Andretić Waldowski
Form A
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Croatian Science Foundation
HRZZ Research Projects
(IP-11-2013)
Research project proposal [Form A]1
(to be evaluated in Step 1)
Defining the role of circadian genes in the
behavioural sensitization to
psychostimulants in Drosophila
melanogaster
FLYHIGH
(see Guide for Applicants for the Research Projects IP-11-2013 Call – instructions
for completing 'Form A' of the project proposal)
Please respect the following formatting constraints: Verdana, font size at least 10,
margins (2.0 side and 1.5 bottom), single line spacing.
Cover Page:
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Rozi Andretić Waldowski
University of Rijeka, Department of Biotechnology
Defining the role of circadian genes in the behavioral sensitization to
psychostimulants in Drosophila melanogaster
36 months
Instructions for completing Form A can be found in the Guide for Applicants for the Research Projects IP-11-2013
Call.
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Andretić Waldowski
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Project proposal summary (half page, possibly copy/paste abstract from the Administrative
form)
Addiction to drugs is a brain disease characterized by changes in the brain functioning caused
by repeated drug taking. Repeated drug taking leads to neuroadaptations which over time
affect neural networks and change behavior. One such change induced by drugs and
commonly studied in lab animals is behavioral sensitization. Regulation of gene expression is
one important mechanism by which drugs change the plasticity of the networks which regulate
behavior. A group of genes which function as transcriptional regulators of the molecular
circadian clock, have been identified as regulators of behavioral sensitization to cocaine in
Drosophila. Subsequent studies in rodents proved the universality of those genes in mediating
drug responses. The genetic pathways and molecular interactions through which circadian
genes regulate drug responses has remained undefined.
We hypothesize that new genes which interact with circadian genes in the regulation of
behavioral sensitization to psychostimulants can be identified in Drosophila. This is a goal for
which Drosophila is perfectly suited, because genetic screens aimed at defining new genes can
be performed relatively easy, fast and cheap. We plan to achiewe this by first, devising a highthroughput metod for measuring behavioral sensitization in flies based on some of the exisiting
methodology. Second, undertaking a directed behavioral screen for mutants with reported
molecular interaction with circadian genes. Third, use of transgenic flies and other genetic
tools to investigate neural mechanisms involved in behavioral sensitization.
The proposed research is innovative and relevant for human health. New gene cadidates
isolated in this screen could easily be translated into mammalian reserch where they will help
further understanding of neuroplastic changes induced by psychostimulants. Given our
expertise and available resources the project has great potential to advance the field.
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Section a: Extended Synopsis of the project proposal (max. 5 pages)
[Concise presentation of the scientific proposal which will allow evaluation panels to assess, in
step 1 of the evaluation, the feasibility of the outlined scientific approach]
Rationale:
Here we propose to use Drosophila to identify and describe new genes which interact with
circadian genes to regulate behavioural sensitization to psychostimulants. As the pioneering
work on the role of circadian genes in regulating cocaine induced behaviours has shown
(ANDRETIC et al. 1999), new genes discovered in Drosophila can lead to important advances in
the understanding of addictive process in mammals. The mechanism through which circadian
genes regulate drug responses remains unclear, although the initial report sparked numerous
studies in rodents which showed universality of those genes in mediating drug responses
(partially reviewed in (FALCON and MCCLUNG 2009). Circadian genes are involved in the action
of not only psychostimulants but also ethanol and opiates, and aside from mediating
behavioural sensitization to cocaine they also mediate the rewarding properties of drugs
(ABARCA et al. 2002; SPANAGEL et al. 2005; WANG et al. 2006). Furthermore, in rodents
circadian genes show region-specific and drug-specific induction indicating that they likely
function as transcriptional regulators, similar to their function in the regulation of the circadian
clock (SPENCER et al. 2013; UZ et al. 2005; WANG et al. 2006). Circadian gene induction has
been linked to changed properties of several cellular pathways which regulate neuronal
functioning in response to addictive drugs (LIU et al. 2007; SPANAGEL et al. 2005). Better
understanding of the molecular interactions between circadian genes and their partners in
transcriptional regulation will help in the understanding of consequences that such induction
has on different systems which regulate brain physiology.
Our hypothesis is that the regulation of behavioural sensitization to psychostimulants can be
studied in Drosophila as it is an excellent model organism for discovering new genes and
investigating their function.
The main aim, identification of new genes which interact with circadian genes in regulating
behavioural sensitization, will be achieved through several sub aims. First is the development
of a high-throughput method for measurement of behavioural sensitization to
psychostimulants. Current methods for measuring behavioural sensitization to cocaine are
time-consuming, not amenable for large genetic screens and do not allow selection of affected
flies for further analysis. The newly designed assay will be used in the second aim, in a
directed behavioural screen for identifying genes which participate in the development of
behavioural sensitization to psychostimulants. This screen will be performed using a selection
of mutant flies in genes with reported interaction with circadian genes which regulate
behavioural sensitization: period (per), Clock (Clk) and cycle (cyc; Bmail1 homologue).
Mutants with an altered phenotype in behavioural sensitization will then be further
characterized in the third aim. The emphasis will be placed on defining neural networks in
which circadian genes and newly identified genes operate and their interaction with
dopaminergic system.
Drosophila is an ideal model organism for such a study because the cost and time involved to
perform similar study in rodents is prohibitive. On the other hand, significant advance in the
understanding of the genetic regulation of sensitization, and addictive process in general, is
not possible without identification of new molecules which regulate neural plasticity in response
to repeated drug taking. Once these genes are identified using relatively simple model
organism they can then be further characterized in mammals.
The successful completion of this project will advance future studies in Drosophila and
mammals for two reasons. First, a high-throughput method for measuring behavioural
sensitization would enable future screens in Drosophila because new gene discovery is not
possible without an objective and valid method for measuring behaviour. Second, new genes
identified in the screen proposed here, and in the future screens which will use this assay, will
aid investigation of molecular and cellular functions which underlie neuroplasticity induced by
repeated drug use. Such work will advance the field of addiction research in this model
organism and also serve to direct and advance addiction research in mammals. Validity of this
argument is exemplified by progress made in fields as diverse as development, immunity or
circadian rhythms where initial discoveries came from Drosophila.
Background :
Drug addiction is a complex disease with number of psychological and social causes and
consequences. The addicted state is characterized by compulsive drug taking in spite of
adverse consequences and by high rates of relapse during abstinence. The persistence of a
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dependent state characterized by craving for drug indicates that repeated drug taking leads to
long term changes in the brain functioning. At the core of this behavioural abnormality is a
biological process characterized by a neuroadaptations at the level of synapse and neural
networks in response to drug exposure (KALIVAS and VOLKOW 2005) (HYMAN et al. 2006) (RUSSO
et al. 2010). One of the big challenges in understanding addiction is to understand how changes
at the level of molecules and neurons lead to the changes in the functioning of the nervous
system which contributes to addiction. Particular focus in the field of addiction has now been
directed to the understanding of genetic mechanisms which participate in neuronal plasticity
caused by drug taking. One of the mechanisms responsible for plastic changes and responsible
for maintaining addicted state is the regulation of gene expression (NESTLER 2012).
Sensitization to the psychomotor properties of drugs is extensively studied in model organisms
because of its relevance for the understanding of craving and sensitized response to incentive
value of drugs. Behavioural sensitization refers to progressive increase in species-specific
behavioural responses to repeated drug administrations, commonly psychostimulants that can
in rodents persist even months after withdrawal.
In Drosophila, behavioural sensitization can occur with only two doses of cocaine when the
second dose is given more than 6 hours, but less than 48 hours after the first (MCCLUNG and
HIRSH 1998). Such temporal profile makes it likely that cocaine exposure stimulates gene
expression or changes the activity of the downstream signalling molecules. A group of
circadian genes which function as transcriptional regulators and which were identified as
regulators of behavioural sensitization in flies, fit perfectly with the hypothesis that behavioural
sensitization requires change in the gene expression as a pre-requisite for the long term
changes in neuronal functioning. Better understanding of the role that circadian genes play
becomes then very relevant for the understanding of addiction process in general.
In Drosophila three out of four core clock genes (per, Clk and cyc, but not tim) participate in
the regulation of behavioural sensitization to cocaine, a finding which was subsequently
supported with results from rodent studies (ANDRETIC et al. 1999; FALCON and MCCLUNG 2009).
Studies done in rodents emphasized the importance of circadian genes in addiction by showing
that: a) circadian gene mutants produce altered responses to drugs of abuse, b) the brain
expression of circadian genes is altered by addictive drugs and c) the circadian gene induction
by drugs is mediated through dopamine receptors, while circadian genes in turn can regulate
dopamine receptors and dopaminergic system function in general. Furthermore, those studies
linked circadian gene induction by drugs with changed properties of several cellular pathways
which regulate neuronal functioning in response to addictive drugs.
This and other findings support the notion that a subset of core circadian genes regulates drug
induced behaviour, and that the role that they play in this process is separate from their
regulation of circadian clock. Furthermore, it is likely that in the regulation of drug induced
behaviours circadian proteins form homo- and heterodimers among themselves and also
engage other partners. This emphasizes the need to identify novel genes whose products
might interact with per or another circadian gene. Better understanding of the molecular
interactions between circadian genes and their partners in transcriptional regulation will help in
the understanding of consequences that such induction has on different systems which
regulate brain physiology.
Methodology :
1. Development of a high-throughput method for measurement of behavioural
sensitization
We propose to measure behavioural sensitization using an automated method which allows for
simultaneous monitoring of a large number of flies with individual resolution. In such a way
we will quantify changes in the locomotor activity (motor-activating effects) and we can also
quantify the changes in the amount of sleep (arousing effects). Because of the objective and
quantifiable nature of such approach it is ideal for screening of large numbers of flies,
particularly in genetic screens. Flies which fit certain criteria could then be selected and used
for potential further analysis.
In our approach we will start with Drosophila Activity Monitoring System (DAMS) which was
developed in the past to measure changes in circadian activity, and was later successfully
adapted to quantify amount of sleep (ANDRETIC and SHAW 2005). From the raw data for a single
fly, it is possible to extrapolate a series of parameters about activity and sleep, such as:
amount of sleep/activity per unit time, frequency and duration of sleep/activity episodes, etc.
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To induce behavioural sensitization to cocaine we will transfer flies from regular food to food
with cocaine and back, and vary: length of exposure to cocaine, cocaine concentration, length
of time without cocaine and the number of repetitions. Behavioural sensitization to volatilized
cocaine is induced when the period between two exposures is at least 6 hours apart, thus the
shortest time off the drug will be at least 6 hours. Behavioural sensitization in DAMS will be
defined as a significant increase in the amount of activity, or a decrease in the amount of
sleep, per unit time. The prerequisite is that a fly shows initial response, i.e., sensitivity
(increase in activity or decrease in sleep), which on the second exposure should be followed by
stronger response to the same drug dose.
This aim is high risk- high payoff. If we are not successful in adapting DAMS for monitoring of
behavioural sensitization this will somewhat dampen the potential impact of this work,
however the realization of this project could still be possible. If ingestion of drugs proves to be
the obstacle in inducing sensitization, we will have to resort to volatilizing cocaine and then
optimize the method which was used in the behavioural screen for the acute sensitivity to
cocaine (BAINTON et al. 2005; HEBERLEIN et al. 2009). As that method was used successfully in
screening large number of flies to a single exposure, we predict that we will be able to adapt it
for monitoring behaviour of flies after multiple exposures.
This aim will be performed under the guidance by R.A.W. with major contribution of Doctoral
student A.F. and H.D. and contribution and of Master’s degree students.
2. Mini screen for per, Clk and cyc interacting proteins
In an attempt to identify a novel proteins with a role in behavioural sensitization we decided to
avoid an open-ended genetics screen, as it requires testing of hundreds single mutants
(thousands of flies) for which we are not currently equipped. To limit our screen to the genes
of interest for us, we will first use bioinformatics in order to pre-select potential candidates,
which we know that interact directly with circadian genes. We performed a preliminary search
using DroID -The Drosophila Interactions Database, a searchable database of protein
interactions based on two-hybrid screen from Drosophila and interactions predicted from other
organisms. Search for proteins that interact with per, Clk and cyc, results in 34 unique proteins
for which Drosophila mutants are available and which we plan to test in our screen. Mutants
will be ordered either from Bloomington Drosophila Stock Center at Indiana University or from
different labs which created mutants or work with them.
In preparation for testing we will first place all mutants in the same wild type, Canton S,
genetic background. This will be followed by screening of single mutants in DAMS in response
to intermittent doses of cocaine, based on a protocol developed in the previous aim. Of interest
for further testing will be mutants which either show: a) no sensitized response, b) have
different initial sensitivity (either more or less sensitive), and c) have stronger sensitization
(percent difference between initial and subsequent exposures).
We plan to do testing in two steps. In the first we will use at least 16 flies of each sex per
genotype, as males and females differ in their sensitivity to drugs. We will monitor their
baseline activity for at least one whole day in 12 hrs light: 12 hrs dark, and stable temperature
and humidity. On the second day they will be given a single administration of drug (time of
day, concentration and length will be determined in previous aim). Based on this we will have
initial indicator of sensitivity to drugs, and sexual dimorphism in the response. In the second
phase (separate experiment), same mutants of a single sex will be monitored again. Based on
the results of phase one, we will decide which sex to use and we will also be able to optimize
drug concentration, in situations where flies showed changed initial responsiveness. In this
phase we will use at least 32 flies per genotype and record one baseline day, followed by at
least two administrations of drug. We currently possess 10 monitors, which means that in
either phase we can test 10 genotypes in one session.
We do not foresee any major problems in the realization of this aim. Ordering and
Cantonization of flies are straightforward procedures. Behavioural screen will be performed
according to parameters set in the previous aim. This aim will be performed under the
guidance by R.A.W. with major contribution of Doctoral student A.F. and H.D., and contribution
by Master degree students.
3. Characterization of mutant(s)
There are two parts to this Aim that will mostly run in parallel at two different locations,
University of Rijeka, Croatia (3.a) and University of California, San Diego (3.b). We will
characterize the role of the newly identified gene from Aim2 and compare its phenotype and
neuronal characteristics with previously identified circadian gene mutants and wild type flies.
In the second part of this aim we will use genetically encoded sensors to characterize
dopaminergic activity in wild type and mutant flies.
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3.a) Genetic experiments to identify the neuronal circuit in which new gene controls
behavioural sensitization
Experiments in this aim will depend on the nature of mutant that gets isolated in the previous
aim. We will use available transgenic constructs because there is a wide range of them
available. There are several main points that we will address: a) verification of the genes role
through rescue experiments and b) defining the minimal neuronal circuit where the gene has
to be expressed for behavioural sensitization.
The cellular specificity of gene expression can be achieved using bipartite expression system
UAS/GAL4, where transcription factor GAL4 is expressed in spatially restricted pattern to drive
the expression of gene of interest placed downstream of GAL4 biding sequence UAS. By
crossing flies carrying GAL4 driver with those with UAS construct and gene of interest, the
precise expression is achieved. Currently there exists a selection of 7000 GAL4 lines which
allow for manipulation of specific brain areas or neuronal types (JENETT et al.). With this
approach we will ask in which neuronal circuit is the gene of interest expressed to regulate
behavioural sensitization. First, to show the rescue we will drive the wild type version of the
gene in the neurons which normally express it, of otherwise mutant fly. Second, we will start
expressing the functional copy of the gene in a smaller area in order to identify minimal
neuronal circuit sufficient for behavioural sensitization. Subsequently, we will change the
activity of those neurons by changing their electrical properties, making them either inactive or
hyperactive. There is a wide array of UAS constructs with modified activity of ion channels
(HODGE 2009). The results from these experiments will complement findings in the following
section.
3. b) Characterization of DA neuronal activity in normal and mutant flies
We will characterize neuronal activity in the dopaminergic network over circadian time in
normal flies before and after sensitization, and in any new mutants that emerge from the
previous Aim. We will use optical brain recording with the newly improved genetically encoded
sensors of intracellular Ca (GCaMP6) (AKERBOOM et al. 2012) and membrane potential (Arclight)
(CAO et al. 2013), under light field microscopy to determine the neurons that respond to
sensitization and to chart how their circuit level activity changes as behaviour changes. By
driving the Arclight sensor specifically in the dopaminergic neurons, using TH-Gal4, we can
focus selectively on that network of cells. Recordings will be made at mid-day, mid-night, and
at lights-on and lights-off circadian times.
We will perform in vivo imaging in an immobilized fly held in a 250 μl pipette tip as the head
capsule is dissected off and a transparent silicon window is placed over the opening in the
head capsule.
After GCaMP or Arclight monitoring, cells with elevated activity are marked by RFP
photoactivation that is performed by scanning from the top to the bottom slices of the defined
volume using a two-photon laser. RFP is initially dark but becomes red fluorescent after 543
nm light irradiation. Living brains freshly dissected from flies carrying GCaMP and a
photoactivable dark-to-red RFP protein (SHCHERBAKOVA et al. 2012) will be fixed, degassed and
mounted for confocal imaging.
The role of these regions for both behavior and physiology will also be tested by manipulating
their activity using genetically encoded channel-rhodopsin (ChR2) or halorhodopsin (HaR)
proteins (SMEDEMARK-MARGULIES and TRAPANI 2013)that alter neuronal activity to make them
either over- or under-active.
R.J.G. is an expert in brain imaging and these experiments will be performed at R.J.G.’s lab at
UCSD with expertise of dr sc. Sophie Aimon and assistance of R.A.W. to learn the technique
and perform the accompanying behavioural experiments.
Team members:
1. R.A.W. is a PI and will devote 70% of her time to the realization of Aim 1-3. Her tasks will
include: directing experimental work on the project, overseeing all aspects of the project,
mentoring doctoral and master’s degree students, writing publications and reports to
HRZZ.
2. A.F. is a doctoral student who will join the project from its start and devote 60% percent of
her time to the project.
3. H.D. will become will join the project in the summer 2014 when she becomes a doctoral
student. She is currently working toward a M.Sc. in the PI's lab at the Department of
Biotechnology, University of Rijeka. She after graduating and will participate with 60% of
her time.
4. R.J.G. is a researcher at UCSD who will participate with 30 % of his time through his lab
members. He will manage experiments on neural imaging of neurons involved in
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behavioural sensitization to cocaine. He has both the expertise and equipment for
realization of Aim 3.
5. Master degree students will be working through this project. Students are required to
spend a semester in a laboratory working on their thesis, as a prerequisite for graduating,
we predict that each year R.A.W. will be hosting at least 2-3 students.
References :
ABARCA, C., U. ALBRECHT and R. SPANAGEL, 2002 Cocaine sensitization and reward are under the
influence of circadian genes and rhythm. Proc Natl Acad Sci U S A 99: 9026-9030.
AKERBOOM, J., T. W. CHEN, T. J. WARDILL, L. TIAN, J. S. MARVIN et al., 2012 Optimization of a
GCaMP calcium indicator for neural activity imaging. J Neurosci 32: 13819-13840.
ANDRETIC, R., S. CHANEY and J. HIRSH, 1999 Circadian genes are required for cocaine
sensitization in Drosophila. Science 285: 1066-1068.
ANDRETIC, R., and P. J. SHAW, 2005 Essentials of sleep recordings in Drosophila: moving beyond
sleep time. Methods Enzymol 393: 759-772.
BAINTON, R. J., L. T. TSAI, T. SCHWABE, M. DESALVO, U. GAUL et al., 2005 moody encodes two
GPCRs that regulate cocaine behaviors and blood-brain barrier permeability in
Drosophila. Cell 123: 145-156.
CAO, G., J. PLATISA, V. A. PIERIBONE, D. RACCUGLIA, M. KUNST et al., 2013 Genetically targeted
optical electrophysiology in intact neural circuits. Cell 154: 904-913.
FALCON, E., and C. A. MCCLUNG, 2009 A role for the circadian genes in drug addiction.
Neuropharmacology 56 Suppl 1: 91-96.
HEBERLEIN, U., L. T. TSAI, D. KAPFHAMER and A. W. LASEK, 2009 Drosophila, a genetic model
system to study cocaine-related behaviors: a review with focus on LIM-only proteins.
Neuropharmacology 56 Suppl 1: 97-106.
HODGE, J. J., 2009 Ion channels to inactivate neurons in Drosophila. Front Mol Neurosci 2: 13.
HYMAN, S. E., R. C. MALENKA and E. J. NESTLER, 2006 Neural Mechanisms of Addiction: The Role
of Reward-Related Learning and Memory. Annu Rev Neurosci.
JENETT, A., G. M. RUBIN, T. T. NGO, D. SHEPHERD, C. MURPHY et al., 2012 A GAL4-driver line
resource for Drosophila neurobiology. Cell Rep 2: 991-1001.
KALIVAS, P. W., and N. D. VOLKOW, 2005 The neural basis of addiction: a pathology of motivation
and choice. Am J Psychiatry 162: 1403-1413.
LIU, Y., Y. WANG, Z. JIANG, C. WAN, W. ZHOU et al., 2007 The extracellular signal-regulated
kinase signaling pathway is involved in the modulation of morphine-induced reward by
mPer1. Neuroscience 146: 265-271.
MCCLUNG, C., and J. HIRSH, 1998 Stereotypic behavioral responses to free-base cocaine and the
development of behavioral sensitization in Drosophila melanogaster. Curr. Biol. 8: 109112.
NESTLER, E. J., 2012 Transcriptional mechanisms of drug addiction. Clin Psychopharmacol
Neurosci 10: 136-143.
ROBISON, A. J., and E. J. NESTLER, 2011 Transcriptional and epigenetic mechanisms of addiction.
Nat Rev Neurosci 12: 623-637.
RUSSO, S. J., D. M. DIETZ, D. DUMITRIU, J. H. MORRISON, R. C. MALENKA et al., 2010 The addicted
synapse: mechanisms of synaptic and structural plasticity in nucleus accumbens.
Trends Neurosci 33: 267-276.
SHCHERBAKOVA, D. M., O. M. SUBACH and V. V. VERKHUSHA, 2012 Red fluorescent proteins:
advanced imaging applications and future design. Angew Chem Int Ed Engl 51: 1072410738.
SMEDEMARK-MARGULIES, N., and J. G. TRAPANI, 2013 Tools, methods, and applications for
optophysiology in neuroscience. Front Mol Neurosci 6: 18.
SPANAGEL, R., G. PENDYALA, C. ABARCA, T. ZGHOUL, C. SANCHIS-SEGURA et al., 2005 The clock gene
Per2 influences the glutamatergic system and modulates alcohol consumption. Nat Med
11: 35-42.
SPENCER, S., E. FALCON, J. KUMAR, V. KRISHNAN, S. MUKHERJEE et al., 2013 Circadian genes Period
1 and Period 2 in the nucleus accumbens regulate anxiety-related behavior. Eur J
Neurosci 37: 242-250.
UZ, T., R. AHMED, M. AKHISAROGLU, M. KURTUNCU, M. IMBESI et al., 2005 Effect of fluoxetine and
cocaine on the expression of clock genes in the mouse hippocampus and striatum.
Neuroscience 134: 1309-1316.
WANG, X., Y. WANG, H. XIN, Y. LIU, H. ZHENG et al., 2006 Altered expression of circadian clock
gene, mPer1, in mouse brain and kidney under morphine dependence and withdrawal. J
Circadian Rhythms 4: 9.
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Section b: PI’s Curriculum vitae (max 2 pages)
Rozi Andretić Waldowski
Radmile Matejčić 2, Department of Biotechnology,
University of Rijeka, 51 000 Rijeka, Croatia
Work: randretic@uniri.hr
Date of birth 04 February 1963
Nationality Croatian, American
Education:
1995 – 2000 PhD in Biology
Department of Biology, University of Virginia, Charlottesville, VA, USA
1981 – 1985 B.A. in Psychology
Department of Psychology, University of Rijeka, Rijeka, Croatia
Training:
1996 Drosophila Neurobiology: Genes, Circuits, Behavior, Cold Spring Harbor Laboratory
course, Cold Spring Harbor, NY, USA
Professional experience:
October 2010 – Present Assistant Professor and Head of Laboratory for Behavioral Genetics
Department of Biotechnology, University of Rijeka, Rijeka, Croatia
2008 – 2010 Assistant Professor and independent researcher
Department of Psychology, University of Rijeka, Rijeka, Croatia
2006 – 2008 Research Fellow in experimental neurobiology
The Neurosciences Institute, San Diego, CA, USA
2000 – 2006 Postdoctoral Fellow in experimental neurobiology
The Neurosciences Institute, San Diego, CA, USA
1993 – 1995 Research Assistant in the lab of Dr Craig Heller, Department of Biology
Stanford University, Palo Alto,CA, USA
1987 – 1991 Resident Psychologist in Elementary School and Pre-schools, Croatia
Elementary school and preschools in Lovran (1987-89) and preschool in Zagreb (Jarun (198990) and Trešnjevka (1990-91)
Publications:
1. “Dopamine in Drosophila: setting arousal in a miniature brain”, van Swinderen B.
Andretic R., Proc. Biol Sci. 2011 Mar 22;278(1707):906-13.
2. ˝Caffeine modulates dDA1 dopamine receptor to promote arousal in Drosophila˝,
Andretic R., KimY-C, Jones F.S., Han K-A and Greenspan R.J., PNAS, 2008,
105(51);20392-7.
3. ˝Genetics of Sleep“, R.Andretic, P.Franken and M. Tafti, Annual Reviews in Genetics,
2008, 42;261-388.
4. ˝Neurohormonal and neuromodulatory regulation of sleep in Drosophila˝, Foltenyi K.,
Andretic R., Newport J.W. and Greenspan R.J., Cold Spring Harb Symp Qaunt Biol,
2007, 72, 565-71.
5. “Dopaminergic Regulation of Arousal in Drosophila”, R. Andretić, B. van Swinderen, R.
J.Greenspan, Current Biology, 2005, 15(13):1165-75.
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6. “Essentials of Sleep Recording in Drosophila: Moving Beyond Sleep Time”, R. Andretić
and P. J.Shaw, Methods in Enzymology, 2005, 393; 759-772.
7. “Arousal in Drosophila”, R. Andretić and B. van Swinderen, Behavioural Processes,
2003, 64(2):133 144.
8. “Circadian Modulation of Dopamine Receptor Responsiveness
melanogaster”, R.Andretić and J. Hirsh, PNAS, 2000, 97(4); 1873-8.
in
Drosophila
9. “A Role for Circadian Genes in Cocaine Sensitization in Drosophila melanogaster”, R.
Andretić, S.Chaney, J. Hirsh, Science, 1999, 285;1066-68.
10.“Developmental Changes in Nicotinic Receptor mRNAs and Responses to Nicotine in the
Suprachiasmatic nucleus and Other Brain Regions”, B.F. O'Hara., E. Macdonald, D.
Clegg, S.W. Wiler, R. Andretić, V.H. Cao, J.D. Miller, H.C. Heller, T.S. Kilduff ,
Molecular Brain Research, 1999, 66;71-82.
11.“Daily Variation of CNS Gene Expression in Nocturnal vs. Diurnal Rodents and in the
Developing Rat Brain”, B.F. O’Hara, F.L. Watson, R. Andretić, S.W. Wiler, K.A. Young, L.
Biting, H.C. Heller, T.S.Kilduff, Molecular Brain Research, 1997, 48(1); 73-86.
12.“GABAA, GABAC and NMDA Receptor Subunits Expression in the Suprachiasmatic
Nucleus and Other Brain Regions”, B.F. O’Hara, R. Andretić, H.C. Heller, D.B. Carter,
T.S. Kilduff; Molecular Brain Research, 1995, 28; 239-250
Principal investigator on scientific projects:
1998 , "Novel role for circadian genes in cocaine responsiveness in Drosophila"
National Institute of Drug Abuse / National Institute of Health pre-doctoral fellowship, 2 years
2008 ,: "Action of psychostimulants on the CNS of Drosophila relevant for addiction and
relapse" Croatian Science Foundation grant for Croatian scientists returning to Croatia, 1 year
Participation in Scientific Projects
1993-1995, Stanford University, Research Assistant:
Circadian, sleep and hibernation related changes in the gene expression in mammalian CNS.
1996-2000, University of Virginia, PhD research:
Circadian regulation of biogenic amines in Drosophila.
2000-2008, The Neurosciences Institute, postdoctoral and research fellow:
Genetic regulation of sleep and arousal in Drosophila.
Genetic and neural mechanisms of sexual dimorphism in sleep regulation in Drosophila.
Honors:
1995 University of Virginia pre doctoral fellowship, 1 year.
1996 Cold Spring Harbor Course, NIH Scholarship, Drosophila Neurobiology
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Section c: PI’s 5-year track-record (max 2 pages)
My track record has a gap for personal reasons, as well as professional.
From August 2008, when I returned to Croatia after studying and working in USA, until August
2009 I was temporarily at the Department of Psychology, University of Rijeka, because
Department of Biotechnology was not yet formed.
Furthermore, during that time and subsequent to being approved for a funding by Croatian
Science Foundation in 2008, for a project for Croatian scientists returning to Croatia, there was
no other funding opportunities in Croatia for which I was eligible.
From August 2009 to October 2012 I was on maternity leave and I took two years of unpaid
leave to stay at home with my new-born son.
During that time I continued reviewing scientific manuscripts for different journals.
This resulted in a significant gap where I could not be perform experiments in the laboratory,
which is evident on my track record.
For those reasons I included scientific achievements which are older than 5 years.
Reviewing of scientific manuscripts for following journals:
PlosOne
Journal of Sleep Research
Sleep
Alcoholism, clinical and Experimental Research
Major Publications:
1. “A Role for Circadian Genes in Cocaine Sensitization in Drosophila melanogaster”, R.
Andretić, S.Chaney, J. Hirsh, Science, 1999, 285;1066-68.
Citations: 222
2. “Circadian Modulation of Dopamine Receptor Responsiveness in Drosophila melanogaster”,
R. Andretić and J. Hirsh, PNAS, 2000, 97(4); 1873-8.
Citations: 82
3. “Dopaminergic Regulation of Arousal in Drosophila”, R. Andretić, B. van Swinderen, R. J.
Greenspan, Current Biology, 2005, 15(13):1165-75.
Citations: 150
4. ˝Genetics of Sleep“, R. Andretic, P. Franken and M. Tafti, Annual Reviews in Genetics, 2008,
42;261-388.
Citations: 51
5. ˝Caffeine modulates dDA1 dopamine receptor to promote arousal in Drosophila˝, Andretic
R., KimY-C, Jones F.S., Han K-A and Greenspan R.J., PNAS, 2008, 105(51);20392-7.
Citations: 27
Chairing and organising the Symposia:
• 2008 ˝Contributions of adenosine and dopamine in mediating arousal: Inseparable
partners?”, European Sleep Research Society Meeting, Glasgow, Scotland
Symposia Participant:
• 2006 „Sleep Deprivation in Animals and Humans – Methodological Issues“, World
Federation of Sleep Research Societies, Cairns, Australia
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Andretić Waldowski
Form A
FLYHIGH
Presentations at the meetings and conferences (1998-2008):
• 2008 Presentation: ˝Interactions between caffeine and dopamine in arousal and
sleep in Drosophila˝ R. Andretic, J.-C. Kim, K.-A. Han, F. Jones and R.J. Greenspan,
European Sleep Research Society Meeting, Glasgow, Scotland
• 2007 "D1-like dopamine receptors mediate the wake-promoting effects of
psychostimulants in Drosophila”, R. Andretic, J.-C. Kim, K.-A. Han, F. Jones and R.J.
Greenspan, World Federation of Sleep Research Societies, Cairns, Australia, poster
presentation.
• 2006 “Dopaminergic Role in the Arousing Effects of Methamphetamine and Caffeine
in Drosophila”,R. Andretić and R. J. Greenspan, European Drosophila Neurobiology
Conference, Leuven, Belgium,oral presentation.
• “The Role of Acetylcholine in Sleep Regulation in Drosophila”, R. Andretić and R. J.
Greenspan, European Sleep Research Society Meeting, Innsbruck, Austria, oral
presentation.
• “Dopaminergic Role in the Arousing Effects of Methamphetamine and Caffeine in
Drosophila”, R. Andretić and R. J. Greenspan, European Sleep Research Society
Meeting, Innsbruck, Austria, poster presentation.
• 2005 “How Arousing is Dopamine in Drosophila”, R. Andretić, B. van Swinderen, R.J.
Greenspan, Neurobiology of Drosophila, Cold Spring Harbor, NY, oral presentation.
• 2004 “Brain Mechanisms Regulating Sexually Dimorphic Sleep in Drosophila”, R.
Andretić, R. J. Greenspan, P. J. Shaw, European Sleep Research Society Meeting,
Prague, Czech Republic, oral presentation.
• 2004 “Role of Dopamine in Methamphetamine-Induced Arousal in Drosophila”, R.
Andretić, B. van Swinderen, R.J. Greenspan, European Sleep Research Society
Meeting, Prague, Czech Republic, poster presentation.
• 2004 “ Brain mechanisms regulating sexually dimorphic sleep in Drosophila”,
R.Andretić, R. J. Greenspan, P.J.Shaw, Gordon Research Conference, Genes and
Behavior, Ventura, CA, poster presentation.
• 2003 “Flies on METH: Behavioral and Physiological Studies of Arousal”, R. Andretić,
B. van Swinderen, R.J. Greenspan, Cold Spring Harbor Meeting on Neurobiology of
Drosophila, Cold Spring Harbor, NY, poster presentation.
• 2003 “Sexual Dimorphism and Critical Periods Influence Sleep in D.melanogaster”,
R. Andretić and P. J. Shaw, Associated Professional Sleep Societies Meeting, Chicago,
IL, oral presentation.
• 2000 “Molecular Mechanisms Linking Circadian Genes and Cocaine Responsiveness
in D. melanogaster”, R. Andretić and J. Hirsh, Society for Research on Biological
Rhythms, Amelia Island, FL, oral presentation.
• 1999 “Circadian Genes do More than Keep Time", R. Andretić and J. Hirsh, Cold
Spring Harbor Meeting on Neurobiology of Drosophila, Cold Spring Harbor, NY, oral
presentation.
• 1999 "Circadian Genes are Required for Sensitization to Cocaine", R. Andretić, S.
Chaney, J. Hirsh, Gordon Research Conference on Chronobiology, Il Ciocco, Italy,
poster presentation.
• 1999 “A Novel Role for Circadian Genes in Cocaine Responsiveness in
D.melanogaster”, R. Andretić, S. Chaney, J. Hirsh, 40th Annual Drosophila Research
Conference, Seattle, WA, poster presentation.
• 1999 “Bugs on Drugs: Molecular Genetics of Cocaine Responsiveness”, R. Andretić,
C. McClung, H. Lee, S. Park, S. Chaney, J. Hirsh, 40th Annual Drosophila Research
Conference, Seattle, WA, workshop presentation.
• 1998 “Dopamine Receptors - Components of the Circadian Output Pathway in
Drosophila”, R. Andretić and J. Hirsh, Society for Research on Biological Rhythms,
Amelia Island, FL, poster presentation.
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