FIGURE 2 Functionally defined model of the circadian system. An entrain-ment pathway that consists of a photoreceptor and coupling mechanism (input) synchronizes a self-sustaining oscillator (pacemaker) to the external light/dark cycle. The output of the pacemaker regulates the timing of various processes (e.g., activity) via coupling to the effector mechanisms.

by which the pacemaking system regulates the various processes under its control?

Circadian Oscillations Are Generated by Discrete, Localized Populations of Cells

Studies on pacemaker localization in insects have largely focused on behavioral rhythms (locomotor activity or eclosion) and their control by the nervous system. Compelling evidence that the brain is the site of generation of circadian timing signals for rhythms in behavior has been obtained in several species, with much of the early work involving studies on the locomotor activity rhythm of the cockroach.

In 1968 it was first discovered that surgical removal of both optic lobes or disconnecting them from the rest of the brain by section of the optic tracts abolished the activity rhythm of the Madeira cockroach, Leucophaea maderae. Results of lesion studies on other cockroach species, several species of crickets, and beetles have also suggested that the optic lobes might contain the pacemaker. Compelling evidence arose from the observation that it is possible to transplant optic lobes between cockroaches whose activity rhythms had quite different free-running periods. Animals that received transplanted optic lobes recovered rhythmicity in a few weeks with regeneration of the optic tracts, and the preoperative period of the donor and the postoperative period of the host were strongly correlated. Thus, the transplantation of the optic lobes not only restored the rhythm of locomotor activity but also, critically, imposed the period of the donor animal's rhythm on the activity of the host. Other studies involving small electrolytic lesions indicated that the cells responsible for generating the circadian signal have their somata and/or processes in the proximal half of the optic lobe, likely in a group of cells located ventrally near the medulla.

In contrast to cockroaches, crickets, and beetles, in a variety of other insects the optic lobes do not appear to be required for rhythmicity and the pacemaker appears instead to reside in the cerebral lobes (midbrain). In a classic series of experiments by James Truman and colleagues it was shown that the circadian pacemaker that controls the timing of eclosion in two silkmoth species, Hyalophora cecropia and Antheraeapernyi, is located in the cerebral lobes of the brain. The time of day at which eclosion occurs is different for the two species. When the insects are maintained in a photoperiod of 17:7 (L:D) hours, H. cecropia emerges shortly after lights-on while A. pernyi emerges just before lights-off. Removal of the brain did not prevent eclosion, but did disrupt its timing. However, if the brain was reimplanted in the abdomen, normal rhythmicity was restored under both entrained and free-running conditions. When brains were transplanted between species, individuals exhibited normal species-specific eclosion behavior, but the phase of the rhythm was characteristic of the donor and not the host. The demonstration that the transplanted brains restored rhyth-micity and determined the phase of the rhythm left little doubt that the circadian pacemaker that regulates the timing of the eclosion rhythm is located in the brains of these moths. The fact that the pacemaker was located in the cerebral lobes and not the optic lobes was demonstrated by subdividing the brain prior to transplantation. It was found that the optic lobes were unnecessary and that transplantation of the cerebral lobes alone was sufficient to restore rhythmicity.

Similarly, in a variety of dipterans, including the fruit fly, the house fly, the blow fly, and the mosquito, regions of the nervous system controlling locomotor activity rhythms have been dissected with both surgical and genetic lesions, and in each instance the pacemaking oscillation appears to be generated in the cerebral lobes. In the fruit fly, Drosophila melanogaster, extensive behavioral and genetic evidence demonstrates a crucial role for the period (per) gene in the circadian pacemaker controlling locomotor activity and eclosion rhythms (see later). The per gene is widely, and in some cell types rhythmically, expressed in the fly, including the head, thorax, and abdomen; thus, its spatial expression pattern in wild-type flies provided no definitive localization of the central pacemaker. However, the expression pattern has been altered by numerous genetic and molecular manipulations and it has been possible to determine the identity of the pacemaker cells in Drosophila by correlating per expression in specific cell types with the presence or absence of behavioral rhythmicity. The results suggest that only a few neurons between the lateral protocerebrum and the medulla of the optic lobes, the lateral neurons, are necessary for the generation of a circadian rhythm in locomotor activity.

The potential for further cellular identification of pacemaker neurons in insects was provided by an observation that in cockroaches and crickets optic lobe neurons that fulfilled the predicted anatomical criteria to be pacemaker cells were labeled by an antibody to crustacean pigment-dispersing hormone (PDH). When anti-PDH was applied to Drosophila brains, it labeled a ventral subset (LNv) of theper-expressing lateral neurons that were identified as pacemaker neurons in genetic studies. Taken together, the results indicated that the PDH-immunoreactive neurons are strong candidates for pacemaker neurons in insects and raise the possiblity that the insect version of crustacean PDH (called pigment-dispersing factor or PDF) may be an important temporal signaling molecule.

Interestingly, the numbers and projection patterns of PDF neurons in cockroaches and crickets are strikingly similar to those of the LNv of Drosophila, suggesting that they are functionally homologous. The most salient difference in the morphology of these neurons is in the locations of their somata: between the lobula and the medulla of the optic lobes in cockroaches and crickets, as opposed to between the medulla of the optic lobes and the lateral margin of the cerebral lobes in fruit flies. This difference may be sufficient to account for the fact that the lesion and transplant studies suggested different anatomical organizations for pacemaker structures in the central nervous system in different insects.

Circadian Pacemakers Are Also Found in Tissues outside the Nervous System in Insects

The localization of circadian pacemakers that regulate behavioral rhythms to the brain raised the question of whether other rhythms are controlled by the same clock. In crickets, beetles, and cockroaches studies indicate that the pacemaker regulating the daily rhythm in retinal sensitivity to light as measured by electroretinogram (ERG) amplitude is located in the optic lobe and suggest that the same pacemaker controls both the ERG amplitude and the activity rhythms. However, in other cases rhythms have been found to be regulated by pacemakers outside the nervous system. These include rhythmic secretion of cuticular layers in newly molted cockroaches, the release of sperm from the testis into the seminal ducts in gypsy moths, and the timing of ecdys-teroid release from the prothoracic gland of the cynthia moth, Samia cynthia. In each of these examples the rhythms were shown to persist in vitro in the absence of neural pacemaking structures.

These results indicate that the distribution of circadian pacemaking centers may be widespread in insects. In support of this view, one recent study by Plautz and co-workers with Drosophila, in which the per promoter was coupled to the coding sequence for luciferase, indicated that rhythmic promoter activity could be detected in a variety of tissues, including the wing, leg, proboscis, and antennae maintained in isolation in tissue culture.

The fact that the circadian system in the individual may be composed of several widely distributed oscillators raises the question of whether there is communication between component oscillators. In general the answer is uncertain. Work on cockroaches has shown that the bilaterally distributed oscillators in the two optic lobes are connected to one another (mutually coupled) and suggested that the coupling was relatively strong. In contrast, both in the beetle, Blaps gigas, and in crickets the data indicated that coupling between optic lobe pacemakers is either absent or weak. Coupling relationships among other oscillators have not yet been systematically explored.

Photoreceptors for Entrainment

Extraretinal photoreceptors are typically involved in entrainment of behavioral rhythms. The classical example is the silkworm, in which it was shown that the photoreceptor for entrainment of the eclosion rhythm resides in the brain. Brains were removed from silkworm pupae and were either replaced in the head region or transplanted to the abdomen. The pupae were then placed in holes in a partition that separated two chambers in which the light/dark cycles were out of phase. Whether the pupae entrained to the light cycle to which the anterior end of the pupae was exposed or entrained to the light cycle at the posterior end corresponded to the location of the brain.

Additional evidence for extraretinally mediated entrainment of pacemakers that are located in the nervous system has been obtained in a variety of other insects, including other lepidopterans, dipterans, and orthopterans. In those instances in which there is evidence on the location of the photoreceptor, the brain appears to be the most likely site. However, more precise identification of the cells involved in the phototransduction has not been accomplished.

Even though the compound eyes may not be necessary for entrainment, they may nevertheless participate. In Drosophila, for example, genetic lesions to the eyes or the phototransduction pathway can alter the entrainment pattern. Further, there are at least two insects, the cockroach and the cricket, in which the compound eyes appear to be the exclusive photoreceptors for entrainment because sectioning the optic nerves between the eyes and the optic lobe or painting over the compound eyes eliminated entrainment of the locomotor activity rhythm to light cycles.

As noted above, there are several instances in which there is convincing evidence for circadian pacemakers outside the nervous system. In the case of the moth testis, since the rhythm measured in vitro responds to light, some cells in the testis—seminal duct complex must be photosensitive. Similarly, in the saturnid moth S. cynthia, the photoreceptor for entrainment of the pacemaker in the prothoracic glands appears to be in the gland itself.

Signals to Communicate Timing Information

Another important issue is how circadian oscillators impose periodicity on the various physiological and behavioral processes they control. A priori, several alternative mechanisms are plausible. Timing information within the individual could be represented by the level of a circulating hormone, impulse frequency in specific neural circuits, changes in general levels of neural excitability through neuromodulation, or, as the weight of the available evidence suggests, some combination of these mechanisms.

There are a large number of studies that suggest that secretion of a variety of insect hormones, including ecdysone, prothoracicotropic hormone, and eclosion hormone, is under the control of the circadian system during development. The experiments involving the transplantation of the silkworm brain, described above, provide the clearest demonstration of a hormonal link in the control of behavior by the circadian system. The signal for the eclosion behavior is the eclosion hormone that is produced in neurosecretory cells located in a region near the midline of the brain, the pars intercerebralis, and released via the neurohemal organs, the corpora cardiaca. The release of the hormone triggers release of two other peptide hormones, pre-ecdysis-triggering hormone (PETH) and ecdysis triggering hormone (ETH). PETH and ETH act on the central nervous system to initiate a stereotyped sequence of behavior that ultimately results in the emergence of the adult moth from the pupal case.

The role of humoral factors in the regulation of adult behaviors in insects (e.g., locomotor activity) is less clear. In cockroaches and crickets, the timing signal that originates in the optic lobe is transmitted to the brain via the optic tracts, and transmission from the brain to the activity centers in the thorax requires that the connectives of the ventral nerve cord be intact. Nerve impulse activity is rhythmic in both the optic tracts and the cervical connective.

In summary, the mechanism by which circadian phase information is transmitted to behavioral effectors in insects is generally not well understood. The emerging picture is that temporal regulation of behavior involves a modulation of excitability in the central nervous system. Axonal connections between the brain and the lower elements of the central nervous system are clearly required for the maintenance of some behavioral rhythms (e.g., cockroach locomotor activity), whereas others appear to rely heavily on hormonal mechanisms (moth eclosion). An important step in understanding how temporal information is transmitted will be the identification of the signal molecules involved.

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