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Towards a Behavioral Genetics of Zebrafish
August 28-29, 1999
Marine Biological Laboratories, Woods Hole, MA
The zebrafish is a well-characterized genetic organism that is
currently being exploited primarily to analyze vertebrate development.
The utility of this organism for understanding the genetics of behavior
and disorders with behavioral components, such as addiction, sensory
deficits, or neurological and psychiatric disorders, is not known
because of the lack of behavioral screens.
The purpose of this meeting was to bring together investigators
who are zebrafish geneticists with investigators who have developed
methodologies for studying behavior in a wide variety of fish species.
It is anticipated that the outcome will be new genetic screens for
mutations in zebrafish that affect behavior. This two-day meeting
was held in conjunction with the MBL course "Neural Development
and Genetics of Zebrafish" and was attended by about 90 people.
The meeting was organized by Susan Volman and Jonathan Pollock,
NIH/National Institute on Drug Abuse and sponsored by many of the
NIH Institutes involved in the Trans-NIH Zebrafish Initiative with
additional assistance from Aquatic Habitats for the poster session.
List of Speakers and Poster
Presentations
Speaker Abstracts
Poster Abstracts
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LIST OF SPEAKERS AND POSTER PRESENTATIONS
Poster Presentations |
|
James C. Beck, A.O. Dennis Willows, and Mark
S. Cooper* |
Univ. of Washington |
Computer-Assisted
Visualizations of Neural Networks: Expanding the Field of View
using Seamless Confocal Montaging |
Kohei Hatta* and Henri Korn |
Institut Pasteur, INSERM, Paris |
Crossed Modulation of
Inhibitory Synaptic Inputs in Left-right Decision Neurons |
PoKay M. Ma |
Queens College |
The Anatomical Organization of
the Locus Coeruleus in the Zebrafish |
Adam Miklos* and Richard J. Andrew |
University of Sussex |
Behavioral screening
techniques for larval and adult zebrafish with special reference
to behavioral lateralization |
Seth A. Budick and Donald M. OMalley*
|
Northeastern University |
Locomotive Repertoire
of the Larval Zebrafish: Swimming, Turning and Prey Capture
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R.E. Rodriguez*, A. Barrallo and R. Gonzalez
Sarmiento |
Univ. of Salamanca |
Cloning
and Characterization of a Gene Homologous to the Delta Opioid
Receptor in Zebrafish |
R. Schmid*, G. Pradel, O. Heller, and M. Schachner |
Justus-Liebig-Univ. University of Hamburg
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Involvement
of Adhesion Molecules in Plasticity of Zebrafish Brain After
Avoidance Conditioning |
Henning Schneider* and Kim H. Eliasz |
William Paterson Univ. |
Fighting Behavior
in Zebrafish, Danio rerio |
Henning Schneider*, Beth M. Sulner, Elisabeth
Abbiati |
William Paterson Univ |
Innervation
of Fin Muscles in Zebrafish, Danio rerio |
Nichole Korpi and Brian Wisenden* |
Moorhead State Univ. |
Learned Recognition
by Zebrafish (Brachydanio rerio) of Novel Predatory Odor Following
Non-simultaneous Presentation of Alarm Pheromone in Skin Extract
and Predator Odor |
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SPEAKER ABSTRACTS
Establishing the Genetic Basis of Vertebrate Oculomotor Behaviors
in Zebrafish
Robert Baker
NYU Medical Center
The central nervous system of all vertebrate embryos is derived
from a series of conspicuous embryonic segments, called neuromeres,
that are particularly visible in the mid- and hindbrain areas that
give rise to the brainstem sensory and motor nuclei. This presentation
focuses on a series of eight hindbrain rhombomeric segments that
represent domains of unique gene expression and lineage restriction
responsible for the development of equally unique neuronal subgroups
producing eye motion. In all vertebrates, columns of vestibular
and reticular subnuclei delineated by Hox gene clusters extend throughout
this segmented hindbrain. The function of each oculomotor related
subgroup is hypothesized to be determined by specific genetic and
molecular signaling mechanisms. Establishing
the role of single gene loci in the development of physiologically-characterized
oculomotor related phenotypes will be addressed based on a genetic
analysis of the anatomical framework underlying three dimensional
oculomotor behavior in zebrafish. For example, the detailed neuronal
map of the circuitry underlying both vestibuloocular and optokinetic
reflexes including eye fixation will be shown to consist of subnuclei
so precisely segregated within distinct rhombomeric segments that
one must conclude that, each "functional" neuronal group
is likely produced by spatially restricted expression of developmental
regulatory genes. The zebrafish brainstem is, therefore, well suited
for a genetic analysis of neuronal circuits responsible for oculomotor
behavior. Individual physiological phenotypes identified within
unique rhombomeric morphogenetic units will be used to show how
a structural and behavioral analysis of single gene mutations in
the zebrafish might distinguish single genes correlated to single
behaviors. A prospective strategy for identifying single genes that
act to determine unique neuronal signal processing elements contributing
to a particular behavior will be illustrated by the oculomotor neural
integrator which is a model system to investigate the genetic basis
of persistent neural activity in vertebrates. In conclusion, the
goal is to demonstrate that genes identified by disruption of oculomotor
behaviors in zebrafish can provide an efficient way of assigning
roles to orthologous human genes as they become recognized by sequence
from the human genome project.
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Neuroethology of social behavior and reproductive plasticity
among teleost fish.
Andrew H. Bass
Dept. of Neurobiology and Behavior
Cornell University
Ithaca, NY
Vertebrate social behavior may be characterized by several
types of sexual plasticity. Perhaps the most frequently described
among these behaviors are alternative mating tactics where one sex,
usually males, displays two or more classes of social reproductive
behaviors. Among vertebrates, teleost fishes are perhaps the "champions"
of alternative tactics, because of the extreme range in reproductive
plasticity they exhibit . In some cases, alternative male phenotypes
or morphs originate from two distinct life history trajectories.
For other species that exhibit adult sex reversal, alternative male
tactics represent sequential life history stages for an individual
that is initially either a reproductively active male (role change)
or female (sex change); yet other sex changing species exhibit a
single male reproductive tactic. Lastly, some teleosts may exhibit
some form of reproductive suppression where a non-reproductive individual
can transform into a reproductive one given the appropriate social
conditions. All of these examples represent points on a continuum
of sexual plasticity with yet other species exhibiting intermediate
conditions.
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A Behavioral Screen for Zebrafish Circadian Clock Mutants.
Gregory M. Cahill
Dept. of Biology and Biochemistry
University of Houston
Houston, TX
A wide variety of biological processes are regulated by circadian
clocks. These clocks are systems of cellular oscillators that can
self-generate circa-24 hour rhythmicity in the absence of external
timing cues. Under natural conditions, the timing of a circadian
clock is set by the environmental light:dark cycle. This results
in appropriately-timed, daily rhythms in gene expression, metabolism,
neural and hormonal activity, and many aspects of behavior.
Recently, there has been rapid progress in identification of the
molecules that make up circadian clocks, and in our understanding
of how these molecules interact to produce circadian rhythmicity.
Most of this progress has come, directly or indirectly, from studies
of genes that were originally identified by mutations. Clock mutant
screens approaching saturation have been performed in cyanobacteria,
Arabidopsis, Neurospora, and Drosophila. Screens for clock mutants
that affect behavior in vertebrates have been more limited, but
have already contributed much to our understanding of clock mechanisms.
In each of these systems, the core of the clock mechanism is made
up of clock-specific proteins that interact in negative feedback
loops to produce rhythms in gene expression. The specific molecules
that make up these loops differ across kingdoms. Vertebrate circadian
oscillator mechanisms clearly include molecular elements that are
evolutionarily conserved with Drosophila. However, the vertebrate
system is more complex and differs in several ways from the Drosophila
system. Clock mutant screens in zebrafish are likely to identify
new vertebrate circadian clock genes, as well as provide insights
into the functions of known clock genes.
Before zebrafish genetics could be exploited in studies of circadian
clock mechanisms, it was necessary to develop efficient measures
of circadian rhythmicity that can be used to screen for mutants,
as well as information on the organization of the circadian system
that can be used to interpret mutant phenotypes. Studies from this
laboratory and others on molecular, cellular, and behavioral circadian
rhythmicity in zebrafish have begun to fulfill these requirements.
The most efficient measure of circadian clock function that we
have found in zebrafish is the circadian rhythm in spontaneous swimming
activity of 9-18 day old larvae. We measure these rhythms with an
automated infrared video image analysis system that tracks the movements
of fish housed in individual 0.7 ml wells. With this system, a single
video camera can monitor the activity of up to 150 individuals simultaneously
for up to a week in constant conditions. Over 95% of larval zebrafish
express robust circadian behavioral rhythms under these conditions,
with highest activity during the subjective day. For our purposes,
the time of the activity peak at the end of a week in constant conditions
is the most precise measure of the circadian timing of individual
fish.
We have also examined locomotor activity rhythms of adult zebrafish,
using a recording system based on infrared beam detectors. We observe
more variability in the activity patterns of adults than those of
larval zebrafish. Patterns range from a single, robust circadian
rhythm, to splitting of the activity rhythm into two rhythms with
different periods, to complete arrhythmicity. Under the best conditions
that we have devised, this system can detect significant circadian
rhythmicity in ~70% of adult zebrafish. With this kind of variability,
the adult behavioral rhythm would not be useful for screening purposes,
but we have found it to be useful for some physiological experiments.
Zebrafish pineals, cultured in a flow-through superfusion system
in constant darkness, produce exceptionally robust circadian rhythms
of melatonin release for at least seven days. This organ, like the
pineals of many other non-mammalian vertebrates, is directly photosensitive;
exposure to light in vitro suppresses pineal melatonin production
and resets the phase of the circadian oscillator. Therefore, the
cultured pineal performs all of the basic functions of a circadian
clock, and it provides an easy and reproducible physiological assay
for cell- and tissue-level rhythmicity. The zebrafish retina also
contains a circadian oscillator that regulates melatonin synthesis.
Although pineal and retinal circadian clocks contribute to the regulation
of behavioral rhythms in other species, we have been unable to detect
any effect of pineal or ocular ablation on the swimming rhythms
of zebrafish. Therefore, the behavioral rhythm and the melatonin
rhythms of cultured pineal and retina are independent measures of
circadian clock function in this species.
We have begun a screen for ENU-induced mutations that alter the
timing of the larval swimming rhythm. Because recording of these
activity rhythms is largely automated, it requires relatively little
human effort to test a few hundred animals every week. Measurement
of circadian rhythmicity is time-consuming, however, and the limiting
factor in the screen is the rate at which animals can be tested.
Therefore, we chose to screen for dominant mutations, which can
be done with a relatively small breeding facility and limited screening
capacity. We test the progeny of ENU-treated males, each of which
is heterozygous for a unique set of mutations. Individuals with
out-of-phase activity peaks after a week in constant conditions
are selected and raised, and their progeny are tested to determine
whether they are authentic mutants. In a pilot screen of 1275 mutagenized
genomes, we identified two semi-dominant mutations that shorten
the free running period of the larval behavioral rhythm by 0.5-0.8
h in heterozygotes and 1.0-1.5 h in homozygotes. We have so far
confirmed that one of these mutations also shortens the period of
the melatonin release rhythms measured from cultured pineal glands,
indicating that the mutant gene product affects tissue-level rhythmicity
as well as behavior. We have used microsatellite markers to map
these mutations, and found that they identify two genes that are
located on different linkage groups.
We are now working toward high-resolution mapping and phenotypic
characterization of these mutations, and we are mapping candidate
cloned genes to determine whether any are linked to our mutations.
We are also continuing to screen for new mutants, with the goal
of identifying additional alleles of these genes, as well as mutations
in other vertebrate clock genes. Our experience so far indicates
that this will be a productive approach to identification of vertebrate
circadian clock genes.
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Searching for Visual System Mutations in Zebrafish
John E. Dowling
Dept. of Molecular and Cell Biology
Harvard Univ.
Cambridge, MA
Zebrafish are highly visual animals, exhibiting light responses
after just 3 days of development, making them ideal for the genetic
analysis of visual behavior. They have large eyes and are tetrachromatic,
possessing ultraviolet- sensitive cones as well as red-, green -,
and blue-sensitive cones. They also have abundant rods, and like
other fish, their retinas continue to grow for the life of the animal.
Our group has recently developed two behavioral tests that can be
used to uncover visual system specific mutations in zebrafish.
One test, based on the optokinetic reflex, enables us to isolate
recessive visual system mutations in larval fish. The optokinetic
response is first evident at 3-4 days; by 5 days of age 98% of wild-type
fish will respond optokinetically to a moving stripe pattern with
a smooth pursuit eye movement followed by a rapid saccade in the
opposite direction. Analysis of the optokinetic responses of larval
fish takes on average no more than one minute which includes time
spent placing the fish in the test apparatus, aligning and observing
them, and returning them to their tank. Thus, one investigator can
examine up to 500 larvae per day.
The second behavioral test is based on the escape response exhibited
by zebrafish when they encounter a threatening object. This test
is used to isolate dominant visual system mutations in adult fish.
Individual fish are placed in a round container with clear sides
that has a post in the middle. Surrounding the container is a rotating
drum on which is marked a black segment that serves as the threatening
object. The fish will respond positively to the threatening object
about 85% of the time when the drum is rotating at a rate such that
the fish encounter the threatening object 20-25 times a minute.
In other words, with this test we can evaluate whether a fish is
seeing the black segment in 5-10 seconds, which enables us not only
to measure absolute thresholds for the rod and cone systems, but
also to measure the course of dark adaptation of zebrafish.
Once an animal with defective vision is identified behaviorally,
the next task is to localize the mutation within the visual system.
Since we are primarily interested in ocular abnormalities, we use
electrophysiological recordings from the eye as secondary screens
to localize mutations within the retina. The electroretinogram,
a field potential recorded from the surface of the eye, provides
information with regard to outer retinal mutations, whereas ganglion
cell recordings made from the optic nerve can indicate both outer
and inner retinal abnormalities. Once a mutation is localized to
a particular retinal locus, singe cell recordings using patch electrodes
and retinal slices, can be made. In this talk, I shall describe
these techniques and several of the mutants we have isolated.
References
Brockerhoff, S. E., Hurley, J. B., Janssen-Bienhold,
U., Neuhauss, S. C., Driever, W. and Dowling, J. E. A behavioral
screen for isolating zebrafish mutants with visual system defects.
Proc. Natl. Acad. Sci., 92, 10545-10549, 1995.
Brockerhoff, S. E., Hurley, J. B., Niemi, G. A.
and Dowling, J. E. A new form of inherited red-blindness in zebrafish.
J. Neurosci., 17, 4236-4242, 1997.
Li, L. and Dowling, J. E. A dominant form of inherited
retinal degeneration caused by a non-photoreceptor cell-specific
mutation, Proc. Natl. Acad. Sci., 94, 11645-11650, 1997.
Li, L. and Dowling, J. E. Zebrafish visual sensitivity
is regulated by a circadian clock, Visual Neurosci., 15, 851-857,
1998.
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The Sense of Hearing in Fishes: Methodologies and Results
Richard R. Fay
Parmly Hearing Institute and Dept. of Psychology
Loyola University of Chicago
Chicago, IL
The methods of psychophysics have been formally applied to
nonhuman animals for over 75 years. Psychophysics measures the performance
of an organism in detecting, discriminating, and otherwise obtaining
information from sensory stimuli, and can be used to determine the
stimulus values required to produce a given level of behavioral
performance. In animal psychophysics, performance is usually measured
as the latency, magnitude, or probability of a conditioned response
that is signaled or controlled by a sensory stimulus. The focus
of psychophysical studies is to determine what the animal can detect
and discriminate rather than how the animal normally behaves with
respect to the stimuli. Thus, psychophysics can reveal what values
and dimensions of a stimulus could be used as information, but it
does not predict behavior in the animals usual world.
This presentation describes some of the conditioning and psychophysical
methods that have been used in studies of the sense of hearing of
goldfish, that like zebrafish, are otophysan "hearing specialists."
Fish detect sound using one or more of the otolith organs (saccule,
lagena, and utricle) found in all fish species. These organs contain
a patch of hair cell receptors overlain by a solid otolith having
a density of about 3. As sound passes through a fish and brings
its tissues into motion, the otoliths move at a different amplitude
due to their greater inertia. In this way, a relative displacement
of the otolith occurs that is in proportion to acoustic particle
motion. All otolith organs in all species will tend to respond to
sound-induced motions of the fish's body. Otolith organs can respond
with reasonable sensitivity to accelerations due to gravity (zero
Hz), and to translatory motions up to several hundred Hz. Behavioral
and neurophysiological thresholds for whole-body, sinusoidal motions
range between 0.1 and 1 nanometer at 100 Hz.
In many fish species, including goldfish and zebrafish, the
otoliths may also receive a displacement input from the swimbladder,
or other gas-filled chamber near the ears. Since motions of the
swimbladder wall are created by changes in the bladder's volume
as sound pressure fluctuates, this input to the ears is proportional
to sound pressure. Thus, many fishes may respond to both acoustic
pressure and particle motion. Species having a particularly efficient
mechanical coupling between the gasbladder and the otolith organs
(e.g., the Weberian ossicles of goldfish and zebrafish) may have
very high sensitivity to sound pressure and may hear in a frequency
range up to 3-5 kHz.
We study the sense of hearing in the goldfish using two methodologies:
Classical, or Pavlovian, conditioning and various psychophysical
methods. In these experiments, goldfish are gently restrained in
a cloth bag in the center of a small, cylindrical water tank with
an underwater loudspeaker at the bottom. A mild electric shock through
a restrained fish's body causes a brief, unconditioned suppression
of respiration and bradycardia. An auditory signal that terminates
with the shock becomes a conditioned stimulus after 10 to 20 delay
conditioning trials, evoking a conditioned respiratory suppression.
Respiration is measured with a thermistor near the fish's mouth
that responds to the cooling effects of respiratory water flow.
Respiratory suppression is quantified as a ratio of the respiration
during the stimulus divided by the activity preceding and during
the stimulus. The experiments are entirely computer-automated.
Psychophysical methods are selected depending on the goals
of the experiment. These include a method of limits, automated staircase
tracking, a method of constant stimuli giving psychometric functions,
and novel methods such as stimulus generalization.
Stimulus protocols are selected according to the goals of the
experiment. In detection studies, a pure tone of 6 sec duration
(continuous or in bursts) is presented against a background of silence,
or of some controlled masking noise. In discrimination studies,
a background sound is presented as brief, repeating bursts and the
conditioning stimulus is a change in some acoustic feature (e.g.,
frequency, level, temporal pulse pattern). In stimulus generalization
studies, animals are given conditioning trials to a single stimulus,
and then tested for response to novel stimuli. The usefulness of
the generalization method is that goldfish apparently learn the
characteristics of conditioned stimuli very specifically, and tend
not to generalize (respond) to novel stimuli differing in frequency
and other stimulus dimensions that lead to qualitatively different
perceptions in human listeners. This pervasive failure to generalize
permits the identification and analysis of stimulus dimensions that
are normally salient or "information-bearing" for goldfish.
These, in turn, permit the analysis of corresponding perceptual
dimensions.
Psychophysical studies have been carried out on sensitivity
and the frequency range of hearing, the effects of signal duration
and noise masking on signal detection, frequency, intensity, and
temporal pattern discrimination, amplitude modulation detection
and discrimination, and other discriminations among spectrally and
temporally complex sounds. Using stimulus generalization paradigms,
goldfish have been shown to behave as if they had internal perceptual
dimensions corresponding to pure tone pitch, complex pitch, timbre,
and roughness. In addition, goldfish have been shown capable of
analytic and synthetic listening, and of auditory scene analysis
for simultaneous complex sound sources. Thus, these methods have
shown the sense of hearing in goldfish to be qualitatively indistinguishable
from that revealed in psychophysical studies of avian and mammalian
listeners. We have tentatively concluded that in spite of the fact
that the goldfishs primary auditory receptor organs are the
saccular otolith organs, its central auditory system and sense of
hearing appear to be primitive vertebrate characters in the sense
that they are generally shared with all other vertebrates investigated,
including humans.
Essentially nothing is known about the sense of hearing of
zebrafish. However, since their peripheral auditory system is essentially
similar to that of goldfish, it would be expected that their sense
of hearing is similar to that of goldfish. In this sense, goldfish
would probably be a good model for zebrafish hearing. At the same
time, the behavioral methods outlined here would seem to be potentially
useful for studies on the sense of hearing (and other sensory modalities)
of zebrafish. One potential problem in applying these particular
methods to zebrafish, however, is animal size. The goldfish used
in these experiments are three inches or more in standard length,
and zebrafish, particularly juveniles, tend to be much smaller than
this. Animal restraint and measurement of respiration could be a
new problem for the considerably smaller animals. I would suggest
attempting to measure respiration or the EKG by monitoring the electric
field surrounding the animal.
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Imaging, ablations and behavior: Optical studies of neuronal
circuits in zebrafish
Joseph Fetcho
Dept. of Neurobiology and Behavior
SUNY At Stony Brook
Stony Brook, NY
One of the key problems in neurobiology is to monitor activity
in single neurons non-invasively during behavior, so that the pattern
of active cells can be correlated with the behavior. We have taken
advantage of the transparency of larval zebrafish and used calcium
imaging and confocal microscopy to study which neurons are active
during escape behaviors. We have also developed approaches for using
lasers to kill individual neurons in intact fish so that we could
study the behavioral consequences of these ablations. Our work has
focused on descending reticulospinal neurons that interact with
spinal circuits to produce the rapid escape movements fish use to
avoid predators. The reticulospinal neurons we studied include the
Mauthner cell, MiD2cm and MiD3cm, which form a serially repeated
set of neurons in hindbrain segments 4, 5 and 6. Our functional
imaging and ablation experiments support the hypothesis that high
performance escape movements are produced by this segmentally repeated
set of hindbrain neurons. Many hindbrain neurons are segmentally
arranged, so it is likely that there are other segmentally repeated
functional groups. The approaches we have used to study hindbrain
cells can be applied to studies of the behavioral roles of neurons
throughout the brain and spinal cord of both normal and mutant lines
of zebrafish.
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Genetic analysis of neural circuit formation in the zebrafish
embryo
Michael Granato*, Christine Davis, Gerald Downes, Kristin Lorent,
Nini Malayaman, Julie Waterbury, Joerg Zeller, Jing Zhang and
Shelley Zhang
Dept. of Cell & Developmental Biology
University of Pennsylvania
Philadelphia, PA
Behavior, including complex behaviors such as curiosity, passion
and aggression, rely on the functionality of the nervous system.
In vertebrates, this functionality is provided by a large number
of neuronal connections which are organized in intricate neural
circuits. Formation of neural circuits occurs extensively during
embryogenesis and includes a variety of specialized processes, such
as neural specification, axonal guidance and synapse formation.
Although molecular insights into each of these processes are available,
it remains a major challenge to understand the molecular mechanisms
by which neural circuits underlying a particular behavior, be it
even very simple, are established during embryogenesis.
Our lab is interested to understand the molecular and cellular
mechanisms by which simple neural circuits develop during embryogenesis.
We are specifically interested in circuits generating locomotion,
in particular alternating, rhythmic movements. Rhythmic movements
are widespread among vertebrates, and provide the basis for more
complex movements, including swimming, crawling and walking. We
focus on mutations in 10 zebrafish genes, which we identified in
a large scale genetic screen. The initial phenotypic analysis of
the mutant phenotypes suggests that these 10 genes as crucial components
for the proper development of the circuits producing undulating,
rhythmic tail movements of the zebrafish embryo and larvae. The
ten genes can be subdivided in two group, according to their mutant
phenotypes.
Mutant embryos for any of the 7 accordion group
genes do not display the normal, alternating pattern of left/right
tail movements, but instead the embryos contract and expand along
their anterior- posterior body axis (like an accordion).
This abnormal phenotype can be mimicked in wild type embryos by
drugs known to antagonize the reciprocal inhibition circuit, indicating
that the accordion group genes are essential components
of the neural circuit underlying reciprocal inhibition. Mutant larvae
for any of the 3 twitch twice group genes do not display
the normal, alternating pattern of left/right tail movements, but
instead the mutant larvae flip their tail repetitively to the same
side, causing the larvae to rotate around its own axis. The
mutant phenotype indicates that the twitch twice genes
are required to generate alternating, rhythmic tail movements during
swimming. Moreover, separating the left half from the right half
of the hindbrain causes an identical twitch twice phenotype
in wild type larvae. This suggests that the neural circuits underlying
coordinated tail movements are at least in part located in the hindbrain,
and that the three twitch twice group genes are essential
components of this neural circuit. We will present progress on how
some of the genes contributes to the formation of the neural circuit,
and on the molecular cloning of the affected genes.
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Zebrafish Embryos: Conservation and Research Resource Applications
Mary Hagedorn
Smithsonian Institution
National Zoological Park and Conservation and Research Center
Washington, D.C. 20008
Our long range goal is to cryopreserve
teleost embryos successfully, specifically zebrafish embryos. The
availability of viable embryos after cryopreservation could have
a profound influence on the conservation of rare or threatened species
and on the extension of medical research resources. In medicine,
the zebrafish has become one of the more important vertebrate models
for the study of development and genetics. The preservation of important
genetic lines is essential, because these lines will play an important
role in future studies on human health and disease. Systematic germ
plasm cryopreservation can have a major impact on the management
of NIH resources, such as the Zebrafish Stock Center by: i) reducing
the size and production costs of facilities; ii) allowing the maintenance
of large gene pools and reducing inbreeding, while minimizing the
amount of space required to hold living animals; iii) reducing pressure
on wild populations from collection activities; and iv) facilitating
global, regional, and institution- to-institution transport of genetic
material. For conservation, the development of frozen or 'insurance'
populations would preserve genetic diversity and assist efforts
to prevent extinction of wild fish species in natural aquatic ecosystems.
Although freezing teleost spermatozoa is commonly practiced,
to date, fish embryos have never been cryopreserved successfully.
The zebrafish (Brachydanio rerio) is an excellent model for basic
studies of cryobiology because they breed regularly providing embryos
daily, and a great deal is known about their normal development
and physiology. For successful cryopreservation, cellular permeability
to water and cryoprotectants must be understood. Ideally, water
must exit, and an appropriate cryoprotectant enter all the cells.
Using a wide variety of techniques, such as electron-spin resonance,
biophysical modeling, and magnetic resonance imaging and spectroscopy,
we have found a major permeability barrier in the zebrafish embryo:
the yolk syncytial layer (YSL), which develops between the yolk
and blastoderm. Due to its low permeability, the YSL blocks water
exit from, and entry of some cryoprotectants into, the yolk. Specifically,
electron microscopy has shown that cryopreservation destroys the
YSL, presumably because ice-crystals form in the yolk and destroy
the YSL. Thus, standard techniques of immersing the embryos in various
cryoprotectants allowing passive permeation over time may not be
practical in this system. Therefore, we are examining ways to modify
membrane permeability to water and cryoprotectants using molecular
and biochemical techniques.
This work was supported by grants from the National Institutes
of Health (R01 RR08769), the Smithsonian Institution, and Maryland
Sea Grant College.
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Asymmetric Gene Expression in the Developing Zebrafish Brain
Marnie E. Halpern1, Scott J. Nowak1, Juan-Carlos
Ispizua-Belmonte2, and Jennifer O. Liang1;
1. Carnegie Institution of Washington, Dept. of Embryology, Baltimore,
MD 21210;
2. Salk Institute, La Jolla, CA 92037
Analyses of zebrafish mutations may provide insight into the
molecular mechanisms that underlie left-right differences in the
vertebrate brain. Zebrafish cyclops (cyc) mutants
lack the ventral brain and floor plate. The cyc gene was
recently found to encode a nodal-related TGF-b
family member that is expressed in the midline mesendoderm and prechordal
plate during gastrulation but is down regulated by early somite
stages. However, a new wave of asymmetric expression appears in
the left lateral plate mesoderm and in the dorsal diencephalon during
mid-somitogenesis. Expression in the dorsal brain may correlate
with the left habenular nucleus and the epiphysis (pineal organ)
also derives from this approximate region. To better characterize
the patterning of the diencephalon, we are examining the relationship
of cyc expression with other developmental (i.e. floating
head, one-eyed pinhead) and pineal-specific (i.e. visual pigments)
genes that are expressed in the dorsal forebrain. Zebrafish antivin
(similar to mouse lefty) is transcribed in the left side
of the dorsal diencephalon, as is the bicoid homeobox gene
Pitx2. Together with cyc, these genes are bilaterally
expressed in the brains of zebrafish mutants that exhibit midline
or other gastrulation defects, consistent with an early specification
of asymmetry that is later manifested in the dorsal brain. We are
exploring how asymmetric gene expression is regulated in the diencephalon
through RNA misexpression studies. By transient rescue of mutant
embryos through gastrulation, we aim to uncover the function of
left-sided Nodal signalling in the developing zebrafish forebrain
and the anatomical and/or behavioral consequences on the adult.
Supported by the NSF (MEH), NIH (J-C I-P and JOL) and the Pew Scholars
Program (MEH and J-C I-P).
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Odorant receptors and olfactory system function in goldfish
and zebrafish
John Ngai
Dept. of Molecular and Cell Biology
University of California, Berkeley
The detection and discrimination of the multitude of environmental
stimuli by the vertebrate olfactory system results from the activation
of olfactory neurons in the nose. The first step in olfactory processing
resides at the level of the interaction of odorous ligands with
odorant receptors. A large multigene family thought to encode odorant
receptors was initially identified in the rat by Buck and Axel in
1991. These receptors are predicted to exhibit a seven transmembrane
domain topology characteristic of the superfamily of G protein-coupled
receptors. The sizes of the receptor repertoires of different vertebrate
species are extremely large and are estimated to contain between
100 and 1000 individual genes. These observations suggest that olfactory
discrimination is accomplished by the integration of signals from
a large number of specific receptors, each capable of binding only
a small number of structurally-related odorants. Other olfactory
G protein-coupled receptors unrelated to the first family of odorant
receptors described have been identified in the vomeronasal organ
(VNO) of mammals as well as in the olfactory epithelium of fish.
These receptors are encoded by two unrelated gene families: the
VNR family and the V2R family; the V2R receptors are structurally
related to the CaSR and mGluR families. While it has been proposed
that the VNR and V2R receptors are pheromone receptors (based on
their expression in the mammalian VNO), the actual function of these
orphan receptors awaits a direct demonstration of their molecular
specificities.
As an approach toward identifying ligands for olfactory receptors,
we have pursued an expression cloning strategy using the goldfish
as a model system. The odorants that fish detect are water soluble,
and include amino acids (feeding cues), bile acids (nonreproductive
social cues with possible roles in migration), and sex steroids
and prostaglandins (pheromonal cues). Electrophysiological studies
have characterized the sensitivities of fish olfactory systems to
specific ligands, demonstrating, for example, thresholds for detection
in the picomolar (for sex steroids) to nanomolar (for amino acids)
range. Thus, the defined properties of odorant-evoked pathways in
vivo provide an excellent starting point for the molecular and biochemical
characterization of fish odorant receptors. We have recently succeeded
in expression cloning a cDNA encoding a goldfish odorant receptor
preferentially tuned to recognize basic amino acids. This receptor,
called receptor 5.24, that shares significant similarity to receptor
families that include the CaSR, mGluR, and V2R class of VNO receptors.
The affinity and specificity of the cloned goldfish odorant receptor
for basic amino acids are remarkably similar to basic amino acid
sensing pathways characterized in vivo. Degenerate polymerase chain
reaction (PCR) reveals other related sequences that are expressed
in the goldfish olfactory epithelium. Our results provide the first
direct evidence that these receptors in fact comprise a family of
odorant receptors. A number of questions can now be pursued based
on our demonstration of receptor 5.24s functional characteristics.
For example, can receptor expression or the behavior of cells expressing
this receptor be altered by odorant-induced activity or experience?
What are the molecular specificities of other olfactory CaSR-like
receptors related to receptor 5.24? Do closely related receptors
recognize other amino acid odorants? Do more distantly related receptors
recognize other classes of odorants, such as pheromones or migratory
cues? Future studies will address these questions using both the
goldfish as well as the closely related zebrafish as model systems.
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Development and Early Behavior in Fishes: a comparative approach
David L. G. Noakes
Department of Zoology & Axelrod Institute of Ichthyology
University of Guelph, Guelph
Ontario N1G 2W1 Canada
Fishes have a greater variety of species, and hence early development
and behavior, than other vertebrates. Reproduction and early development
are the key features to understand this variation. I will propose
a unifying scheme to incorporate this variation, with examples including
zebrafish. The scheme integrates early development (ontogeny), evolution
(function, phylogeny), and causation (physiological mechanisms).
The life history of any species consists of a maximum of four developmental
intervals. The number of these developmental intervals for any species,
and the features of each interval are the keys to understanding
the patterns of early development in fishes. The timing of key events
during development characterizes this pattern of ontogeny. I will
explain this scheme with examples of avoidance and preference tests,
social and feeding behavior, and sexual development.
Key words: toxicology, avoidance responses, locomotor
behavior, choices, receptor development, social behavior, genetics,
feeding, sex
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Zebrafish Touch-Insensitive Mutants
Angeles Ribera
Dept. of Physiology & Biophysics
Univ. of Colorado Health Sciences Center
Denver, CO
Developmental changes in neuronal connectivity, synaptic function
and excitable membrane properties underlie stage-specific appearance
of embryonic behaviors. Several zebrafish embryonic mutants display
abnormal motility (*Granato et al., 1996) and provide opportunities
for identification of genetic, molecular and cellular mechanisms
generating specific behaviors. One group of mutants swims spontaneously
but not in response to touch (touch-insensitive mutants). The specificity
of the behavioral phenotype suggests that the defect arises at the
level of the relevant sensory neurons, mechanosensory Rohon-Beard
cells. Whole cell recording using patch clamp techniques was performed
to examine the excitable membrane properties of Rohon-Beard cells.
Wildtype zebrafish embryos respond to touch at 27 hours post fertilization.
Electrophysiological analysis of Rohon-Beard cells of wild type
embryos indicates that, during the transition from a touch-insensitive
to a touch-sensitive embryo, action potentials acquire larger overshoots
and briefer durations. During the same period, amplitudes of sodium
and potassium currents both increase. In contrast, Rohon-Beard cells
of several homozygous touch insensitive mutant embryos (macho,
mao; alligator, ali; steifftier, ste)
fail to fire action potentials. The strongest defects are found
in homozygous mao mutants. Similar to wildtype embryos, mechanosensory
neurons of unaffected sibling embryos of all mutant lines fire action
potentials with overshoots of normal amplitude. General measures
of membrane properties, such as resting membrane potential and input
resistance, are not affected in homozygous mutants excluding the
possibility of nonspecific effects. Examination of voltage-dependent
currents that underlie action potentials demonstrates that impulses
are not generated because of a specific reduction in sodium current.
The amplitude of voltage-dependent potassium current increases normally,
consistent with the fact that developmental regulation of the duration
of the action potential is unaffected in mechanosensory neurons
of mutants. However, sodium current remains small, thereby preventing
the normal increase in overshoot of the action potential. These
results indicate that developmental regulation of sensory neuron
sodium current plays an essential role in acquisition of embryonic
touch sensitivity.
*Granato et al., 1996, Development 123: 399-413.
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Chemically-mediated predator-prey interactions in Ostariophysan
fishes
Brian D. Wisenden
Dept. of Biology
Moorhead State University
Moorhead, MN 56560
Animal behavior evolves, like any other trait, by natural selection
rewarding individuals with appropriate behavior with greater rates
of reproductive success than individuals with inappropriate or ill-timed
behavior. Genes that contribute to the physiology (sensory receptors)
and inclination (neural processing) to perform effective behavior
are thusly promoted at the expense of genes for less effective behavior.
Antipredator behavior serves as a good model for the effect of environmental
selection gradients on the evolution of behavior. Predation exerts
a steep selection gradient on shaping behavior.
The expression of behavior is an interaction between the genes
that code for the behavior and environmental cues that signal the
appropriate timing and degree of response. In aquatic environments,
visual information is not available under conditions of high turbidity,
areas of structural complexity, or at night. Chemical cues are not
affected by these limitations. In addition, water is the universal
solvent and ideal for the solution and dispersal of chemical cues.
Chemical cues are released during predation events - at the point
of initial detection, during attack and capture and even post-ingestion
of prey. Thus, publicly available chemical information is a reliable
indicator of predation risk and should be used widely by aquatic
animals for risk assessment.
For example, larval damselflies (Enallagma sp.) are aquatic insects.
They respond with antipredator behavior to injury-released chemical
cues from conspecifics. These cues are released when a predator
grasps its prey. Fishes too, including zebra danios (Brachydanio
rerio), exhibit clear antipredator responses to injury-released
chemical cues from their own species. Typically, these cues elicit
a reduction in overall activity, movement to the substratum, increased
shoal cohesion and area avoidance (when possible).
Injury-released chemical cues occur in relatively late stages of
a predation event. These cues indicate that the predator is foraging
actively. However, prey can detect the predator's presence at a
much earlier stage by detecting the odor of the predator itself.
This affords prey more time to initiate crypsis or escape behavior.
How do prey come to recognize the odor of their predators? This
is not a trivial question because the suite of predators to which
a prey population is subjected can vary widely over space and time.
A growing body of evidence indicates that genetically-based recognition
templates for every potential predator was not the parsimonious
solution favored by natural selection. Instead, a robust learning
paradigm has evolved whereby novel stimuli, such as an odor or image,
come t o be associated with predation risk when presented simultaneously
with injury-released chemical cues. Thereafter, the novel stimulus
on its own elicits a full antipredator response.
This phenomenon has been demonstrated in a number of aquatic taxa,
including, damselfly larvae, fathead minnows (Pimephales promelas)
and zebra fish. Recognition learning of indicators of predation
risk is remarkable in that only a single trial is required. Memory
of the novel cue's riskiness is retained for at least a year by
minnows. Minnows and zebra fish are so adept at recognition learning
that they can be easily tricked to fear non-biological stimuli.
Recent data indicate that recognition learning occurs even if there
is a slight delay between introduction of injury-released cues and
the novel cue.
Zebra fish are in the superorder Ostariophysi. This group includes
the cyprinidae (minnows, of which the zebra fish is one), the characidae
("tetras"), the siluridae (catfish), the catastomidae (suckers)
and sundry other minor families. Altogether, they represent 64%
of all freshwater fish species. One defining character of this group
is the presence of specialized club cells in their epidermis, known
as alarm substance cells (ASC). They are fragile, thin-walled cells
on the surface of the epithelium and are easily ruptured when the
prey is grasped by a predator. Alarm substance, thought to be hypoxanthine-3(N)-oxide
or some close derivative, greatly enhances the general phenomena
associated with injury-released chemical cues, including learning.
Production and maintenance of ASCs have a high metabolic cost.
Minnows invest facultatively into ASCs in response to perceived
levels of predation risk. For example, minnows fed high rations
develop a thicker epidermis with disproportionately more ASCs than
minnows fed a low ration. This cost is offset by the fitness advantage
of attracting additional predators to a predation event in progress.
This helps the prey because predators threaten and bully each other
providing the prey with an opportunity for escape. Shoalmates from
a minnow's own shoal ("familiar shoalmates") execute more effective
group-level antipredator responses than those executed by non-familiar
shoalmates. High-ration minnows make more ASCs when held with non-familiar
shoalmates than when held with familiar shoalmates. This latter
finding suggests a trade-off between the cost of making ASCs and
predation risk. When in the company of familiar shoalmates minnows
rely more on effective group antipredator behavior and spend less
energy making ASCs. When in the company of non-familiar shoalmates,
minnows invest more in ASCs to better attract secondary predators.
This system is ripe for developmental biologists using zebra fish
as a model. Virtually nothing is known about the olfactory receptors
involved in this response, the neural processing for learned recognition
of novel stimuli, the production of ASCs nor the nature of the cue
itself.
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POSTER ABSTRACTS
Computer-Assisted Visualizations of Neural Networks: Expanding
the Field of View using Seamless Confocal Montaging
James C. Beck, A.O. Dennis Willows, and Mark S. Cooper
Department of Zoology
University of Washington
Seattle 98195-1800, U.S.A.
Microscopic analysis of structure/function relationships within
the neural networks of adult and developing tissues often requires
visualization of large regions of neuronal architecture. To accomplish
this, there are two visualization approaches: (1) image the entire
area at once with low spatial resolution; or (2) image small areas
at higher magnification/resolution, and then piece the regions back
together using a mosaic reconstruction (i.e. photomontaging).
Low magnification imaging is relatively rapid to perform, resulting
in a visualization that encompasses a large field of view with an
extended depth of field. However, for fluorescence microscopy, low
magnification visualization is often plagued by poor spatial resolution.
Although high magnification imaging has superior spatial resolution,
it produces a visualization with limited depth of field. Moreover,
when creating a larger field of view, the visualization is fragmented
at the seams where multiple images must be stitched together. Using
confocal microscopy as well as common image-processing functionalities,
we outline a new visualization approach that transforms a montage
of spatially-contiguous z-series (i.e. vertical optical sections)
into a large visualization with a seamless field of view and an
extended depth of field. We illustrate our method for visualizing
neural networks using tissues from the adult gastropod mollusc,
Tritonia diomedea, and the developing zebrafish, Danio
rerio.
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Crossed Modulation of Inhibitory Synaptic Inputs in Left-Right
Decision Neurons
Kohei Hatta and Henri Korn
Institut Pasteur, Biologie Cellulaire et Moléculaire du
Neurone, INSERM U261
Département des Biotechnologies
75015 Paris, France
e-mail: khatta@pasteur.fr
Continuous fluctuations of membrane potential or synaptic noise
in central neurons are caused by spontaneous impulses in presynaptic
afferents. The paired Mauthner (M-) cells of teleosts are a part
of the brain stem escape network and the direction of escape of
fish is determined by which one reaches threshold first. In these
cells, synaptic noise is mostly inhibitory (ISN) and it influences
the neuron's input-output relation. We found that in the adult zebrafish
M-cells ISN exhibits two distinct patterns, i.e. a "noisy" state,
made of bursts of fast strychnine-sensitive inhibitory postsynaptic
potentials, and a "quiet" state, with less inhibition, both of which
can last from 14 msec to several minutes, and then spontaneously
flip-flop. Simultaneous recordings have revealed that these patterns
are complementary in the left and right M-cells. An action potential
induced by a brief current injection transiently turns on the noisy
state in the activated neuron, while it suppresses the ISN in the
other side. Finally the noisy state contains periodic components
the frequency of which is also up-regulated after an action potential.
Our data suggest that this asymmetric modulation of ISN contributes
to the "choice" of the direction of the escape most appropriate
for animal.
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The Anatomical Organization of the Locus Coeruleus in the Zebrafish
PoKay M. Ma
Department of Biology
Queens College, The City University of New York
Flushing, New York 11367
The locus coeruleus (LC) is a brainstem noradrenergic nucleus present
in all vertebrates. In the zebrafish, this nucleus contains an average
of only 6.8 ± 1.5 (range 3 - 10) neurons. Both genetic and epigenetic
factors are implicated in the regulation of LC cell number. The
low neuron number offers an opportunity for elucidating the organization,
development and function of the entire nucleus. Preliminary results
suggest that subsets of distinct LC neurons exist. (i) On the basis
of dendritic morphology, three types of neuron can be distinguished.
The relative proportion of the three types of cells appears to depend
on the total number of neurons in the LC. (ii) Retrograde tracing
experiments show that LC neurons project to many targets in the
forebrain, midbrain and cerebellum. Double-labelling studies reveal
the existence of neurons with distinct projection patterns. No target
receives input from all neurons, and each neuron projects to a combination
of targets. The combination of targets each neuron projects to also
appears to depend on the total number of cell in the LC. These observations
suggest that in the LC, anatomical organization is subject to adjustment
based on cell number.
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Behavioral screening techniques for larval and adult zebrafish
with special reference to behavioral lateralization
Adam Miklosi and Richard J. Andrew
Centre for Neuroscience, School of Biology
University of Sussex
Brighton, UK
We have developed behavioral tests, which allow screening of large
numbers of zebrafish larvae and systematic testing of adults. These
are suitable to identify mutations affecting visual mechanisms as
well as behavioral lateralization.
In adult tests, fish live singly in a small tank, with a test compartment,
where they are fed, and which they visit frequently and spontaneously.
They respond to colored beads initially with bites and after habituation
across trials with viewing alone. The right eye is used when a bite
will be performed, and there is a shift to left eye use in viewing
after habituation. Dishabituation is clear as a result of color
change between trials. This would allow measurement of the ability
to discriminate hue and of perceptual memory, as well for screening
fish with reversed bias.
The screening tests for fry uses a small swimway, divided into
compartments, each with an exit to the next trough a vertical slit.
Positive phototaxis is used to cause the larva to move to the next
compartment, by darkening the one the larva is in, and illuminating
the next. Larvae aged 4, 6, and 8 days show a turning bias which
in wild type is leftward on day 4 but shifts toward rightward by
day 8. These age-dependent changes are very similar to shift described
in chicks; the underlying mechanism is unknown but their discovery
in a teleost confirms evidence that they may be widespread in vertebrate
development. In a further application of this procedure a black
stripe (1.2 cm) was placed on the side walls of the compartments.
We found that naive, 6 day old larvae turned away from the stripes
(avoidance), however larvae that were exposed to the stripes on
day 5 in their home environment showed either no avoidance or even
some preference (approach) to the stripes. This test presents an
good opportunity to screen for mutations affecting early avoidance
and approach responses in larvae, as well as long term memory and
lateralization.
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Locomotive Repertoire of the Larval Zebrafish: Swimming, Turning
and Prey Capture
Seth A. Budick and Donald M. OMalley*
Department of Biology
414 Mugar Hall
Northeastern University
Boston, MA 02115
*corresponding author, email: domalle@lynx.neu.edu
Larval zebrafish are a popular model system because of their transparency
and relative simplicity. They have a total of about 160 neurons
in the brainstem that project to the spinal cord, many of which
can be individually identified and laser-ablated in intact larvae.
This should facilitate cellular-level characterization of the descending
control of larval behaviors. As a first step, we attempted to delimit
the range of locomotive behaviors exhibited by zebrafish larvae.
Using high-speed digital imaging, a variety of swimming and turning
behaviors were analyzed in 6-to-9 day old larval fish. Swimming
episodes appeared to fall into two categories, with the center of
bend of the larvas body occurring either near the mid-body
(burst swims) or closer to the caudal fin (slow swims).
Burst swims also involved larger amplitude bending, faster speeds
and greater yaw then slow swims. Turning behaviors clearly fell
into two distinct categories: fast, large-angle escape turns
characteristic of escape responses, and much slower routine turns
lacking the characteristic "return tail flip" that accompanied
the escape turns. Prey-capture behaviors were also recorded and
appeared to be comprised of two more elemental locomotive behaviors:
routine turns and slow swims. The different behaviors observed were
analyzed with regard to possible underlying neural control systems.
Our analysis suggests the existence of discrete sets of controlling
neurons and helps to explain the need for the 160 or so spinal-projecting
nerve cells in the larval zebrafish brainstem.
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Cloning and Characterization of a Gene Homologous to the Delta
Opioid Receptor in Zebrafish
R.E. Rodriguez, A. Barrallo and R. Gonzalez Sarmiento
University of Salamanca, Salamanca, Spain
e-mail: requelmi@gugu.usal.es
A full-length cDNA, ZFOR1, has been isolated from the teleost
zebrafish (Danio rerio) using a probe from rat m opioid receptor.
ZFOR1 encodes a 373 amino acid protein with seven potential transmembrane
domains that shows a high degree of homology to mammalian d opioid
receptor. We have also isolated a genomic clone which contains two
exons of ZFOR1, homologous to exons 2 and 3 in mouse and human d
opioid receptor. Expression of ZFOR1 appears to be restricted to
nervous tissue as assessed by Northern blot. In situ hybridization
in zebrafish brain with specific probes revealed several discrete
areas of ZFOR1 expression; higher levels are detected in dorsal
telencephalic areas, periventricular layer of the optic tectum,
and granular layer of the cerebellum. To characterize the receptor
and to compare its pharmacological profile with the delta opioid
receptor we stably expressed ZFOR1 in HEK 293 cells. Competitive
binding studies with several opioid ligands presented low affinity
for the classical opioid selective ligands , but showed high affinity
for the d -agonist BW373U86 (Ki 89nM). In functional GTPgS studies,
BW373U86was found to be a good agonist. These are the first molecular
evidences on the presence of a functional d opioid receptor-like
in the zebrafish.
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Involvement of Adhesion Molecules in Plasticity of Zebrafish
Brain After Avoidance Conditioning
R. Schmidt1*, G. Pradel1, O. Heller1,
M. Schachner2
Biotechnology Center, Justus-Liebig-University, Giessen (1) and
Molecular Neurobiology Center, University, Hamburg (2), Germany
We have developed an active shock avoidance paradigm for goldfish
and zebrafish that favorably lends itself towards behavioral analyses
in theses cyprinids. Application of [
14C] -deoxyglucose revealed
a significantly increased energy demand in the zebrafish optic tectum
after learning of the avoidance response. In situ hybridizations
with antisense probes against the cell adhesion molecules L1.1 and
NCAM located both messages to specific neuronal populations in several
distinct layers of the optic tectum. Expression of L1.1 and NCAM
mRNA was significantly enhanced 3 hours after learning. In order
to analyze, whether induction of these cell adhesion molecules is
a prerequisite for long-term memory formation, HNK-1 antibodies,
directed against a specific carbohydrate epitope on L1.1, NCAM and
ependymins, were injected between training and test. Various other
antibodies served as controls. In the test, fish injected with HNK-1
antibody exhibited significantly reduced retention scores (RS =
0.30) as compared with controls (RS = 0.77). The HNK-1 antibody
had no influence on the performance of the avoidance response, when
injected into overtrained animals (RS = 0.80). Similar results where
obtained with specific antibodies directed against the deglycosylated
(i.e., HNK-1-free) form of ependymins (RS = 0.24). Our results point
towards a pivotal role of cell adhesion molecules in long-term memory
formation in the cyprinid brain. - Financial Support by the Deutsche
Forschungsgemeinschaft is gratefully acknowledged (Schm 478/10-1).
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Fighting Behavior in Zebrafish, Danio rerio
Henning Schneider and Kim H. Eliasz
Dept. of Biology
William Paterson University of NJ
Wayne, NJ 07470
Zebrafish establish dominant hierarchies that are maintained
for long periods of time. To study the behavioral elements that
lead to these social hierarchies, we analyzed the interactions of
groups of two fish of similar body mass and size.
Behavioral patterns such as lateral displays, frontal displays,
nipping, and chasing were observed and recorded over a 30 min period.
Hierarchies appeared to be established within a fighting period
of up to 5 min. This fighting period is characterized by frequent
lateral and frontal displays that are accompanied by nipping. Following
this fighting period, dominant fish chased subordinate fish for
the rest of the observational period. A second encounter of the
same pair of fish on the following day (day 2) showed similar characteristics.
The winner of the fight on day 1 also was the winner on day 2.
Our studies indicate that zebrafish can establish dominant
hierarchies during a short period of intense fighting. Whether the
social status of zebrafish leads to molecular or physiological changes
in the nervous system will be addressed in future studies. Supported
by Center for Research, WPUNJ.
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Innervation of Fin Muscles in Zebrafish, Danio rerio
Henning Schneider, Beth M. Sulner, and Elisabeth Abbiati
Dept. of Biology
William Paterson University of NJ
Wayne, NJ 07470
Sets of fin muscles control movements of dorsal, caudal, and
pectoral fins in adult zebrafish. In order to gain insight into
the control of these fin muscles, we characterized the organization
of fin motorneurons (MNs) anatomically.
Application of neurobiotin to dorsal fin depressor and erector
muscles labeled 10 to 12 secondary MNs per hemisegment. These MNs
(10 - 15 µm in diameter) are located in a ventro-lateral motor column
and tend to appear in groups of up to 4 MNs. Double labeling with
fluorescent tracers showed that dorsal fin MNs can be distinguished
from secondary axial MNs by position of cell bodies. Dye injections
into the protractor muscle revealed a new type of zebrafish neuron
with contralateral projections.
Caudal fin muscles are innervated by nerves that originate
in the last 5 segments of the spinal cord. Six of eight caudal fin
muscles are located in a dorsal, median, and ventral region of the
tail. Dye injections into these three regions yielded staining of
up to 66 MNs for the dorsal group, up to 71 MNs for the medial group,
and up to 33 MNs for the ventral group.
The MNs innervating pectoral fin muscles are located in the
first segments of the spinal cord. Application of tracers to adductor
and abductor muscles yielded up to 40 neurons for each muscle. MNs
of each pectoral fin muscle are located in separate clusters.
Future investigations will be concerned with physiological
activities of mapped fin MNs.
Supported by Center for Research, WPUNJ.
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Learned Recognition by Zebra Fish (Brachydanio Rerio)
of Novel Predator Odor Following Non-Simultaneous Presentation of
Alarm Pheromone in Skin Extract and Predator Odor
Nichole Korpi and Brian Wisenden
Department of Biology
Moorhead State University
Moorhead, MN, 56560
Fishes in the superorder Ostariophysi (minnows, characins, catfish,
etc) possess specialized epidermal cells that contain an alarm pheromone.
These cells lack a duct to the skin surface. The alarm pheromone
is released when the skin is damaged, such as occurs when the fish
is grasped by a predator. Thus, the pheromone serves as a reliable
indicator of predation risk. Fish can learn to recognize and associate
novel odors, such as the odor of a predator, when a novel odor is
encountered simultaneously with alarm pheromone. Thereafter, the
novel cue is recognized as an indicator of risk and induces antipredator
behavior. In nature, the odor of a novel predator may not be detected
simultaneously with alarm pheromone. Can a delay between the presentation
of alarm pheromone and novel predator odor still result in associative
learning? We presented zebra danios with the odor of northern pike
(Esox lucius) 5 min after presenting them with either alarm
substance or water (control). During a predation event, 5 min is
a long time. When later retested with pike odor alone, zebra fish
conditioned with alarm substance significantly increased antipredator
behavior (z = 1.91, P = 0.024) but control fish did not (z = 0.10,
P = 0.464). These data show 1) that learned recognition of predation
risk is robust enough to accommodate ecologically realistic temporal
shifts in stimulus presentation, and 2) confirm that zebra danios
are good test organisms for learned risk aversion and potentially
the developmental and genetic mechanisms that support these behaviors.
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