Postdoc with confessed aversion to genetics

“… now inspiring a new generation of neurophysiologists”

There’s a nice story on the ADInstruments website about Stefan Pulver (PhD ’09) and Nick Hornstein (’11) and the tools they developed in the Griffith lab for “Optogenetics in the Teaching Laboratory” using Drosophila and channelrhodopsin-2. Stefan is currently in Cambridge (England) doing a postdoc, and Nick is starting his MD/PhD at Columbia real soon now.

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.

Brandeis Profs are Pretty Fly

Last week the Genetics Society of America (or GSA) held their annual Drosophila Research Conference in sunny San Diego.  Following a 52 year tradition, the meeting brought together some of the world’s greatest scientific minds to discuss all things fruit fly (formally known as Drosophila melanogaster).  Brandeis Professor Leslie Griffith and alumnus Giovanni Bosco (PhD ’98), now at the University of Arizona, were among the meeting’s head organizers, and were visible figures throughout the course of the entire conference.

Brandeis was also a commanding presence throughout the keynote talks, with Biologist Michael Rosbash kicking off the first night’s festivities.  His lecture, which documented the history of fruit fly behavioral research, recounted a number of both professional and personal experiences with some of history’s most renowned Drosophila researchers, including Seymour Benzer and Brandeis’ own Jeff Hall.  Neuroscientist Paul Garrity further represented Brandeis with his keynote address, titled “From the Cambrian to the Sushi bar: TRPA1 and the Evolution of Thermal and Chemical Sensing”.   The talk, which discussed the molecular underpinnings of thermosensation in fruit flies, also demonstrated that these mechanisms are well conserved between many invertebrate and vertebrate species, and likely date back to a common ancestor that walked (crawled?) the earth millions of years before humans existed.  Other presentations encompassed a number of exciting topics, including aging, immunity, population genetics, evolution, and models of human disease.

Brandeis Professors Michael Rosbash (left) and Paul Garrity (right), both of whom were featured in this year’s Drosophila Research Conference Keynote Lectures.

 

The next meeting will be held on March 7-11, 2012 in Chicago, Illinois.  For more information, visit http://www.drosophila-conf.org/2012/.

Getting a Leg Up on Movement Disorders

Over 40 million people worldwide suffer from movement disorders, which are clinically defined as any type of affliction that affects the speed, fluency, ease, or quality of motion. The symptoms of these disorders can manifest in many different ways (the most common being tics, tremors, dystonia, and chorea), and treatment is still elusive for a large number of these often debilitating diseases.  The past several decades, however, have seen enormous advances in our understanding of the genes and proteins underlying these conditions, and what remains to be determined is the way in which these molecules interact with each other to produce either normal or pathological locomotor patterns.

Scaffolding proteins have recently become a point of interest in the field of movement disorders.  As their name implies, these proteins act as “scaffolds” to tether other proteins together, thus facilitating protein-protein interactions.  It has long been thought that scaffolding protein dysfunction could disrupt the formation of protein complexes critical for the production normal locomotion, but evidence for such conjectures has remained elusive.

in a recent article in the journal GENETICS, Dr. Leslie Griffith’s lab at Brandeis University published work implicating one such scaffolding protein of the MAGUK family, known as CASK-b, in locomotor pathology. Using the fruit fly Drosophila melanogaster as a model system, researchers in the lab combined recently-developed genetic tools with cutting-edge computer behavior analysis software to demonstrate that knocking out this protein produces a complex motor deficit (see figure below).  Furthermore, this deficit appears to stem from a loss of CASK-b in the central nervous system, suggesting it plays a role in higher-order regulation of motor output.  Interestingly, both the major locomotor control center of the insect brain (known as the ellipsoid body), as well as the motor neurons which the locomotor control center regulates, do not appear to require this protein to produce normal locomotor patterns.  This finding implies that a novel region or regions of the fly brain may be contributing to central locomotor control.  Understanding both the specific mechanism through which this protein acts, as well as the underlying circuitry responsible for this deficit, could contribute largely to the field of movement disorders as a whole.

Another surprising finding to come out of this study was the discovery of an additional mRNA transcript that arises from an alternative promoter in the CASK locus.  Although similar to CASK-b in many ways, this alternative protein is actually most homologous to another member of the same family in vertebrates, known as MPP1.  MPP1, like most of its MAGUK cousins, is also a scaffolding protein that plays a vital role in bringing various proteins together into signaling complexes, thus providing more opportunities for complex interactions to take place.  The Drosophila genome has many fewer MAGUK proteins than most mammalian genomes.  This finding implies that through utilization of alternative start sites that generate multiple proteins, the fly can still end up with a wide array of subcellular interactions.  It is this underlying diversity of molecular interactions that is thought to allow the fly to produce to a variety of unusually complex behaviors, such as courtship, aggression, flight, and in this case motor control.

2010 Beckman Scholars

active site of thymidylate kinase colored by conservation of residues between humans and Cryptosporidium parvumBrandeis was recently awarded a grant from the Arnold and Mabel Beckman Foundation through their Beckman Scholars Program. This grant will support two students each through two summers and one academic year of undergraduate research.

Philip Braunstein and Jessica Hutcheson have been named the 2010 Beckman Scholars.  Braunstein (class of 2012) is a Biochemistry major identifying parasite-selective inhibitors of pyrimidine biosynthesis in the Hedstrom laboratory.  Hutcheson (2011) is a Biochemistry/Neruoscience major investigating the molecular processes that determine memory in the Griffith laboratory.

Congratulations to the winners!

BIOL 99 AND NEUR 99 Senior Honors Talks

Senior honors presentations and defenses for Biology and Neuroscience are this week and next Monday.

Name      Faculty Sponsor & Committee  Time & Location of Talk

Biol 99

Alicia Bach Dagmar Ringe, Neil Simister, Liz Hedstrom May 10   3 pm    Bassine 251
Kristin Little Bruce Goode, Joan Press, Satoshi Yoshida May 6    10 am    Bassine 251
Spencer Rittner KC Hayes, Carolyn Cohen, Larry Wangh May 6    3 pm      Bassine 251
Danielle Saly Michael Rosbash, Mike Marr, Nelson Lau May 10   11 am   Bassine 251
Sue Yen Tay Jim Haber, Sue Lovett, Joan Press  May 7    11am     Bassine 251
Alan Tso Daniela Nicastro, Liz Hedstrom, Greg Petsko May 10   2 pm    Bassine 251
Hannah Worchel Jim Morris, Ruibao Ren, Paul Garrity   May 6    2 pm     Bassine 251

Neur 99
Sarah Pease Sue Paradis, Gina Turrigiano, Paul Miller  May 10   11 am   Volen 201
Solon Schur John Lisman, Eve Marder, Paul Miller May 6    10 am   Volen 201
Alexander Trott Leslie Griffith, Piali Sengupa, Melissa Kosinski-Collins May 6    11 am   Volen 201
Dylan Wolman Sue Paradis, Sacha Nelson, Piali Sengupta May 10   1 pm    Volen 201

Faculty research mentor (emphasized) is chair of the committee.

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