We especially thank Timo Fritsch for excellent animal care and Hermann D&#246;ring, Mohamed Elsaey, Sol Pose-M&#233;ndez, Jakob von Trotha, Komali Valishetti and Barbara Winter for their helpful support. We are also grateful to all the other members of the K&#246;ster lab for their feedback. The project was funded in part by the German Research Foundation (DFG, KO1949/7-2) project 241961032 (to RWK) and the Bundesministerium f&#252;r Bildung und Forschung (BMBF; Era-Net NEURON II CIPRESS project 01EW1520 to JCM) is acknowledged.

All animal work described here is in accordance with legal regulations (EU-Directive 2010/63). Maintenance and handling of fish have been approved by local authorities and by the animal welfare representative of the Technische Universit&#228;t Braunschweig.
1. Preparation of artificial cerebro spinal fluid (ACSF), low melting agarose and sharp glass needles
<ol>
	<li>Prepare the ACSF by dissolving the listed chemicals at following concentrations in distilled water. 134 mM NaCl (58.44 g/mol),...

<ol>
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The presented method provides an alternative approach to brain isolation or the treatment of zebrafish larvae with pharmaceuticals inhibiting pigmentation for recording high resolution images of neurons in their in vivo environment. The quality of images recorded with this method is comparable to images from explanted brains, yet under natural conditions.
Furthermore, a loss in intensity of fluorescence is avoided, because there is no need for treatment with fixatives<sup class="xref"...

Over the recent decades, the zebrafish (Danio rerio) has evolved as one of the most popular vertebrate model organisms for embryonic and larval developmental studies. The large fecundity of zebrafish females coupled with the rapid ex utero development of the embryo and its transparency during early embryonic developmental stages are just a few key factors that make zebrafish a powerful model organism to adress developmental questions1. Advances in molecular genetic technologies combined with high resolution in vivo imaging studies allowed for addressing cell biological mechanisms underlying developmental processes2. In particular, in the field of neuronal differentiation, physiology, connectivity, and function, zebrafish has shed light on the interplay of molecular dynamics, brain functions and organismic behavior in unprecedented detail.
Yet, most of these studies are restricted to embryonic and early larval stages during the first week of development as transparency of the nervous system tissue is progressively lost. At these stages, brain tissue is prevented from access by high resolution microscopy approaches becoming shielded by skull differentiation and pigmentation3.
Therefore, key questions of neuronal differentiation, maturation, and plasticity such as the refinement of neuronal connectivity or synaptic scaling are difficult to study. These cellular processes are important in order to define cellular mechanisms driving, for example, social behavior, decision making, or motivation-based behavior, areas to which zebrafish research on several weeks&#39; old larvae has recently contributed key findings based on behavioral studies4.
Pharmacological approaches to inhibit pigmentation in zebrafish larvae for several weeks are barely feasible or may even cause detrimental effects5,6,7,8. Double or triple mutant strains with specific pigmentation defects, such as casper9 or crystal10, have become tremendously valuable tools, but are laborious in breeding, provide few offspring, and pose the danger of accumulating genetic malformations due to excessive inbreeding.
Here, a minimal invasive procedure as an alternative is provided that is applicable to any zebrafish strain. This procedure was adapted from electrophysiological studies to record neuronal activity in living and awake zebrafish larvae. In teleosts, skin and soft skull cartilage can be carefully removed by micro-peeling these layers, because they are not tightly interwoven with the brain vasculature. This allows for exposing brain tissue containing neurons and axonal tracts without damage and for recording neuronal morphology, including synaptic structures and their molecular contents, which in turn include the observation of physiological changes such as Ca2+ transients or intracellular transport events for up to several hours. Moreover, beyond descriptive characterizations, the direct access to brain tissue enables interrogation of mature neuronal functions by means of neuropharmacological substance administration and optogenetic approaches. Therefore, true structure function relationships can be revealed in the juvenile zebrafish brain using this brain exposure strategy.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Calcium chloride</td>
			<td>Roth</td>
			<td>A119.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Laser scanning microscope</td>
			<td>Leica</td>
			<td>TCS SP8</td>
			<td></td>
		</tr>
		<tr>
			<td>d-Glucose</td>
			<td>Sigma</td>
			<td>G8270-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>d-Tubocurare</td>
			<td>Sigma-Aldrich</td>
			<td>T2379-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Capillary type 1</td>
			<td>WPI</td>
			<td>1B150F-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Capillary type 2</td>
			<td>Harvard Apparatus</td>
			<td>GC100F-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Coverslip</td>
			<td>deltalab</td>
			<td>D102424</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Roth</td>
			<td>9105.4</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Invitrogen (Thermo Fischer)</td>
			<td>H3570</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaging chamber</td>
			<td>Ibidi</td>
			<td>81156</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Normapur</td>
			<td>26764298</td>
			<td></td>
		</tr>
		<tr>
			<td>LM-Agarose</td>
			<td>Condalab</td>
			<td>8050.55</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride (Hexahydrate)</td>
			<td>Roth</td>
			<td>A537.4</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Camera</td>
			<td>Leica</td>
			<td>DFC9000 GTC</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle-Puller type 1</td>
			<td>NARISHIGE</td>
			<td>Model PC-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle-Puller type 2</td>
			<td>Sutter Instruments</td>
			<td>Model P-2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur-Pipettes 3ml</td>
			<td>A.Hartenstein</td>
			<td>20170718</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Roth</td>
			<td>P029.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Normapur</td>
			<td>28244262</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricain</td>
			<td>Sigma-Aldrich</td>
			<td>E10521-50G</td>
			<td></td>
		</tr>
		<tr>
			<td>Waterbath</td>
			<td>Phoenix Instrument</td>
			<td>WB-12</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm petri dish</td>
			<td>Sarstedt</td>
			<td>833900</td>
			<td></td>
		</tr>
		<tr>
			<td>90 mm petri dish</td>
			<td>Sarstedt</td>
			<td>821473001</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo imaging of fully active brain tissue in awake zebrafish larvae and juveniles by skull and skin removal

Understanding the ephemeral changes that occur during brain development and maturation requires detailed high-resolution imaging in space and time at cellular and subcellular resolution. Advances in molecular and imaging technologies have allowed us to gain numerous detailed insights into cellular and molecular mechanisms of brain development in the transparent zebrafish embryo. Recently, processes of refinement of neuronal connectivity that occur at later larval stages several weeks after fertilization, which are for example control of social behavior, decision making or motivation-driven behavior, have moved into focus of research. At these stages, pigmentation of the zebrafish skin interferes with light penetration into brain tissue, and solutions for embryonic stages, e.g., pharmacological inhibition of pigmentation, are not feasible anymore.
Therefore, a minimally invasive surgical solution for microscopy access to the brain of awake zebrafish is provided that is derived from electrophysiological&#160;approaches. In teleosts, skin and soft skull cartilage can be carefully removed by micro-peeling these layers, exposing underlying neurons and axonal tracts without damage. This allows for recording neuronal morphology, including synaptic structures and their molecular contents, and the observation of physiological changes such as Ca2+ transients or intracellular transport events. In addition, interrogation of these processes by means of pharmacological inhibition or optogenetic manipulation is feasible. This brain exposure approach provides information about structural and physiological changes in neurons as well as the correlation and interdependence of these events in live brain tissue in the range of minutes or hours. The technique is suitable for in vivo brain imaging of zebrafish larvae up to 30 days post fertilization, the latest developmental stage tested so far. It, thus, provides access to such important questions as synaptic refinement and scaling, axonal and dendritic transport, synaptic targeting of cytoskeletal cargo or local activity-dependent expression. Therefore, a broad use for this mounting and imaging approach can be anticipated.

Figure 3A,C show a 14 dpf larva of the transgenic line Tg[-7.5Ca8:GFP]bz12[15] with the skull still intact. The pigment cells in the overlaying skin are distributed all over the head and are interfering with the fluorescence signal in the region of interest (here, cerebellum). With the larva in this condition, it is not possible to obtain high resolution images of the brain. F...

Watch this Scientific Journal Video about In vivo Imaging of Fully Active Brain Tissue in Awake Zebrafish Larvae and Juveniles by Skull and Skin Removal at JoVE.com

In vivo Imaging of Fully Active Brain Tissue in Awake Zebrafish Larvae and Juveniles by Skull and Skin Removal

Here we present a method to image the zebrafish embryonic brain in vivo upto larval and juvenile stages. This microinvasive procedure, adapted from electrophysiological approaches, provides access to cellular and subcellular details of mature neuron and can be combined with optogenetics and neuropharmacological studies for characterizing brain function and drug intervention.

Understanding the ephemeral changes that occur during brain development and maturation requires detailed high-resolution imaging in space and time at ...

This method allows the visualization of Zebrafish brain in later larval stages to allow the observation of the neuronal architecture in a highly detailed fashion in vivo that has previously not been possible. Pigment cells, which emerged during later larval development and prevents the brain from clear imaging, are no problem with this method because they are simply removed. With the method, it should be possible to study processes that occur at later stages during larval development of the brain, processes that impact on synaptic plasticity, degeneration of neurons or their regeneration.Before beginning the experiment use a micropipette puller to prepare sharp, thin, glass needles from glass capillaries. Next, use a plastic Pasteur pipette to collect larva into a 90 millimeter diameter Petri dish containing the appropriate solution. Transfer the selected larva to a 35 millimeter diameter Petri dish containing ACSF and place a 24 by 24 millimeter, square, glass cover slip into the Petri dish lid.Then aliquot the volume of ACSF needed for the experiment into an appropriate phial for oxygenation with carbogen. To embed the larva, use a Pasteur pipette to transfer the anesthetized larva to a mounting chamber under a stereomicroscope. If any larva are still able to move, do not use the fish for the experiment until the fish are completely unable to move.When the larva are immobile, carefully remove the excess medium and immediately add at least one milliliter of low melting agarose onto the larva. Orient the Zebrafish with the dorsal region facing up as close to the surface of the agarose as possible. If the larva will be imaged using an inverted microscope, after a solidifying, trim the agarose containing the larva into a small cuboid block.To expose the brain for imaging, trim away the excess agarose over the brain region of interest as necessary and use a glass needle to make a small incision through the skin near, but not over, the region of interest without penetrating too deeply into the tissue. Barely moving the needle just under the skin surface, continue to carefully make very small cuts around the region of interest until the skin over the region of interest can be removed or pushed aside. When the tissue has been removed from all the embryos, add a small drop of low melting agarose to the previously prepared imaging chamber of an inverted microscope and use a small spatula to flip the cuboid agarose block 180 degrees onto the agarose drop in the imaging chamber.When the agarose has solidified, fill the imaging chamber with fresh ACSF and begin imaging the larva. Here, an intact 14 days post-fertilization transgenic larva with the skull still intact can be observed. As shown, the pigment cells within the overlaying skin are distributed all over the head, interfering with the fluorescent signal in the region of interest.After open skull surgery, the area of interest becomes freely accessible for detailed high resolution imaging. The skin, skull, and or blood brain barrier also hinder the penetration of substances into the brain, as illustrated by the robust nuclear dye staining observed in these brain cells only after open skull surgery. These advantages are also observed at 30 days post-fertilization.In transgenic fish containing a fluorescent brain vasculature due to the expression of the red fluorescent protein, mCherry, in endothelial cells, color-coding at the depth values of the image stack demonstrates that even blood vessels deeply buried within the brain to nearly 250 microns are still clearly visible and traceable. When performing this procedure always keep in mind that this is a surgery in a living animal. Therefore, it requires respect and a focused mind.After this procedure, recording of the neuronal morphology, including synaptic structures, physiological and intracellular transport events can be performed in larvae older than seven days post-fertilization. With this technique, we hope to provide an approach that allows to study also cellular processes in the brain that occur at later larval stages, and that are involved in establishing, for example, such complex behaviors as social behavior or decision-making.

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Research

JoVE Journal

Neuroscience

4.5K Views.  Technische Universität Braunschweig. Questo metodo consente la visualizzazione del cervello di Zebrafish in fasi larvali successive per consentire l'osservazione dell'architettura neuronale in modo altamente dettagliato in vivo che in precedenza non era possibile. Le cellule pigmentate, che sono emerse durante il successivo sviluppo larvale e impediscono al cervello di avere immagini chiare, non sono un problema con questo metodo perché vengono semplicemente rimosse. Con il metodo, dovrebbe essere possibile studiare i processi ...