Introduction

The general goal of the laboratory is the comprehensive identification and examination of neural circuits controlling behavior using the larval zebrafish as a model system. To that end, we have established and quantified a series of visually induced behaviors and analyzed the individual resulting motor components. Using these assays in combination with various calcium indicators and two-photon microscopy we have monitored neuronal activity throughout the fish brain in an awake and intact preparation. An extended goal is the study of how changes or variations in the behavior are reflected in changes in the underlying neuronal activity. To that end, we have developed several quantitative learning assays and tools for in vivo monitoring of neural activity in freely swimming larvae.  

Background

Neuroscientists have long been working to understand how biological structures can produce the complex behaviors that are generated by the nervous system. However, even the basic operational principles governing a brain’s interconnected network of cells have remained painfully elusive. My laboratory is working on a scientific strategy focused on building a complete, multi-level picture of simple neural circuits that will advance our basic understanding of brain function and offers a complete view into the neuronal activity underlying a series of relatively complex behaviors.

Generally the question of how the brain, or neural circuits in particular, function ought to be reduced to the question of what the brain or particular parts of the brain are doing. This is best addresses by rigorous and quantitative behavioral assays that allow us to relate a particular set of input stimuli – in our case visual signals that get translated by the retina into action potentials in ganglion cells – to a set of motor actions that are controlled by an array of spikes in motor neurons – the output of the system. Both data-sets can be assessed to a first order by behavioral experiments. The second question then has to be how the neurons and synapses within the circuit actually perform this computation. Important insights into this problem can be gained by recording activity in every neuron of the fish’s brain in the context of a specific behavior. This would be a truly daunting task in any mammalian preparation but the small and transparent brain of the larval zebrafish allowed us to develop and extend the technology that makes it possible to acquire these datasets in fish expressing genetically encoded indicators in all neurons¹. In parallel we have tried to simplify the question by the examination of individual modules in the brain. Here, we have started to make progress by finding specific nuclei in the fish brain - the optic tectum which processes inputs directly from the retina² on the one hand and the reticular spinal formation, a set of identified neurons that exclusively controls behavior^3,4, on the other hand - that provide stepping stones on the long journey to a complete understanding of the brain and its role in producing behavior.

Current and future projects – Neuronal circuits controlling innate behaviors.

The opto-motor response (OMR)

When presented with visual stimuli containing whole field motion, fish will turn and swim to follow the moving patterns. In order to study the neuronal networks controlling this particular behavior we implemented a method to label all neurons projecting into the spinal cord – the reticular spinal formation - with a dextran-conjugated calcium indicator dye. These neurons forms a bottle neck of circa 300 identified cells that have exclusive control over tail motion and therefore the majority of all fish behaviors. Data describing the response properties of these neurons and their role in transducing visual information into motor output is described in Orger et al. ¹¹. Using a combination of quantitative behavioral analysis, in-vivo two photon imaging and targeted ablations we found that a surprisingly small set of identified neurons – not more than twelve cells on each side of the hindbrain – are responsible for right- or left- turning respectively. These spinal projection neurons link sensory processing in the brain to motor output in the spinal cord and, therefore, provide an excellent starting point for studying the 
complete sensorimotor transformations underlying behavior.

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

Having identified which neurons control particular motor patterns, we can now ask how their activity is decoded in the spinal cord to produce the associated behavior and, importantly, start to investigate the upstream circuitry that leads to the selective activation of these descending control neurons. To that end we have generated a series of transgenic fish lines that express genetically encoded calcium indicators in all neurons.   Head-fixed fish that expresses G-CaMP2 in all neurons views OMR inducing stimuli while activity in its neurons is recorded with a two-photon microscope. This technique will allow us to map out the neuronal activity related to this 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 to stimuli that reliably induce forward swimming or turning in the fish and, remarkably, all neurons upstream of the retina that respond to whole field motion stimuli show very distinct directional preference. Complementary to the functional imaging we are using extra- and intracellular electrophysiology recordings for a detailed characterization of the calcium signals and as an important probe to examine the precise temporal patterns of evoked action potentials and synaptic inputs.

Innate attraction and repulsion

We found that larval fish, when presented with small moving objects, will track these visual stimuli or turn away from them depending primarily on the object’s size and contrast. The central module of the set-ups used to quantify these (and several other) behaviors in larval zebrafish is described in Figure 2. A freely swimming fish is monitored by a high-speed camera, its body position and gaze direction extracted online and visual stimuli are presented through a LPD projector onto a 360 degree screen in an online-feedback loop. This “head-fixed” stimulation gives the experimenter absolute control over the visual input and evoked (as well as spontaneous) motor-output can be recorded with millisecond precision and high spatial resolution. A dataset acquired by this method is shown in the right panel. Larval zebrafish will track virtual prey-like stimuli projected 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 elicit a reliable turning away from the stimulus and projection pattern akin to whole field motion (20 degrees) elicit optomotor-like behavior. These are important findings that tell us which parameters in visual stimulus space get mapped onto distinct output patterns. Experiments to study the role of different brain regions in these transformations are currently under way using 2-photon microscopy and transgenic fish-lines that express genetically encoded calcium indicators in all neurons. However, a first examination of the processing of these stimuli in the optic tectum - one of the first processing stations after the retina - was undertaken using bulk labeling by bolus injection of a calcium indicator conjugated to AM-esther. By combining two-photon imaging, binocular motion stimuli and artificial re-wiring of retinofugal projections we could show that this nucleus serves as a module to extract direction selective information from prey like stimuli ¹². This is particularly interesting since it is strongly reminiscent of higher cortical function in mammalian preparations while in lower vertebrates the retina has been thought to be responsible for 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 engine, coupled with a custom real-time CCD-based object tracking system, which allows the online association of zebrafish behaviour and stimulus presentation. Right panel – responses of a zebrafish larva to moving spots of varying sizes. Spots appear at frame zero straight ahead of the fish and are moved at constant speed for the next 100 frames either to the left or the right. Fish respond with whole body tracking motion to spots of 1 degree size and with distinct turns away from stimuli with a diameter of 5 degrees or larger.

Somatosensory stimuli, targeted stimulation and escape

A 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 in this behavior we have generated transgenic fish lines expressing Channelrhodopsin2 (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 behavior under scrutiny. A first study employing this technique probed the role of trigeminal and Rohon-Beard neurons in the context of escape behaviors in the larval zebrafish. Here we could show that single, ChR2 evoked action potentials in individual sensory neurons are sufficient to trigger complex escape reflexes 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 circuits.

Figure 3: (Left panel) Channelrhodopsin expression and bahvioral stimulation.  (Right panel) Channelrhodopsin elicits single spikes.  See Douglass et al., 2008

Current and future projects – recording in freely moving animals

In order to examine response properties of specific neuronal subpopulation in freely swimming larvae we have developed an imaging technique based on the bioluminescence of Aquorin-GFP that does not require excitation light and can therefore operate on a zero background signal ¹⁷. This optical signal can be collected with large angle optics and detected as a one-dimensional temporal signal by a photon multiplier tube. Figure 3 shows data from a first series of experiments in which Aquorin is expressed in the hypocretin system, a small nucleus consisting of less than 20 neurons that are known to regulate the sleep-wake cycle.

The left side shows an in-vivo two-photon image of the transgenic fish and a diagram of the set-up. On the right side bioluminescence photon counts from the same fish are shown in green below traces describing the simultaneously monitored behavior of the fish in black. These data illustrate the feasibility of recording neuronal activity in a small set of identified neurons in a freely behaving animal with high temporal resolution and over many hours.        

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