Current and future projects –
Neuronal circuits controlling innate
The opto-motor response
presented with visual stimuli containing whole field motion, fish will
swim to follow the moving patterns. In order to study the neuronal
controlling this particular behavior we implemented a method to label
neurons projecting into the spinal cord – the reticular spinal
formation - with
a dextran-conjugated calcium indicator dye. These neurons forms a
of circa 300 identified cells that have exclusive control over tail
therefore the majority of all fish behaviors. Data describing the
properties of these neurons and their role in transducing visual
into motor output is described in Orger et al. 11. Using a combination of quantitative
analysis, in-vivo two photon imaging and targeted ablations we found
surprisingly small set of identified neurons – not more than
twelve cells on
each side of the hindbrain – are responsible for right- or left-
respectively. These spinal projection neurons link sensory processing
brain to motor output in the spinal cord and, therefore, provide an
starting point for studying the
complete sensorimotor transformations
Figure 1: (Left panel) Behavioral scheme with OMR
stimulus. (RIght panel) Functional properties of hindbrain
neurons to various moving gratings.
See Orger et al., 2008
identified which neurons control particular motor patterns, we can now
their activity is decoded in the spinal cord to produce the associated
and, importantly, start to investigate the upstream circuitry that
leads to the
selective activation of these descending control neurons. To that end
generated a series of transgenic fish lines that express genetically
calcium indicators in all neurons. Head-fixed
fish that expresses G-CaMP2 in all neurons views OMR inducing stimuli
activity in its neurons is recorded with a two-photon microscope. This
technique will allow us to map out the neuronal activity related to
particular behavior in the complete fish brain with sub-cellular
resolution. So far we find that
surprisingly small sets of neurons show selective response properties
stimuli that reliably induce forward swimming or turning in the fish
remarkably, all neurons upstream of the retina that respond to whole
motion stimuli show very distinct directional preference. Complementary
functional imaging we are using extra- and intracellular
recordings for a detailed characterization of the calcium signals and
important probe to examine the precise temporal patterns of evoked
potentials and synaptic inputs.
attraction and repulsion
We found that larval fish,
when presented with small moving objects, will track these visual
turn away from them depending primarily on the object’s size and
central module of the set-ups used to quantify these (and several
behaviors in larval zebrafish is described in Figure 2. A freely
is monitored by a high-speed camera, its body position and gaze
extracted online and visual stimuli are presented through a LPD
a 360 degree screen in an online-feedback loop. This
gives the experimenter absolute control over the visual input and
well as spontaneous) motor-output can be recorded with millisecond
and high spatial resolution. A dataset acquired by this method is shown
right panel. Larval zebrafish will track virtual prey-like stimuli
onto the screen as long as their size doesn’t exceed 3 degrees,
approximately the size of
paramecia or artemia in a natural hunting situation. Larger stimuli
reliable turning away from the stimulus and projection pattern akin to
field motion (20 degrees) elicit optomotor-like behavior. These are
findings that tell us which parameters in visual stimulus space get
distinct output patterns. Experiments to study the role of different
regions in these transformations are currently under way using 2-photon
microscopy and transgenic fish-lines that express genetically encoded
indicators in all neurons. However, a first examination of the
these stimuli in the optic tectum - one of the first processing
the retina - was undertaken using bulk labeling by bolus injection of a
indicator conjugated to AM-esther. By combining two-photon imaging,
motion stimuli and artificial re-wiring of retinofugal projections we
show that this nucleus serves as a module to extract direction
information from prey like stimuli 12. This is particularly interesting since it
strongly reminiscent of higher cortical function in mammalian
while in lower vertebrates the retina has been thought to be
most of the extraction of direction selective information.
Figure 2: left
panel - a
360° X 90° virtual
environment controlled by a custom DirectX (Microsoft ©) graphics
coupled with a custom real-time CCD-based object tracking system, which
the online association of zebrafish behaviour and stimulus
panel – responses of a zebrafish larva to moving spots of varying
appear at frame zero straight ahead of the fish and are moved at
for the next 100 frames either to the left or the right. Fish respond
whole body tracking motion to spots of 1 degree size and with distinct
away from stimuli with a diameter of 5 degrees or larger.
stimulation and escape
brief touch to the head
or body of a zebrafish larva will elicit a rapid and pronounced escape
response. In order to examine the role of the primary sensory neurons
behavior we have generated transgenic fish lines expressing
(ChR2) and an archaeal light-driven chloride pump
(NpHR) 16 under various promotors which enables
us to selectively excite or inhibit specific neurons involved in the
under scrutiny. A first study employing this technique probed the role
trigeminal and Rohon-Beard neurons in the context of escape behaviors
larval zebrafish. Here we could show that single, ChR2 evoked action
in individual sensory neurons are sufficient to trigger complex escape
in the animal 13. This is a first demonstration of the
feasibility of this novel technique in the zebrafish preparation and reveals
a degree of efficiency in sensory coding that
is normally found only in the central processing components of neural
Figure 3: (Left panel) Channelrhodopsin expression and
bahvioral stimulation. (Right panel) Channelrhodopsin elicits
single spikes. See Douglass et al., 2008
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