The authors would like to thank Xixia Luo for excellent fish husbandry and technical support. This work was funded by the National Institutes of Health grants R21EY019401 (RT) and P30EY04068 (RT), and start-up funds to RT, including an unrestricted grant from Research to Prevent Blindness to the Wayne State University, Department of Ophthalmology. JT was supported by a Thomas C. Rumble Fellowship provided by the Wayne State University Graduate School.

All procedures described in this protocol were approved by the animal use committee at Wayne State University School of Medicine.
1. Dark Adaptation
<ol>
	<li>Transfer ~10 adult albino or pigmented fish from the normal housing system into a dark enclosure. If available, use a dark enclosure that is built into the zebrafish housing module, which allows for normal water flow through the tank. (If such a system is not available, place the fish tank in a completely dark enclosure, making sure to aerate the fish with oxygen).</li>
	<li>Keep the fish in the dark for 10 days. When feeding the animals or adding new fish to the dark box, make sure to move as quickly as possible to avoid exposing the fish to a long period of light.</li>
</ol>
2. UV Light Exposure
<ol>
	<li>Make sure the power to the UV source is OFF. Remove the UV light filament from fluorescent stereomicroscope.
	<ol>
		<li>Use an inverted 15 cm diameter glass Petri dish (or something of a similar 2 cm height) as a stand for the UV filament. Tape the Petri dish to the lab bench.</li>
		<li>Tape the UV light filament to top of the inverted Petri dish. Arrange so that ~2 mm of the end of the filament overhangs the Petri dish.</li>
	</ol>
	</li>
	<li>Obtain a 250 ml glass beaker. Cover &#189; of the bottom, sides, and back of the beaker with aluminum foil, making sure the &#34;shiny&#34; side of the foil faces the interior of the beaker. If the beaker is graduated, cover the worded half of the beaker with foil, leaving the clear half of the beaker exposed.</li>
	<li>Fill the 250 ml beaker with 100 ml of water from the fish facility system.</li>
	<li>Place the 250 ml beaker in a 4 L beaker. Fill the 4 L beaker with water until the water level is even with the 100 ml water line in the 250 ml beaker.</li>
	<li>Add a maximum of 10 dark-treated animals to the 250 ml beaker. Cover the 250 ml beaker with a small piece of aluminum foil.</li>
	<li>Place the entire beaker apparatus immediately adjacent to the UV filament. The filament should be touching the outside of the 4 L beaker and facing the exposed portion of the 250 ml beaker. Make sure that the 250 ml beaker is centered in the bottom of the 4 L beaker.</li>
	<li>Position a large, opaque screen behind the 4L beaker, allowing for the animals to be exposed, but preventing any lab personnel from seeing the tip of the UV filament. WARNING: make sure this barrier is in place BEFORE turning on the power to the UV source.</li>
	<li>Turn on the UV power source. Set timer for 30 min. Place whatever necessary warning labels to ensure that unsuspecting lab personnel do not accidentally expose themselves to UV radiation.</li>
	<li>After 30 min, shut off the UV power source. Remove the 250 ml beaker with the fish. NOTE: if multiple rounds of UV exposure are required, let the power source cool down (~20 min) before repeating the exposure protocol. Make sure to replace the 100 ml of water with fresh system water for each exposure. The water in the 4 L beaker does not need to be replaced.</li>
</ol>
3. Halogen Lamp Light Exposure
<ol>
	<li>Transfer the fish from 250 ml beaker into a 1.8 L clear acrylic fish tank. Fill the tank to the overflow mark.</li>
	<li>Set up the halogen lamp light treatment area. Obtain four 250 W halogen utility lamps from a local hardware store. Facing two lamps in the same direction, arrange 29 cm apart on center. Arrange the other two lamps in a similar fashion.
	<ol>
		<li>Place the second set of lamps so that they are facing the first set of lamps, leaving ~73 cm in between the two sets of lamps. This creates a rectangle-shaped light treatment area of 29 x 73 cm.</li>
	</ol>
	</li>
	<li>Obtain a small fan from a local hardware store. Place the fan equidistant between the two sets of lamps, just outside of the light treatment area.</li>
	<li>Place two 1.8 L tanks full of water in the center of the light treatment area, equidistant between the two sets of lamps. The distance between the lamps and the outside wall of the tank should be ~29 cm. Note: even if only treating 10 fish in one 1.8 L tank, use this arrangement. Both tanks are needed to keep the water temperature of each tank within the appropriate range.</li>
	<li>Place an oxygen aerator in each tank.</li>
	<li>Cover each tank with clear acrylic lids, leaving a ~2 cm gap at the end closest to the fan. Arrange the fan so that it will blow air into this gap. Place a thermometer in one or both of the tanks.</li>
	<li>Turn on the power to the lights, fan, and aerators. Maintain light treatment for up to 4 days. The fish should not be fed during the light treatment, as this will affect water quality and result in more stress to the animals.</li>
	<li>Monitor temperature and water level on a daily basis. Maintain the temperature at 30-33 &#176;C. If necessary, adjust the fan speed and/or the distance between the tanks and the lights.
	<ol>
		<li>Top off each tank with system water as needed, usually daily.</li>
		<li>Always use healthy adult zebrafish (typically 6-9 months of age) to maintain a survival rate of near 100%. However, if a fish is found dead in the tank, remove it immediately and replace the water in the entire tank.</li>
	</ol>
	</li>
</ol>
4. Tissue Collection
<ol>
	<li>48 hr after the onset of the halogen light treatment remove the fish from the treatment area.
	<ol>
		<li>Prepare fresh ethanolic formaldehyde fixative (1 part 37% formaldehyde, 9 parts 100% ethanol).</li>
		<li>Euthanize zebrafish with an anesthetic overdose of 2-phenoxyethanol (0.4 mg/L).</li>
		<li>Transfer the euthanized zebrafish to a paper towel. Enucleate the eye using curved forceps.</li>
		<li>Place enucleated eyes in fixative and store O/N at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Cryoprotect the eyes.
	<ol>
		<li>Wash the eyes in 5% sucrose 1x PBS for 30 min at room temperature, then replace with fresh 5% sucrose 1x PBS for 2 hr. Next, wash the eyes in 30% sucrose 1x PBS O/N at 4 &#176;C. Wash the eyes in a 1:1 (equal portions) of tissue freezing medium and 30% sucrose 1x PBS O/N at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Embed the eyes in 100% tissue freezing medium and store at -80 &#176;C. Orient the eyes so that cryosectioning of the tissue is performed on the dorsal/ventral axis.</li>
	<li>Cryosection the tissue and place on glass slides. Warm slides for 2 hr at 55 &#176;C. Store slides at -80 &#176;C or immediately perform standard immunohistochemistry.</li>
	<li>Perform standard immunohistochemistry on sectioned tissue and image with fluorescent microscopy40,51.</li>
</ol>

The authors have nothing to disclose.

Here we show that combining a short UV exposure with a continual bright light exposure results in widespread photoreceptor loss and a robust regeneration response. Compared with the individual LIRD methods, this combined method is also the most effective protocol to damage both rods and cones in both halves of the retina. Importantly, this treatment is effective in pigmented animals as well as albino animals.
Although we provide evidence that the combined protocol results in more wide...

Light-induced retinal degeneration (LIRD) is commonly used in both rodents and zebrafish to damage rod and cone photoreceptors. In adult zebrafish, photoreceptor degeneration triggers M&#252;ller glial cells to re-enter the cell cycle and produce transient-amplifying progenitors. These progenitors continue to proliferate as they migrate to the damaged area, where they ultimately give rise to new photoreceptors. Currently, there are two widely-used LIRD paradigms, each of which results in varying degrees of photoreceptor loss and corresponding differences in the regeneration response. As more genetic and pharmacological tools are available to test the role of individual genes of interest during regeneration, there is a need to develop a robust LIRD paradigm. Here we describe a LIRD protocol that results in widespread and consistent loss of both rod and cone photoreceptors in which we have combined the use of two previously established LIRD techniques. Furthermore, this protocol can be extended for use in pigmented animals, which eliminates the need to maintain transgenic lines of interest on the albino background for LIRD studies.

<table border="0" cellpadding="0" cellspacing="0" width="586">
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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">UV light source</td>
			<td style="width:215px;">Leica</td>
			<td style="width:155px;">EL600</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass Petri dish (150 x 20 mm)</td>
			<td>Sigma-Aldrich/Pyrex</td>
			<td>CLS3160152BO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">250 ml glass beaker</td>
			<td>Sigma-Aldrich/Pyrex</td>
			<td>CLS1000250</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4 L glass beaker</td>
			<td>Sigma-Aldrich/Pyrex</td>
			<td>CLS10004L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aluminum foil</td>
			<td>Fisher</td>
			<td>01-213-105</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">250 W halogen lamps</td>
			<td>Workforce</td>
			<td>265-669</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.8 L clear acrylic tanks</td>
			<td>Aquaneering</td>
			<td>ZT180T</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.8 L clear acrylic tank lids</td>
			<td>Aquaneering</td>
			<td>ZT180LCL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fan</td>
			<td>Honeywell</td>
			<td>HT-900</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aerator</td>
			<td>Tetra</td>
			<td>77853-900</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermometer</td>
			<td>Cole-Parmer</td>
			<td>YO-08008-58</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bent forceps (5/45)</td>
			<td>World Precision Instruments</td>
			<td>504155</td>
		</tr>
	</tbody>
</table>

a novel light damage paradigm for use in retinal regeneration studies in adult zebrafish

Light-induced retinal degeneration (LIRD) is commonly used in both rodents and zebrafish to damage rod and cone photoreceptors. In adult zebrafish, photoreceptor degeneration triggers M&#252;ller glial cells to re-enter the cell cycle and produce transient-amplifying progenitors. These progenitors continue to proliferate as they migrate to the damaged area, where they ultimately give rise to new photoreceptors. Currently, there are two widely-used LIRD paradigms, each of which results in varying degrees of photoreceptor loss and corresponding differences in the regeneration response. As more genetic and pharmacological tools are available to test the role of individual genes of interest during regeneration, there is a need to develop a robust LIRD paradigm. Here we describe a LIRD protocol that results in widespread and consistent loss of both rod and cone photoreceptors in which we have combined the use of two previously established LIRD techniques. Furthermore, this protocol can be extended for use in pigmented animals, which eliminates the need to maintain transgenic lines of interest on the albino background for LIRD studies.

The heretofore described light treatment protocol was compared to each individual method of LIRD. In dark-treated adult albino animals (Figures&#160;3-5), the individual light treatments resulted in significant loss of rod (Figure&#160;3) and cone (Figure&#160;4) photoreceptors. However, both individual treatments primarily damaged photoreceptors in the dorsal half of the retina, leaving the ventral retina relatively protected from the light treatments (...

Watch this Scientific Journal Video about A Novel Light Damage Paradigm for Use in Retinal Regeneration Studies in Adult Zebrafish at JoVE.com

A Novel Light Damage Paradigm for Use in Retinal Regeneration Studies in Adult Zebrafish

Multiple light damage protocols have been described to damage photoreceptors and consequently induce a retinal regeneration response in adult zebrafish. This protocol describes an improved method that can be used in pigmented animals and that damages the vast majority of rod and cone photoreceptors across the entire retina.

Light-induced retinal degeneration (LIRD) is commonly used in both rodents and zebrafish to damage rod and cone photoreceptors. In adult zebrafish, ...

a-novel-light-damage-paradigm-for-use-retinal-regeneration-studies

Research

JoVE Journal

Neuroscience

10.1K Views.  Wayne State University School of Medicine. Multiple light damage protocols have been described to damage photoreceptors and consequently induce a retinal regeneration response in adult zebrafish. This protocol describes an improved method that can be used in pigmented animals and that damages the vast majority of rod and cone photoreceptors across the entire retina.

Video: A Novel Light Damage Paradigm for Use in Retinal Regeneration Studies in Adult Zebrafish

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Design and Assembly of an Ultra-light Motorized Microdrive for Chronic Neural Recordings in Small Animals

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of neural circuits underlying a variety of natural behaviors, including navigation1 , decision making 2,3, and the generation of complex motor sequences4,5,6. Advances in precision machining has allowed for the fabrication of light-weight devices suitable for chronic recordings in small animals, such as mice and songbirds. The ability to adjust the electrode position with small remotely controlled motors has further increased the recording yield in various behavioral contexts by reducing animal handling.6,7
 Here we describe a protocol to build an ultra-light motorized microdrive for long-term chronic recordings in small animals. Our design evolved from an earlier published version7, and has been adapted for ease-of use and cost-effectiveness to be more practical and accessible to a wide array of researchers. This proven design 8,9,10,11 allows for fine, remote positioning of electrodes over a range of ~ 5 mm and weighs less than 750 mg when fully assembled. We present the complete protocol for how to build and assemble these drives, including 3D CAD drawings for all custom microdrive components.

The design, fabrication and assembly of an ultra-light motorized microdrive is described. The device provides a cost-effective and easy-to-use solution for chronic recordings of single units in small behaving animals.

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of ...

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, including rats, hamsters, and mice, are active during the night and relatively inactive during the day. Many other behavioral and physiological measures also exhibit daily rhythms, but in rodents, running-wheel activity serves as a particularly reliable and convenient measure of the output of the master circadian clock, the suprachiasmatic nucleus (SCN) of the hypothalamus. In general, through a process called entrainment, the daily pattern of running-wheel activity will naturally align with the environmental light-dark cycle (LD cycle; e.g. 12 hr-light:12 hr-dark). However circadian rhythms are endogenously generated patterns in behavior that exhibit a ~24 hr period, and persist in constant darkness. Thus, in the absence of an LD cycle, the recording and analysis of running-wheel activity can be used to determine the subjective time-of-day. Because these rhythms are directed by the circadian clock the subjective time-of-day is referred to as the circadian time (CT). In contrast, when an LD cycle is present, the time-of-day that is determined by the environmental LD cycle is called the zeitgeber time (ZT).
Although circadian rhythms in running-wheel activity are typically linked to the SCN clock6-8, circadian oscillators in many other regions of the brain and body9-14 could also be involved in the regulation of daily activity rhythms. For instance, daily rhythms in food-anticipatory activity do not require the SCN15,16 and instead, are correlated with changes in the activity of extra-SCN oscillators17-20. Thus, running-wheel activity recordings can provide important behavioral information not only about the output of the master SCN clock, but also on the activity of extra-SCN oscillators. Below we describe the equipment and methods used to record, analyze and display circadian locomotor activity rhythms in laboratory rodents.

Circadian rhythms in voluntary wheel-running activity in mammals are tightly coupled to the molecular oscillations of a master clock in the brain. As such, these daily rhythms in behavior can be used to study the influence of genetic, pharmacological, and environmental factors on the functioning of this circadian clock.

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, ...

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs survival and regeneration experiments. Many studies have been performed in mammalian animals to research RGCs survival after optic nerve injury. However, retrograde labeling of RGCs in adult zebrafish has not yet been reported, though some alternative methods can count cell numbers in retinal ganglion cell layers (RGCL). Considering the small size of the adult zebrafish skull and the high risk of death after drilling on the skull, we open the skull with the help of acid-etching and seal the hole with a light curing bond, which could significantly improve the survival rate. After absorbing the dyes for 5 days, almost all the RGCs are labeled. As this method does not need to transect the optic nerve, it is irreplaceable in the research of RGCs survival after optic nerve crush in adult zebrafish. Here, we introduce this method step by step and provide representative results.

We introduce an efficient method to retrograde label retinal ganglion cells (RGCs) in adult zebrafish.

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs ...

The Use of Trace Eyeblink Classical Conditioning to Assess Hippocampal Dysfunction in a Rat Model of Fetal Alcohol Spectrum Disorders

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain undergoes rapid organizational change and is similar to accelerated brain changes that occur during the third trimester in humans. This model of fetal alcohol spectrum disorders (FASDs) produces severe brain damage, mimicking the amount and pattern of binge-drinking that occurs in some pregnant alcoholic mothers. We describe the use of trace eyeblink classical conditioning (ECC), a higher-order variant of associative learning, to assess long-term hippocampal dysfunction that is typically seen in alcohol-exposed adult offspring. At 90 days of age, rodents were surgically prepared with recording and stimulating electrodes, which measured electromyographic (EMG) blink activity from the left eyelid muscle and delivered mild shock posterior to the left eye, respectively. After a 5 day recovery period, they underwent 6 sessions of trace ECC to determine associative learning differences between alcohol-exposed and control rats. Trace ECC is one of many possible ECC procedures that can be easily modified using the same equipment and software, so that different neural systems can be assessed. ECC procedures in general, can be used as diagnostic tools for detecting neural pathology in different brain systems and different conditions that insult the brain.

Trace eyeblink classical conditioning (ECC) was used to assess hippocampal-dependent associative learning in adult rats that were administered a high concentration (11.9% v/v) of alcohol during early neonatal brain development. In general, ECC procedures are sound diagnostic tools for detecting brain dysfunction across many psychological and biomedical settings.

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain ...

Modeling Amyloid-&#946;42 Toxicity and Neurodegeneration in Adult Zebrafish Brain

Alzheimer's disease (AD) is a debilitating neurodegenerative disease in which accumulation of toxic amyloid-&#946;42 (A&#946;42) peptides leads to synaptic degeneration, inflammation, neuronal death, and learning deficits. Humans cannot regenerate lost neurons in the case of AD in part due to impaired proliferative capacity of the neural stem/progenitor cells (NSPCs) and reduced neurogenesis. Therefore, efficient regenerative therapies should also enhance the proliferation and neurogenic capacity of NSPCs. Zebrafish (Danio rerio) is a regenerative organism, and we can learn the basic molecular programs with which we could design therapeutic approaches to tackle AD. For this reason, the generation of an AD-like model in zebrafish was necessary. Using our methodology, we can introduce synthetic derivatives of A&#946;42 peptide with tissue penetrating capability into the adult zebrafish brain, and analyze the disease pathology and the regenerative response. The advantage over the existing methods or animal models is that zebrafish can teach us how a vertebrate brain can naturally regenerate, and thus help us to treat human neurodegenerative diseases better by targeting endogenous NSPCs. Therefore, the amyloid-toxicity model established in the adult zebrafish brain may open new avenues for research in the field of neuroscience and clinical medicine. Additionally, the simple execution of this method allows for cost-effective and efficient experimental assessment. This manuscript describes the synthesis and injection of A&#946;42 peptides into zebrafish brain.

This protocol describes the synthesis, characterization, and injection of monomeric amyloid-&#946;42 peptides for generating amyloid toxicity in adult zebrafish to establish an Alzheimer&#39;s disease model, followed by histological analyses and detection of aggregations.

Alzheimer's disease (AD) is a debilitating neurodegenerative disease in which accumulation of toxic amyloid-&#946;42 (A&#946;42) peptides leads to ...

M&#252;ller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after severe damage. Investigating the regeneration pathways in zebrafish might bring new insights to develop innovative strategies for the treatment of retinal degenerative diseases in mammals. Herein, we focused on the induction of a focal lesion to the outer retina in adult zebrafish by means of a 532 nm diode laser. A localized injury allows investigating biological processes that take place during retinal degeneration and regeneration directly at the area of damage. Using non-invasive optical coherence tomography (OCT), we were able to define the location of the damaged area and monitor subsequent regeneration in vivo. Indeed, OCT imaging produces high-resolution, cross-sectional images of the zebrafish retina, providing information which was previously only available with histological analyses. In order to confirm the data from real-time OCT, histological sections were performed and regenerative response after the induction of the retinal injury was investigated by immunohistochemistry.

The zebrafish is a popular animal model to study mechanisms of retinal degeneration/regeneration in vertebrates. This protocol describes a method to induce localized injury disrupting the outer retina with minimal damage to the inner retina. Subsequently, we monitor in vivo the retinal morphology and the M&#252;ller glia response throughout retinal regeneration.

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after ...

Preparing Fresh Retinal Slices from Adult Zebrafish for Ex Vivo Imaging Experiments

The retina is a complex tissue that initiates and integrates the first steps of vision. Dysfunction of retinal cells is a hallmark of many blinding diseases, and future therapies hinge on fundamental understandings about how different retinal cells function normally. Gaining such information with biochemical methods has proven difficult because contributions of particular cell types are diminished in the retinal cell milieu. Live retinal imaging can provide a view of numerous biological processes on a subcellular level, thanks to a growing number of genetically encoded fluorescent biosensors. However, this technique has thus far been limited to tadpoles and zebrafish larvae, the outermost retinal layers of isolated retinas, or lower resolution imaging of retinas in live animals. Here we present a method for generating live ex vivo retinal slices from adult zebrafish for live imaging via confocal microscopy. This preparation yields transverse slices with all retinal layers and most cell types visible for performing confocal imaging experiments using perfusion. Transgenic zebrafish expressing fluorescent proteins or biosensors in specific retinal cell types or organelles are used to extract single-cell information from an intact retina. Additionally, retinal slices can be loaded with fluorescent indicator dyes, adding to the method&#39;s versatility. This protocol was developed for imaging Ca2+ within zebrafish cone photoreceptors, but with proper markers it could be adapted to measure Ca2+ or metabolites in M&#252;ller cells, bipolar and horizontal cells, microglia, amacrine cells, or retinal ganglion cells. The retinal pigment epithelium is removed from slices so this method is not suitable for studying that cell type. With practice, it is possible to generate serial slices from one animal for multiple experiments. This adaptable technique provides a powerful tool for answering many questions about retinal cell biology, Ca2+, and energy homeostasis.

Preparing Fresh Retinal Slices from Adult Zebrafish for Ex Vivo Imaging Experiments

Imaging retinal tissue can provide single-cell information that cannot be gathered from traditional biochemical methods. This protocol describes preparation of retinal slices from zebrafish for confocal imaging. Fluorescent genetically encoded sensors or indicator dyes allow visualization of numerous biological processes in distinct retinal cell types.

The retina is a complex tissue that initiates and integrates the first steps of vision. Dysfunction of retinal cells is a hallmark of many blinding ...

Ablation of a Neuronal Population Using a Two-photon Laser and Its Assessment Using Calcium Imaging and Behavioral Recording in Zebrafish Larvae

To identify the role of a subpopulation of neurons in behavior, it is essential to test the consequences of blocking its activity in living animals. Laser ablation of neurons is an effective method for this purpose when neurons are selectively labeled with fluorescent probes. In the present study, protocols for laser ablating a subpopulation of neurons using a two-photon microscope and testing of its functional and behavioral consequences are described. In this study, prey capture behavior in zebrafish larvae is used as a study model. The pretecto-hypothalamic circuit is known to underlie this visually-driven prey catching behavior. Zebrafish pretectum were laser-ablated, and neuronal activity in the inferior lobe of the hypothalamus (ILH; the target of the pretectal projection) was examined. Prey capture behavior after pretectal ablation was also tested.

Here, we present a protocol to ablate a genetically labeled subpopulation of neurons by a two-photon laser from Zebrafish larvae.

To identify the role of a subpopulation of neurons in behavior, it is essential to test the consequences of blocking its activity in living animals. Laser ...

A Novel In Vitro Model of Blast Traumatic Brain Injury

Traumatic brain injury is a leading cause of death and disability in military and civilian populations. Blast traumatic brain injury results from the detonation of explosive devices, however, the mechanisms that underlie the brain damage resulting from blast overpressure exposure are not entirely understood and are believed to be unique to this type of brain injury. Preclinical models are crucial tools that contribute to better understand blast-induced brain injury. A novel in vitro blast TBI model was developed using an open-ended shock tube to simulate real-life open-field blast waves modelled by the Friedlander waveform. C57BL/6N mouse organotypic hippocampal slice cultures were exposed to single shock waves and the development of injury was characterized up to 72 h using propidium iodide, a well-established fluorescent marker of cell damage that only penetrates cells with compromised cellular membranes. Propidium iodide fluorescence was significantly higher in the slices exposed to a blast wave when compared with sham slices throughout the duration of the protocol. The brain tissue injury is very reproducible and proportional to the peak overpressure of the shock wave applied.

A Novel In Vitro Model of Blast Traumatic Brain Injury

This paper describes a novel model of primary blast traumatic brain injury. A compressed-air driven shock tube is used to expose in vitro mouse hippocampal slice cultures to a single shock wave. This is a simple and rapid protocol generating a reproducible brain tissue injury with a high throughput.

Traumatic brain injury is a leading cause of death and disability in military and civilian populations. Blast traumatic brain injury results from the ...