Is it the clock’s fault?

Brandeis researchers Jerome Menet and Professor Michael Rosbash (Biology Dept., Natl. Ctr. for Behavioral Genomics, and HHMI) review the relationships between psychiatric disease and the circadian clock in a review entitled “When brain clocks lose track of time: cause or consequence of neuropsychiatric disorders“. This review appeared recently in Current Opinion in Neurobiology. They discuss an increasing body of evidence that disorders in the clock may be directly involved in the etiology of these disorders.

Light buffers the wake‐promoting effect of dopamine

Sleep is driven and regulated by the integration of diverse internal and external (environmental) cues. Light is known to be a potent inhibitor of sleep in diurnal animals (awake during daylight hours and sleep at night), including both humans and fruit flies. Yet wakefulness does not scale linearly with light intensity and a lack of light does not automatically result in sleep. (Evolution seems unlikely to favor animals who become hyperactive in dangerously hot midday sunlight and fall asleep in an uncontrollable narcoleptic fashion when the sun goes down, unable to wake until the next morning.) The sleep regulatory system must be plastic — capable of weighing the relative importance of incoming sleep and wake‐promoting cues, and buffering the effects of those cues on sleep drive accordingly. In a recent Nature Neuroscience paper from a team led by postdoc Yuhua Shang (Rosbash lab), with collaborators from the Griffth, Pollack, and Hong labs at Brandeis, we determined at the cell and molecular level how the fruit fly, Drosophila melanogaster, is able to buffer the wake‐promoting effects of the neurotransmitters dopamine and octopamine in the presence of light in order to maintain a proper sleep:wake balance.

It is known that dopamine and octopamine both promote wakefulness in flies. Previous work in the Rosbash and Griffith labs has shown that 10 neurons in the Drosophila brain that release the neuropeptide pigment‐dispersing factor (PDF), known as the l‐LNvs, are critical for transducing the wake‐promoting effects of light. Quantifying mRNAs from all 18 PDF-expressing neurons revealed an enrichment of octopamine and dopamine receptors specifically in the ten wake‐promoting l‐LNvs. We wondered if the l‐LNvs were also able to respond to and transduce the wake‐promoting effects of dopamine and octopamine, and if so, how these effects were integrated with the wake‐promoting effects of light by these cells.

Figure: The l-LNvs use two parallel intracellular pathways to regulate the stimulating effects of DA and OA. Both DA and OA increase the cAMP levels in the l-LNvs. Light in the housing environment suppresses the effects of both DA and OA, but in different ways. In the case of dopamine, light induces increased expression of an inhibitory D2R receptor and in the case of octopamine, the effect is dependent on the circadian clock (Per.)

Using a fluorescence resonance energy transfer (FRET)‐based cyclic AMP reporter expressed in all 18 Pdf neurons, we were able to see robust responses to both octopamine and dopamine in only the t0 l‐LNvs, confirming the mRNA result. To verify that the l‐LNvs are in fact in close apposition to presynaptic octopaminergic and dopaminergic neurons, we looked for reconstitution of a split GFP protein between pre- and post‐synaptic cells. With different GFP fragments expressed at the membrane of the l‐LNvs and presynaptic dopaminergic or octopaminergic neurons, reconstituted GFP would only be visible if these cell populations were in close contact. Reconstituted GFP was seen in both cases around l‐LNv cell bodies and dendritic areas.

To determine the behavioral effect of increased dopaminergic neuron activity on sleep, we transiently hyper‐excited the dopaminergic neurons in flies using the Garrity lab’s heat‐activated dTrpA1 channel. When the housing temperature of flies expressing dTrpA1 in dopaminergic neurons was increased, activating dTrpA1 activity, flies exhibited increased wakefulness. Interestingly, this increased wakefulness was much greater in flies housed in constant darkness as compared to those housed in light:dark cycling conditions. This suggested that the l‐LNvs are a convergence point for the wakepromoting effects of dopamine and light. FRET analysis confirmed this, showing that the l‐LNv response to both dopamine and octopamine is much weaker in flies kept in light:dark conditions as compared to those kept in constant darkness. We then determined that light causes increased expression of an inhibitory dopamine receptor, resulting in a weaker excitatory response to dopamine by the l‐LNvs. In the case of octopamine, the circadian clock was found to regulate the effects of light. Such plasticity allows flies to maintain similar amounts of total sleep in varying environmental conditions, decreasing the relevance of internally generated wake‐promoting cues, in the presence of stronger environmental cues (light). It will be interesting to see how these results generalize to mammals, since light and dopamine also both promote wakefulness in mammals.

Time for Worms in Circadian Biology

Almost every organism on earth, from archae to humans, exhibits circadian rhythms – periodic cycles of behavior or gene expression that repeat approximately every 24 hours. These rhythms are generated by a circadian clock – an internal time-keeping mechanism – which can be entrained and synchronized by environmental cues such as temperature or light/dark cycles. This clock may provide organisms with an adaptive advantage throughout their life, and disruption of the function of this clock can lead to severe behavioral and metabolic disorders in humans.

For more than two decades researchers have wondered whether the tiny soil-dwelling nematode worm Caenorhabditis elegans, one of the foremost model organisms, contains a circadian clock. Circadian rhythmic behaviors described previously in C. elegans are variable and hard to quantify, and no genes were known to exhibit gene expression oscillations with 24 hr cycles as shown in many other animals.

Now, in a recent study published in the open-access journal PLoS Biology, several students and postdoctoral fellows in the labs of Piali Sengupta and Michael Rosbash joined forces and took on the challenge to identify C. elegans genes under clock control.

Light and temperature cycles both drive and entrain 24 hr oscillations in gene expression in C. elegans.

They showed that indeed C. elegans contains genes whose expression cycles in a circadian manner. They found that light and temperature cycles appear to regulate different sets of genes (see above), indicating that these stimuli may entrain two distinct clocks. Moreover, the underlying clock mechanisms may not be dependent on oscillations of known clock genes. “These findings were surprising to us since Drosophila only has a single conserved clock running in multiple cells and tissues” says Alexander van der Linden – lead author and former postdoctoral fellow in the Sengupta Lab.

C. elegans has a wealth of genetic and behavioral tools. The next critical step will be to identify the mechanisms underlying the C. elegans circadian clock(s). These investigations may also provide information of how the clock evolved since nematodes and humans split about 600-1200 million years ago.

Alexander M. van der Linden is now an Assistant Professor at the University of Nevada, Reno. The work was conducted in the labs of Profs. Michael Rosbash, a member of the Howard Hughes Medical Institute and Piali Sengupta in the Department of Biology. Other authors who contributed to this work include Molecular and Cell Biology graduate students Matthew Beverly, Joseph Rodriquez and Sara Wasserman (now a postdoctoral fellow at UCLA), and Sebastian Kadener, a former postdoctoral fellow who is now an Assistant Professor at the Silberman Institute of Life Science, The Hebrew University of Jerusalem, Israel.

Lots of seminars coming

Whole bunch of seminars and award lectures coming up in the next week. Steven Reppert from U. Mass. talks today at 4 about monarch butterfly migration and its relationship to the circadian clock. On Monday at noon, Giovanni Bosco (PhD in Mol Cell Biol, Brandeis, 1998) will talk about condensins and global chromosome structure.

On Tuesday, we have the 39th Annual Rosenstiel Award lectures at 4. Jules Hoffman and Ruslan Medzhitov will get award “for their elucidation of the mechanisms of innate immunity”.

Next Wednesday we have the Heart Research Series lecture. Monty Krieger, Whitehead Professor of Molecular Genetics at MIT, will talk about cholesterol, genetics, and heart disease. Finally, next Thursday will have Josh Tenenbaum from MIT speaking in the Psychology Colloquium about “How to Grow a Mind”.

Details (time, room number) about upcoming seminars are always available in the Seminars widget in the left-hand column on this blog.

Back online

We’ve been off-line for a while, and now we’ve moved into the new version of WordPress supported by the campus IT folks. It should now be relatively easy for labs to post themselves to this blogs.

What have we missed? Well, for one, Michael Rosbash and Jeff Hall are getting the Gruber Neuroscience Prize, together with Michael Young (Rockefeller U), for their work on genetics of circadian rhythms.

I’ll post some more of the “backdated” news when I get a chance. Feel free to ask for an account so you can do it yourself…

Rise and shine, little fly

Most animals sleep, but why they sleep and how the brain generates sleep is mysterious. In a recent study published in Neuron, postdoc Katherine Parisky and colleagues use genetic tools to manipulate the activity of neurons that control sleep in flies. Their results demonstrate that in the fly sleep is generated by GABAergic inhibition of a small cluster of peptidergic neurons within the circadian clock. Flies carrying mutations in this peptide, PDF, or its receptor, are hypersomnolent, similar to human narcoleptics who have defective signaling by the peptide hypocretin/orexin. These results suggest that the circuit architecture used to control arousal is ancient.

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