gene regulation at the transcriptional level (4-6). Experiments involving mosaics (7) and brain-transplantation experiments (8) show that the focus for the action of PER maps to the brain. PER is found in the nuclei of the photoreceptor cells of the compound eye and the ocelli, in many glial cells throughout the brain, and in a few neurons (9-12). A circadian cycling in the abundance of PER is observed in all these cells (9, 10) and a cycling in the level of overall mRNA of PER can be measured (4, 5). Cell bodies of PER-containing neurons lie in the posterior dorsal cortex of the central brain and in the lateral brain close to the optic lobe (9-12). Because of their position the latter were called lateral neurons (LNs) (9, 10). Circumstantial evidence points to the LNs as the site for circadian pacemakers (9-11, 13). In addition, PER-containing glial cells appear to be important for rhythmicity (11). The neuronal network that comprises the circadian clock, and the pathway by which the pacemaker neurons transfer the circadian information to subsequent cells, is still unknown. A major problem is that antisera raised against PER do not immunostain axons and dendrites of the LNs. Neurons that fulfill the anatomical criteria (i.e., location and arborization pattern) for pacemaker neurons have recently been described for cockroaches (14), crickets (14), and Drosophila (15). These neurons immunoreact with an antiserum (16) against the pigment-dispersing hormone (f3-PDH) of the fiddler crab Uca pugilator. The PDHs, a family of octadecapeptides, were originally isolated from crustacean species (17), where they trigger the light-adaptational migration of chromatophoral and retinal screening pigment (18). Peptides similar to the crustacean PDHs were found in different insects (19), and PDH-immunoreactive neurons could be revealed in the brain of a variety of hemiand holometabolous insect species (14, 15, 20-24). In Drosophila the most prominent PDH-immunoreactive neurons have somata at the anterior margin of the medulla and arborize extensively in the optic lobe and the brain (15). Based on anatomical criteria, they are well suited to modulate the activity of many neurons in the optic lobe and to transfer the circadian information to the midbrain (15). Since their somata are similar in location to the LNs, it was suggested that these are at least partly identical (15). To test this hypothesis, double-label experiments were performed in the present study. MATERIALS AND METHODS To visualize PER-containing cells transformant flies (D. melanogaster) carrying a fusion between a fragment of DNA from the period locus and the sequences encoding the bacterial enzyme 3-galactosidase (12) were used. In these transformants 13-galactosidase expression is a faithful reporter of PER expression (12, 25). 8-Galactosidase histochemistry was carried out on dissected whole-mount brains using the chromogen X-Gal (5-bromo-4-chloro-3-indolyl 3-D-galactopyranoside) as described by Liu et al (12). Flies were maintained in a light/dark cycle (12 h:12 h). They were dissected in the early morning, just after the light turned on, since at that time the amount of PER is rather high (10). To visualize the PDH-immunoreactive neurons, an antiserum raised in rabbit (16) was used. The specificity of the anti-PDH antiserum has been well documented (14-16, 21, 22, 24). Preincubation of the diluted anti-PDH antiserum with 100 ,tM Uca/Cancer 13-PDH for at least 5 h at room temperature abolished all immunoreactive staining in the brain ofDrosophila. For the double-staining, ,B-galactosidase histochemistry was first carried out on dissected whole-mount brains after they had been fixed for 4 min in 4% paraformaldehyde. The stained brains were subsequently fixed for 4 h in 4% paraformaldehyde/7.5% picric acid in 0.1 M phosphate buffer (pH 7.4) and either the brains were immunostained with the anti-PDH antiserum as whole mounts or they were first embedded in gelatin/albumin and sectioned on a Vibratome at 30 ,um. PDH immunocytochemistry was performed on the whole mounts or on the sections by either the indirect immunofluorescence technique with a tetramethylrhodamine f3 isothiocyanateAbbreviations: LN, lateral neuron; PDH, pigment-dispersing hormone; X-Gal, 5-bromo-4-chloro-3-indolyl ,B-D-galactopyranoside; LNd, dorsal LN; LNv, ventral LN; DN, dorsal neuron; SCN, suprachiasmatic nuclei. 612 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Proc. Nati Acad. ScL USA 92 (1995) 613