Circadian Clock Neurons

Here’s another interesting question from our most recent neurobiology exam. With some luck PZ won’t get irritated that I keep recycling my work. This paper was a bit of a brain thumper but also very interesting after deciphering what it’s talking about.

3) Summarize this paper and describe both the neural circuit and the genes underlying this particular rhythm.
Stoleru D, Peng Y, Agosta J, Rosbash M (2004). Coupled oscillators control morning
and evening locomotor behavior of Drosophila. Nature 431:862-868

The roughly one hundred bilaterally arranged circadian clock neurons in adult fly brains occur in six groups: dorsal neurons (DN1, DN2, DN3), dorsal lateral neurons (LNdS), and the PDF neuropeptide expressing small and large ventral lateral neurons (LNvS). Extirpation via proapoptotic genes was used to assess that D.melanogaster lacking LNvS in natural light/dark conditions displayed little change however in continuously dark environments yield arrhythmicity. Time intervals in the light/dark experiment were determined using Zeitgeber time in which lights on (sunrise) corresponds with ZT0 and lights off (sunset) corresponds with ZT12. Although the two LNvS cell groups have an imperative role in rhythmic gene expression, neurons expressing circadian photoreceptor cyrptochrome (cry) genes were also found to assist rhythmicity in natural light/dark conditions.

Green fluorescent protein reporter was used to stain the six clock neuron groups, determining that the cry-GAL4 driver, which facilitates cry gene expression, is present in all dorsal and ventral lateral neurons (LNdS and LNvS) and in two dorsal neurons (DN1). The proapoptotic gene hid was used to excise the cry gene in LNdS and LNvS generating cry-GAL4;UAS-hid flies that were arrhythmic in both natural light/dark and continuously dark environments. The dorsal neuron groups (DN1, DN2, DN3) are mostly unaffected by hid expression meaning that because cry-GAL4;UAS-hid flies are arrhythmic in both environments, these neuron groups are incapable of maintaining circadian rhythms independently.

Crossing D.melanogaster exhibiting the Pdf-GAL80 gene, which represses GAL4-mediated transcriptional activity, with flies exhibiting green fluorescent protein in circadian neuron groups via Pdf-GAL4 and cry-GAL4 drivers yielded flies without green fluorescent protein. This means that the cry and Pdf promotors, segments of DNA that control gene expression, coupled with GAL80 genes override and prevent their corresponding GAL4 drivers from transcribing. With the crossing of cry-GAL4 driver and Pdf-GAL80 repressor, and other mixed crosses, green fluorescent protein was observed only in some circadian neuron groups.

Crossing Pdf-GAL80 with cry-GAL4;UAS-hid allowed researchers to determine the effects of extirpating PDF+, CRY+PDF-, and CRY+ neurons on D.melonogaster circadian rhythm. They found that flies extirpated of CRY+ neurons were phenotypically arrhythmic and flies extirpated of PDF+ neurons had diminished morning lights-on anticipation with normal evening lights-off anticipation. Flies extirpated of CRY+PDF- had diminished evening lights-off anticipation and normal morning lights-on anticipation. These flies maintained a circadian rhythm in continuous darkness indistinguishable from wild type flies based unimodally on the morning oscillator. The phenotypes of these three strains of D.melonogaster are not affected by whether the environment is light and dark or continuously dark meaning that the oscillators are not driven by light. Using deductive logic, the researchers concluded that PDF+ neurons correspond to lights-on behavior and oscillate independently of CRY+PDF- neurons, which correspond to lights-off behavior.

Green fluorescent protein techniques for visualizing neurons confirm that the CRY+PDF- and PDF+ oscillators are coupled through PDF- axonal processes that protrude from the LNd neuron group into the LNv region. Immunostaining visualization techniques reveal that PDF neuropeptide travels in the opposite direction that the PDF- axons extend, that is, from the LNv to LNd neuron group. PDF neuropeptides function to coordinate the lights-on anticipation behaviors in the morning with the independently oscillating lights-off anticipation behaviors in the evening.

Stoleru D, Peng Y, Agosta J, Rosbash M (2004). Coupled oscillators control morning
and evening locomotor behavior of Drosophila. Nature 431:862-868


  1. #1 Brownian, OM
    December 12, 2007

    With some luck PZ won’t get irritated that I keep recycling my work.

    So far, no one here has noticed that I’ve been recycling my comments.

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