Final Report Summary - ZF OPTOGENETICS (Optogenetic analysis of neural circuits controlling behavior in zebrafish)
Project context and objectives
One of the most important questions in current neuroscience research is how groups of neurons in the brain drive behaviour. In order to answer this question we need to study a distinct behaviour, identify the neurons responsible for it, and then understand how the activity of these neurons is translated into movement. This is challenging task, because there is a huge gap between our knowledge at the cellular and behavioural levels. In this project, we propose to gain some insight into this problem by using an optogenetic forward screening assay in zebrafish. Optogenetic tools are proteins which, when inserted in neurons, and illuminated with the appropriate light wavelength, can control neuronal electrical activity with high precision. Zebrafish are easy to manipulate genetically, and therefore to create lines that express optogenetic tools. In addition, young zebrafish (larvae) are transparent, which allows the light used to stimulate the optogenetic tools to go easily into the brain, and at the same to perform optical measures of the neuronal activity.
Project work and results
Therefore, we created zebrafish lines expressing the protein Channelrhodosin-2 (that excites neurons when illuminated with blue light). We screened them for behavioural phenotypes, finding one that perform J-turns, (an orientation towards the prey behaviour) on blue light illumination, and then identified the neural circuit driving these behaviours.
We performed a simple behavioural test: we put zebrafish larvae into a 35-mm petri dishes, swimming freely, and illuminated the whole field with a blue LED while capturing the swimming behaviour with a video camera. Wild-type fish were startled by blue light illumination and increased transiently their swimming speed. We tested three zebrafish lines expressing channelrhodopsin: Dlx4/5:itTA/Tet07:ChR2YFP (which expresses ChR2 in telencephalic GABAergic interneurons), OMP:ChR2YFP(which expresses ChR2 in olfactory sensory neurons), and HuC:itTA/Tet07:ChR2YFP (which expresses ChR2 across the whole brain). When tested with blue light illumination, the two first lines behaved in a similar way to the wild type. HuC:itTA/Tet07:ChR2YFP, however, did not increase the swimming speed with blue light. A close look at the videos revealed that the fish moved backwards when illuminated with blue light. As this behaviour is not common in zebrafish, we decided to study it in more detail.
In order to understand how the fish moved backwards, we performed a detailed video analysis. We found that fish bent the tip of the tail unilaterally while keeping the upper part of the tail stiff. To further characterise this behaviour, we immobilised the fish by embedding it in agarose. In this condition, the fish can still bend the tip of the tail when illuminated with blue light. In addition, other characteristics, such as coordinated pectoral fins movement or convergent eye movements could be observed. All these characteristics are fully congruent with a behaviour already described in zebrafish, called J-turn. J-turns are a movement the fish uses to slowly orientate towards a prey. Therefore, channelrhodopsin-2 stimulation is triggering J-turns. Tracking the angle of the tail during this movement clearly shows that this is a distinct behaviour easily distinguishable from others such as forward swimming.
Next, we asked what brain region is triggering J-turns. First, we introduced agar embedded in fish in an epifluorescence microscope, and systematically illuminated regions in the brain while looking at the tail through the condenser. We found that the anterior part of the midbrain/diencephalon triggered J-turns, although this approach failed to give higher resolution. To achieve better resolution, we coupled optic fibres to a blue laser and performed the screening through the brain again. Using a 200-µm-diameter fibre pointed at the anterior midbrain/diencephalon as the region triggering J-turns. In addition, we realised that illuminating the right side of the brain produced tail bending to the left side and vice versa, meaning that this behaviour was fully lateralised. Probing with a 50-µm-diameter fibre, with the fibre perpendicular to the fish revealed two small places in each side of the brain that triggered J-turns to the contralateral side. If the optic fibre was turned to 45 degrees relative to the skin surface, the illumination point should be more lateral, meaning that the region triggering J-turns was deep in the brain.
To fully identify the group of cells triggering J-turns, we performed kaede photoconversion in HuC-Kaede fish. Kaede is a protein that normally emits green fluorescence and changes to red when illuminated with ultraviolet light. Illuminating the fish head with a 50-µm optic fibre coupled to an UV laser, both vertical and in 45 degrees relative to the fish head, shows a group of photoconverted cells lying in the anterior and ventral part of the optic tectum, and superficial pre-tectum. Optic tectum and superficial pre-tectum are brain regions processing visual information coming from the retina and then transmitting this information to the reticular formation for selection of the appropriate behaviour. Here we demonstrate that a small sub-region of these nuclei can selectively trigger J-turning, a distinct behaviour in zebrafish.
To study the neural activity triggering J-turning, we performed calcium imaging in an in-vitro preparation of zebrafish head with the eyes enucleated. We visualised calcium increases in response to blue-light stimulation in HuC:itTA/Tet07:ChR2YFP fish expressing channelrhodopsin ,but not in wild-type siblings. This method can therefore be applied to understand how sensory information is transformed into motor commands in the zebrafish tectum.
Contact Rainer Friedrich (rainer.friedrich@fmi.ch) and Otto Fajardo (otto.fajardo@fmi.ch) for more information.
One of the most important questions in current neuroscience research is how groups of neurons in the brain drive behaviour. In order to answer this question we need to study a distinct behaviour, identify the neurons responsible for it, and then understand how the activity of these neurons is translated into movement. This is challenging task, because there is a huge gap between our knowledge at the cellular and behavioural levels. In this project, we propose to gain some insight into this problem by using an optogenetic forward screening assay in zebrafish. Optogenetic tools are proteins which, when inserted in neurons, and illuminated with the appropriate light wavelength, can control neuronal electrical activity with high precision. Zebrafish are easy to manipulate genetically, and therefore to create lines that express optogenetic tools. In addition, young zebrafish (larvae) are transparent, which allows the light used to stimulate the optogenetic tools to go easily into the brain, and at the same to perform optical measures of the neuronal activity.
Project work and results
Therefore, we created zebrafish lines expressing the protein Channelrhodosin-2 (that excites neurons when illuminated with blue light). We screened them for behavioural phenotypes, finding one that perform J-turns, (an orientation towards the prey behaviour) on blue light illumination, and then identified the neural circuit driving these behaviours.
We performed a simple behavioural test: we put zebrafish larvae into a 35-mm petri dishes, swimming freely, and illuminated the whole field with a blue LED while capturing the swimming behaviour with a video camera. Wild-type fish were startled by blue light illumination and increased transiently their swimming speed. We tested three zebrafish lines expressing channelrhodopsin: Dlx4/5:itTA/Tet07:ChR2YFP (which expresses ChR2 in telencephalic GABAergic interneurons), OMP:ChR2YFP(which expresses ChR2 in olfactory sensory neurons), and HuC:itTA/Tet07:ChR2YFP (which expresses ChR2 across the whole brain). When tested with blue light illumination, the two first lines behaved in a similar way to the wild type. HuC:itTA/Tet07:ChR2YFP, however, did not increase the swimming speed with blue light. A close look at the videos revealed that the fish moved backwards when illuminated with blue light. As this behaviour is not common in zebrafish, we decided to study it in more detail.
In order to understand how the fish moved backwards, we performed a detailed video analysis. We found that fish bent the tip of the tail unilaterally while keeping the upper part of the tail stiff. To further characterise this behaviour, we immobilised the fish by embedding it in agarose. In this condition, the fish can still bend the tip of the tail when illuminated with blue light. In addition, other characteristics, such as coordinated pectoral fins movement or convergent eye movements could be observed. All these characteristics are fully congruent with a behaviour already described in zebrafish, called J-turn. J-turns are a movement the fish uses to slowly orientate towards a prey. Therefore, channelrhodopsin-2 stimulation is triggering J-turns. Tracking the angle of the tail during this movement clearly shows that this is a distinct behaviour easily distinguishable from others such as forward swimming.
Next, we asked what brain region is triggering J-turns. First, we introduced agar embedded in fish in an epifluorescence microscope, and systematically illuminated regions in the brain while looking at the tail through the condenser. We found that the anterior part of the midbrain/diencephalon triggered J-turns, although this approach failed to give higher resolution. To achieve better resolution, we coupled optic fibres to a blue laser and performed the screening through the brain again. Using a 200-µm-diameter fibre pointed at the anterior midbrain/diencephalon as the region triggering J-turns. In addition, we realised that illuminating the right side of the brain produced tail bending to the left side and vice versa, meaning that this behaviour was fully lateralised. Probing with a 50-µm-diameter fibre, with the fibre perpendicular to the fish revealed two small places in each side of the brain that triggered J-turns to the contralateral side. If the optic fibre was turned to 45 degrees relative to the skin surface, the illumination point should be more lateral, meaning that the region triggering J-turns was deep in the brain.
To fully identify the group of cells triggering J-turns, we performed kaede photoconversion in HuC-Kaede fish. Kaede is a protein that normally emits green fluorescence and changes to red when illuminated with ultraviolet light. Illuminating the fish head with a 50-µm optic fibre coupled to an UV laser, both vertical and in 45 degrees relative to the fish head, shows a group of photoconverted cells lying in the anterior and ventral part of the optic tectum, and superficial pre-tectum. Optic tectum and superficial pre-tectum are brain regions processing visual information coming from the retina and then transmitting this information to the reticular formation for selection of the appropriate behaviour. Here we demonstrate that a small sub-region of these nuclei can selectively trigger J-turning, a distinct behaviour in zebrafish.
To study the neural activity triggering J-turning, we performed calcium imaging in an in-vitro preparation of zebrafish head with the eyes enucleated. We visualised calcium increases in response to blue-light stimulation in HuC:itTA/Tet07:ChR2YFP fish expressing channelrhodopsin ,but not in wild-type siblings. This method can therefore be applied to understand how sensory information is transformed into motor commands in the zebrafish tectum.
Contact Rainer Friedrich (rainer.friedrich@fmi.ch) and Otto Fajardo (otto.fajardo@fmi.ch) for more information.