The brain of larval Rhodnius prolixus releases neurohormones with a circadian rhythm, indicating that a clock system exists in the larval brain. Larvae also possess a circadian locomotor rhythm. The present paper is a detailed analysis of the distribution and axonal projections of circadian clock cells in the brain of the fifth larval instar. Clock cells are identified as neurons that exhibit circadian cycling of both PER and TIM proteins. A group of eight lateral clock neurons (LNs) in the proximal optic lobe also contain pigment-dispersing factor (PDF) throughout their axons, enabling their detailed projections to be traced. LNs project to the accessory medulla and thence laterally toward the compound eye and medially into a massive area of arborizations in the anterior protocerebrum. Fine branches radiate from this area to most of the protocerebrum. A second group of clock cells (dorsal neurons [DNs]), situated in the posterior dorsal protocerebrum, are devoid of PDF. The DNs receive two fine axons from the LNs, indicating that clock cells throughout the brain are integrated into a timing network. Two axons of the LNs cross the midline, presumably coordinating the clock networks of left and right sides. The neuroarchitecture of this timing system is much more elaborate than any previously described for a larval insect and is very similar to those described in adult insects. This is the first report that an insect timing system regulates rhythmicity in both the endocrine system and behavior, implying extensive functional parallels with the mammalian suprachiasmatic nucleus. J. Comp. Neurol. 518:1264 -1282, 2010. INDEXING TERMS: insect; clock; lateral neurons; PDF; neuroarchitecture; timing network; PERIOD; TIMELESS Circadian timing systems have been found in all organisms that have been studied, from bacteria to humans. They enable organisms to anticipate the arrival of favorable times of day (or seasons of the year) for execution of diverse rhythmic activities ranging from gene expression to hormone secretion, growth, reproduction, and behaviours. These circadian rhythms are driven by endogenous biological clocks centered primarily in groups of nerve cells. These cells generate rhythms with a periodicity of about 24 hours that become synchronized (entrained) to the precisely 24-hour external world by time signals (Zeitgebers) arising principally from components of the light/ dark cycle, such as dusk and dawn.The molecular machinery with which cells generate circadian rhythmicity was first elucidated in the fruit fly, Drosophila melanogaster. Several genes have been found that are central to the generation of circadian oscillations. These oscillations fundamentally comprise feedback loops between transcription of the genes and their protein products (reviewed by Hall, 2005;Taghert and Lin, 2005) and summarized by Nitabach and Taghert (2008) and Dubruille and Emery (2008). Rhythmic transcription of the canonical clock genes in Drosophila, period (per), and timeless (tim) leads to rhythmic formation of PE...

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This paper reports the localization in the Rhodnius prolixus brain of neurons producing the key neuropeptide that regulates insect development, prothoracicotropic hormone (PTTH) and describes intimate associations of the PTTH neurons with the brain circadian timekeeping system. Immunohistochemistry and confocal laser scanning microscopy revealed that the PTTH-positive neurons in larvae are located in a single group in the lateral protocerebrum. Their number increases from two in the last larval instar to five during larval-adult development. In adults, there are two distinct groups of these neurons composed of two cells each. A daily rhythm in content of PTTH-positive material occurs in both the somata and the axons in both larval and adult stages. These rhythms correlate with previous evidence of a circadian rhythm of PTTH release from brains in vitro. The key circadian clock cells of Rhodnius are eight neurons, which co-express pigment-dispersing factor (PDF) and the canonical clock proteins PER and TIM; PDF fills the axons. Equivalent cells control behavioral rhythms in other insects. Double labeling revealed intimate associations between axons of larval PTTH neurons and clock neurons, indicating a neuronal pathway from the brain timekeeping system for circadian control of PTTH release. Additional PDF neurons appear in the adult, associated with the second group of PTTH neurons. These findings provide the first direct evidence that neurons of the insect brain timekeeping system control hormone rhythms. The range of functions regulated by this timekeeping system is quite similar to those of the vertebrate suprachiasmatic nucleus, for which the insect system is a valuable model.

Prothoracicotropic hormone (PTTH) is the central cerebral neurohormone in insect development. Its release has been believed for decades to be confined to one (or two)

The rhythmic phenomena expressed by organisms change over their lifetimes, but little is known of accompanying reorganization of the central circadian timing system in the brain. Especially dramatic changes in overt rhythms and morphology occur during transformation of larval insects into the adult form (metamorphosis). In Rhodnius prolixus, both the physiology of metamorphosis and its hormonal control are known in detail. Here we report changes in the brain timing system as revealed by pigment dispersing factor immunohistochemistry and confocal microscopy. Most of the features of the larval system are retained, but new clock cells differentiate and the arborizations of their axons increase in complexity, as do pathways connecting the lateral (LNs) and dorsal (DNs) groups of clock neurons. Early in metamorphosis, the LNs increase from 8 to 11 in number, becoming five small and six large LNs. Two large LNs then migrate to new positions in the protocerebrum. Another clock cell differentiates in the posterior protocerebrum. Each change occurs at a characteristic concentration of the ecdysteroid molting hormones that regulate metamorphosis. Clock cell axons invade the mushroom body and corpus allatum and travel down the ventral nerve cord. New overt rhythms develop during metamorphosis, in which these structures participate. The neuroendocrine cells of the brain receive more extensive branches of clock cell axons than in larvae. These increases in size and complexity of the circadian system during metamorphosis imply a greater complexity and diversity of outputs from it to both behavioral and hormonal rhythms in the adult.