This work was supported by DZNE and the Helmholtz Association (VH-NG-1021), CRTD, TU Dresden (FZ-111, 043_261518), and DFG (KI1524/6) (C.K.); and by the Leibniz Association (SAW-2011-IPF-2) and BMBF (BioLithoMorphie 03Z2E512) (Y.Z.). We would also like to thank Ulrike Hofmann for peptide synthesis, and to Nandini Asokan, Prayag Murawala, and Elly Tanaka for help during filming the procedure.

This protocol is a standard procedure suggested by the EU guidelines (2010/63) and the European Society for Fish Models in Biology and Medicine (EuFishBioMed) in Karlsruhe Insitute of Technology (KIT). All methods described after here have been approved by the ethics commission (Landesdirektion Dresden; document number TVV-52/2015).
1. Preparation of A&#946;42 Peptide
<ol>
	<li>Synthesize peptides (see Table 1) using the standard 9-fluorenylmethoxycarbonyl (Fmoc) chemistry with 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronoiumhexafluorphosphate (HBTU) as the coupling reagent on an automated solid-phase peptide synthesizer52. The scale of the synthesis was 100 &#181;mol.</li>
	<li>Load the automated peptide synthesizer with 500 mg of the Fmoc-protected resin as the solid phase (loading capacity of 0.2 mmol/g). Load the dissolved Fmoc-protected amino acids at a concentration 0.5 M in the volume required and as calculated for the respective synthesizer. 
	NOTE: The calculations are made to enable the coupling of each Fmoc-protected amino acid twice with 5 times excess of each building block to the resin47.</li>
	<li>Dissolve the required reagents for peptide synthesis in dimethlyformamide (DMF). For example, prepare the activator, HBTU, at a concentration of 0.48 M, 45% v/v N-Methylmorpholine (NMM) (the base), and 5% v/v Acetic Anhydride (the capping mixture) to cap the non-reacted amino groups.</li>
	<li>Cleave all the peptides from the resin by continuously mixing the solid support using an agitator in a freshly prepared cleavage mixture consisting of Trifluoroacetic acid (TFA): Triisopropylsilane(TIS): water: Dithiothreitol (DTT) at 90 (v/v): 5 (v/v): 2.5 (v/v): 2.5 (m/v), for 4 h. Use 10 mL for the synthesis scale of 100 &#181;mol.</li>
	<li>Precipitate the cleaved product by adding the cleavage mixture to 100 mL ice-cold diethyl ether. Pass through a filtration unit containing a Polytetrafluoroethylene (PTFE) filter with a pore size of 0.45 &#181;m and wash with 20 mL ice-cold diethyl ether.</li>
	<li>Collect the filtered peptide from the filter paper and dissolve 100 mg of precipitated peptide in 5 mL distilled deionized water: acetonitrile at 1:1.</li>
	<li>Purify via reverse-phase high-pressure liquid chromatography (HPLC) on a semi-preparative HPLC equipped with a porous polystyrene divinylbenzene column of bead size 10 &#181;m.
	<ol>
		<li>Pre-heat the column and maintain at 50 &#176;C using a column heating device. Collect all the major fractions using an automated fraction collector by applying a gradient from 5% to 100% solvent B over 25 min at 4 mL/min. 
		NOTE: Solvent A is 0.1% TFA in water and solvent B is 0.1% TFA in acetonitrile52.</li>
		<li>Monitor the chromatogram at 220 nm, collect the appropriate peaks, and analyze using the LC-MS.</li>
	</ol>
	</li>
	<li>Confirm the purity by analytical reverse phase ultra-high Pressure Liquid Chromatography (UPLC) at 220 nm by monitoring with a UV Detector while passing the sample through an analytical C18 column (bead size 1.7 &#181;m). Confirm the peptide product by mass spectrometry. 
	NOTE: The UPLC is coupled to an electrospray ionization mass spectrometry (ESI-MS) and Tandem Quadrupole Detector.</li>
	<li>Lyophilize the correct fractions of the desired peptide in a round bottom flask, by applying a vacuum of 0.052 mbar, to a fluffy powder. Couple the vacuum pump to a freezing unit maintained at -78 &#176;C. Store at -80 &#176;C, indefinitely.</li>
	<li>Use the lyophilized peptide to prepare a stock solution of 1 mM in a mixture of acetonitrile: DMF: analytical grade water at 1:1:1 for the experiments. This solution can be stored for at least 6 months, as the peptides do not aggregate in this solution.</li>
</ol>
2. Preparation of the Injection Mixture
<ol>
	<li>Prepare phosphate-buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4) for diluting the lyophilized peptides.</li>
	<li>Dissolve the peptide to a final concentration of 20 &#181;M in PBS. Prepare this solution fresh. Mix well and store on ice until the injection. Do not exceed 30 min to prevent aggregation in solution.</li>
</ol>
3. Anesthesia
<ol>
	<li>Prepare the stock solution of the anesthetics, 0.1% ethyl-m-aminobenzoate methanesulphonate (MESAB), in regular fish water from the circulating system. Prepare the anaesthetization solution to a final concentration of 0.0025% (v/v).</li>
	<li>Remove the desired number of fish from their tanks into a transport container with 5 L of system water.</li>
	<li>Half-fill a plastic Petri dish (90 mm) with 40 mL anesthetics. Use this dish for injections.</li>
	<li>Incubate the fish in the anesthetics until the opercular movement has ceased.</li>
</ol>
4. Cerebroventricular Microinjection
<ol>
	<li>Preparation of injection apparatus
	<ol>
		<li>Prepare the glass injection capillaries using a needle puller with the following parameters: heating cycle, 537; pulling cycle, 250; velocity, 1.5 s; time, 80 ms.</li>
		<li>Bring the pressure setting on the pressure source to 25 psi.</li>
		<li>Set the microinjector parameters to the following: hold pressure 20 psi; eject pressure 10 psi; period value 2.5; gating value 100 ms.</li>
		<li>Load the glass capillary with the injection solution. Insert the glass capillary into the microinjection holder. Adjust the injection angle to 45&#176;. 
		NOTE: More detailed protocols can be found as described in references27,46.</li>
	</ol>
	</li>
	<li>Place one fish into a new Petri dish filled with anaesthetization solution (as in step 3.3).</li>
	<li>Hold the fish with the forceps and orient for injection.</li>
	<li>Generate a slit using the tip of a 30 G needle in the skull over the optic tectum where the two lateral plates meet. Do not insert the tip into the brain tissue, this would cause bleeding and damage. See references27,45,46 for additional details.</li>
	<li>Insert the glass capillary into the slit.
	<ol>
		<li>Use only the tip of the needle and do not penetrate more than 1 mm through the skull.Keep holding the fish and insert the tip of the glass capillary through the incision site.</li>
		<li>Orient the tip of the glass capillary towards the telencephalon at a 45&#176; angle. Inject 1 &#181;L of the solution. The liquid disperses immediately after injection.</li>
	</ol>
	</li>
</ol>
5. Recovery
<ol>
	<li>Place the fish back to a transport container until it recovers. Connect the container to the regularly circulating fish water to ensure optimum water quality. 
	NOTE: The recovery should normally take 1 min. If it takes longer, the fish must be kept in the anesthetics for a shorter time (this needs to be optimized by the experimenter).</li>
</ol>
6. Tissue Preparation and Sectioning
<ol>
	<li>Wait for a desired period of time before sacrificing the fish. 
	NOTE: This depends on the experimental question. Amyloid deposition can be seen as early as 1 day after injection.</li>
	<li>Sacrifice the fish using the appropriate method depending on the ethical regulations (e.g., treat the fish with 0.1 M MESAB).</li>
	<li>Cut open the skull above the optic tectum using pointed forcep on the dorsal side, and dissect the head just behind the pectoral fin using a scalpel.</li>
	<li>Fix the heads using 2% paraformaldehyde (PFA) overnight at 4 &#176;C. Use 3 mL of PFA per head in a plastic tube with a screw lid. 
	CAUTION: Wear the appropriate personal protective equipment when handling PFA.</li>
	<li>For cryoprotection and decalcification, wash the heads thrice in 0.1M Phosphate Buffer (pH 7.4), then transfer them into 20% sucrose/20% ethylenediaminetetraacetic (EDTA) solution and incubate overnight at 4 &#176;C.</li>
	<li>To embed the tissue into sectioning resin, freeze the heads in 7.5% gelatin/20% sucrose solution in plastic histology molds on dry ice (approximately 3-5 min to freeze a block). Store the samples at -80 &#176;C or continue to the next step (cryosectioning).</li>
	<li>Section the heads into 12 &#181;m thick cryosections using a cryostat as described28,42. Transfer the cryosections directly onto glass slides and store at -20 &#176;C for long-term usage.</li>
</ol>
7. Immunohistochemical Staining and Microscopy
NOTE: Perform all incubation steps in a humidified chamber. And, all the washing steps are for 10 min each.
<ol>
	<li>Dry the sections for 30 min at room temperature. After thawing, wash the sections twice in PBS and once in PBSTx (PBS with 0.03% Triton-X-100).</li>
	<li>Incubate with the primary antibody (anti-A&#946;42 antibody; 1:500 dilution in PBSTx) at 4 &#176;C overnight.&#160; Following day, wash once in PBS and twice in PBSTx.</li>
	<li>Incubate the sections with the secondary antibody (fluorescence-coupled detection, 1:500 dilution) along with DAPI (1 &#956;g/mL) in PBSTx for 2 h at room temperature.</li>
	<li>Wash three times in PBSTx. After the final washing, mount the slides with a coverslip&#160; using 100 &#956;L 70% glycerol.</li>
	<li>Acquire fluorescent images using a confocal microscope (for example with 20X PL APO N.A. 0.7 objective with 488 excitation range and a standard green filter; Figure 2)39.</li>
</ol>

The authors have nothing to disclose

The amyloid peptides can be modified to include sequence variations or various tags. For instance, a scrambled amyloid peptide can be generated, and the peptides can be labeled with fluorescent tags at the N-terminus of the peptide end or tagged with carrier peptides39. Similarly, in this protocol, the carrier peptide is the cell-penetrating peptide TR because of its effectiveness to transport cargo deep into the brain tissue39. Additionally, our method allows for injection...

AD is a chronic progressive disease characterized by the loss of neurons and synapses in the cerebral cortex1,2,3,4,5. The classical neuropathological hallmarks of AD are the deposition of amyloid peptides and formation of the neurofibrillary tangles (NFTs)6. Senile plaques, also known as amyloid plaques, are composed of amyloid-&#946; (A&#946;) peptides that form &#946;-pleated structures in the brain parenchyma5. The accumulation of A&#946;42 in AD patients has an early and critical role in disease progression. AD triggers a cascade of events leading to synaptic dysfunction, impaired plasticity, and neuronal loss7,8,9,10.
The adult brain of teleost zebrafish serves as an excellent model to study the regulation of stem cell plasticity11,12,13,14,15,16,17,18,19,20 and various diseases in the central nervous system (CNS), including AD21,22,23,24. Owing to a vast array of available experimental methods19,20,25,26,27,28,29,30,31, these studies are informative and feasible. Zebrafish can replenish the CNS13,15,32,33,34,35,36,37,38, in part by using molecular programs activated after neuronal loss19,39,40,41,42,43,44. Therefore, establishing a neurodegenerative disease model in zebrafish can help address novel questions regarding regenerative ability and stem cell biology in vertebrate brains.
Recently, we developed an amyloid toxicity model in adult zebrafish brain by injecting synthetic A&#946;42 peptides (Table 1)39. This injection caused neurodegeneration phenotypes reminiscent of human brain pathology (e.g., cell death, microglial activation, synaptic degeneration, and memory deficits), indicating that zebrafish can be used for eliciting neurodegeneration in zebrafish brain, A&#946;42 peptides can be detected with immunohistochemical stainings, and molecular mechanisms of regeneration in adult zebrafish CNS can be identified39. In this protocol, we demonstrate the injection of synthetic amyloid peptides into the zebrafish brain using a cerebroventricular injection (CVMI) method27,39,45,46 to mimic amyloid deposition (Figure 1). CVMI provides a novel way of delivering the peptides, which aggregate upon injection as &#946;-sheet structures and exert toxicity. The peptides are distributed evenly throughout the brain, targeting the ventricular area along the entire rostro-caudal axis45. Additionally, this method allows for analyzing the morphological and molecular response of the NSPCs in adult zebrafish brain following amyloid inclusions. Such studies will provide us an insight for successful brain repair in mammals. Our method can be used to understand the necessary molecular mechanism of a successful regeneration response after AD-like symptoms to induce replenishment of lost neurons and functional recovery.

<table>
	<tbody>
		<tr>
			<td>Fmoc-protected amino acids</td>
			<td>IRIS Biotech GmbH (Marktredwitz, Germany)</td>
			<td></td>
			<td>Fmoc-based amino acids for solid phase peptide synthesis (SPPS)</td>
		</tr>
		<tr>
			<td>N,N,N&#8242;,N&#8242;-Tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU)</td>
			<td>IRIS Biotech GmbH (Marktredwitz, Germany)</td>
			<td>RL-1030</td>
			<td>Activator</td>
		</tr>
		<tr>
			<td>Oxyma</td>
			<td>IRIS Biotech GmbH (Marktredwitz, Germany)</td>
			<td>RL-1180</td>
			<td>Racemization supressor</td>
		</tr>
		<tr>
			<td>N,N-Diisopropylethylamine</td>
			<td>IRIS Biotech GmbH (Marktredwitz, Germany)</td>
			<td>SOL-003</td>
			<td>Base</td>
		</tr>
		<tr>
			<td>Dimethylformamide</td>
			<td>IRIS Biotech GmbH (Marktredwitz, Germany)</td>
			<td>SOL-004</td>
			<td>Solvent</td>
		</tr>
		<tr>
			<td>N-Methylmorpholine</td>
			<td>Thermo Fisher (Kandel) GmbH, Germany</td>
			<td>A12158</td>
			<td>Base</td>
		</tr>
		<tr>
			<td>1-Hydroxybenzotriazole hydrate (HOBT)</td>
			<td>Sigma-Aldrich Co. LLC. (St. Louis, MO, USA)</td>
			<td>157260 ALDRICH</td>
			<td>Activator</td>
		</tr>
		<tr>
			<td>Piperidine</td>
			<td>MERCK KGaA (Darmstadt, Germany)</td>
			<td>822299</td>
			<td>Fmoc deprotection reagent</td>
		</tr>
		<tr>
			<td>Dichlormethane (DCM)</td>
			<td>MERCK KGaA (Darmstadt, Germany)</td>
			<td>106050</td>
			<td>Solvent</td>
		</tr>
		<tr>
			<td>Formic acid (FA)</td>
			<td>MERCK KGaA (Darmstadt, Germany)</td>
			<td>100264</td>
			<td>Buffer component for HPLC</td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid (TFA)</td>
			<td>MERCK KGaA (Darmstadt, Germany)</td>
			<td>808260</td>
			<td>Clevage Mixture reagent</td>
		</tr>
		<tr>
			<td>Triisopropylsilane(TIS)</td>
			<td>MERCK KGaA (Darmstadt, Germany)</td>
			<td>233781 ALDRICH</td>
			<td>Clevage Mixture reagent</td>
		</tr>
		<tr>
			<td>Acetonitrile (for UPLC/LCMS)</td>
			<td>Sigma-Aldrich Laborchemikalien GmbH</td>
			<td>34967-1L</td>
			<td>Solvent</td>
		</tr>
		<tr>
			<td>Acetonitrile (for HPLC)</td>
			<td>VWR International Ltd, England</td>
			<td>83639.320</td>
			<td>Solvent</td>
		</tr>
		<tr>
			<td>Diethylether</td>
			<td>VWR International Ltd, England</td>
			<td>23811.326</td>
			<td>Solvent for peptide precipitation</td>
		</tr>
		<tr>
			<td>Dithiotritol (DTT)</td>
			<td>VWR International Ltd, England</td>
			<td>0281-25G</td>
			<td>Clevage Mixture reagent</td>
		</tr>
		<tr>
			<td>TentaGel S RAM Fmoc rink amide resin</td>
			<td>Rapp Polymere GmbH (Tuebingen, Germany)</td>
			<td>S30023</td>
			<td>Solid phase for SPPS</td>
		</tr>
		<tr>
			<td>Peptide synthesis 5 ml syringes with included filters</td>
			<td>Intavis AG (Cologne, Germany)</td>
			<td>34.274</td>
			<td>Reaction tube for SPPS and for clevage from the Solid Phase</td>
		</tr>
		<tr>
			<td>Polytetrafluoroethylene (PTFE) filter</td>
			<td>Sartorius Stedtim (Aubagne, France)</td>
			<td>11806-50-N</td>
			<td>Filteration of precipitated peptides</td>
		</tr>
		<tr>
			<td>Polyvinylidenefluoride (PVDF) syringe filter</td>
			<td>Carl Roth GmbH + Co. KG Karlsruhe</td>
			<td>KC78.1</td>
			<td>Pre-filteration for HPLC</td>
		</tr>
		<tr>
			<td>Peptide Synthesizer</td>
			<td>Intavis, Cologne, Germany</td>
			<td>ResPep SL</td>
			<td>Automated solid-phase peptide synthesizer</td>
		</tr>
		<tr>
			<td>Water Alliance HPLC</td>
			<td>Waters, Milford Massachusetts, USA</td>
			<td>Waters 2998, Waters e2695</td>
			<td>Semi-preparative reverse-phase high pressure liquid chromatography (HPLC)</td>
		</tr>
		<tr>
			<td>PolymerX, bead size 10&#956;m, 250x10 mm</td>
			<td>Phenomenex Ltd. Germany</td>
			<td>00G-4328-N0</td>
			<td>Porous polystyrene divinylbenzene HPLC column</td>
		</tr>
		<tr>
			<td>Milli-Q Advantage A10, with a Milli-Q filter</td>
			<td>EMD Millipore Corporation, Billerica, MA, USA</td>
			<td>LCPAK0001</td>
			<td>Water purification system</td>
		</tr>
		<tr>
			<td>Filtration Unit</td>
			<td>Sartorius Stedtim (Aubagne, France)</td>
			<td>16307</td>
			<td>Filtration unit for peptide precipitation</td>
		</tr>
		<tr>
			<td>UPLC Aquity with UV Detector</td>
			<td>Waters, Milford Massachusetts, USA</td>
			<td>M09UPA 664M</td>
			<td>Analytical reverse phase ultra HPLC for LC-MS</td>
		</tr>
		<tr>
			<td>ACQUITY UPLC BEH C18, bead size 1.7 &#956;m, 50x2.1 mm</td>
			<td>Waters, Milford Massachusetts, USA</td>
			<td>186002350</td>
			<td>Analytical C18 column</td>
		</tr>
		<tr>
			<td>ACQUITY TQ Detector</td>
			<td>Waters, Milford Massachusetts, USA</td>
			<td>QBB908</td>
			<td>Electrospray ionization mass spectrometry (ESI-MS)</td>
		</tr>
		<tr>
			<td>CHRIST ALPHA 2-4 LD plus + vacuubrand RZ6</td>
			<td>Martin Christ Gefriertrocknungsanlagen GmbH, Germany</td>
			<td>16706, 101542</td>
			<td>Lyophilizer with vaccum pump</td>
		</tr>
		<tr>
			<td>Paradigm plate reader</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MESAB (ethyl-m-aminobenzoate methanesulphonate)</td>
			<td>Sigma-Aldrich</td>
			<td>A5040</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Sarstedt</td>
			<td>821.472</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline</td>
			<td>Life Technologies, GIBCO</td>
			<td>10010-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle</td>
			<td>Becton-Dickinson</td>
			<td>305178</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Olympus, Leica, Zeiss</td>
			<td>Varies with the manufacturer</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont Tweezers</td>
			<td>World Precision Instruments</td>
			<td>501985</td>
			<td></td>
		</tr>
		<tr>
			<td>Gillies Dissecting Forceps</td>
			<td>World Precision Instruments</td>
			<td>501265</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass injection capillaries</td>
			<td>World Precision Instruments</td>
			<td>TWF10</td>
			<td></td>
		</tr>
		<tr>
			<td>PicoNozzle</td>
			<td>World Precision Instruments</td>
			<td>5430-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Pneumatic PicoPump</td>
			<td>World Precision Instruments</td>
			<td>SYS-PV820</td>
			<td></td>
		</tr>
		<tr>
			<td>Ring illuminator; Ring Light Guide</td>
			<td>Parkland Scientific</td>
			<td>ILL-RLG</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica</td>
			<td>CM1950</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

HPLC was used to purify the synthesized peptide and mass spectrometry has been used to characterize the purified amyloid &#946; peptides. The HPLC column was heated to 50 &#176;C to improve the separation of the A&#946; peptides and all the fractions were collected. To identify the correctly synthesized peptide, mass spectroscopy analysis was performed for all fractions. The UPLC chromatogram shows the purity of the compound. The HPLC fraction that yielded one peak on the UPLC (i.e.</...

Watch this Scientific Journal Video about Modeling Amyloid-ÃŽÂ²42 Toxicity and Neurodegeneration in Adult Zebrafish Brain at JoVE.com

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

modeling-amyloid-42-toxicity-neurodegeneration-adult-zebrafish

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JoVE Journal

Neuroscience

11.1K Views.  German Centre for Neurodegenerative Diseases (DZNE) Dresden within Helmholtz Association. This protocol describes the synthesis, characterization, and injection of monomeric amyloid-β42 peptides for generating amyloid toxicity in adult zebrafish to establish an Alzheimer's disease model, followed by histological analyses and detection of aggregations.

Video: Modeling Amyloid-β42 Toxicity and Neurodegeneration in Adult Zebrafish Brain

In vivo Laser Axotomy in C. elegans

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron is damaged by injury or disease, it may regenerate. Cell-intrinsic and extrinsic factors influence the ability of a neuron to regenerate and restore function. Recently, the nematode C. elegans has emerged as an excellent model organism to identify genes and signaling pathways that influence the regeneration of neurons1-6. The main way to initiate neuronal regeneration in C. elegans is laser-mediated cutting, or axotomy. During axotomy, a fluorescently-labeled neuronal process is severed using high-energy pulses. Initially, neuronal regeneration in C. elegans was examined using an amplified femtosecond laser5. However, subsequent regeneration studies have shown that a conventional pulsed laser can be used to accurately sever neurons in vivo and elicit a similar regenerative response1,3,7.
We present a protocol for performing in vivo laser axotomy in the worm using a MicroPoint pulsed laser, a turnkey system that is readily available and that has been widely used for targeted cell ablation. We describe aligning the laser, mounting the worms, cutting specific neurons, and assessing subsequent regeneration. The system provides the ability to cut large numbers of neurons in multiple worms during one experiment. Thus, laser axotomy as described herein is an efficient system for initiating and analyzing the process of regeneration.

In vivo Laser Axotomy in C. elegans

A protocol to cut neurons in C. elegans with a MicroPoint pulsed laser is presented. We describe setting up the system, immobilizing worms, and severing labeled neurons. Advantages include a relatively low-cost system and the ability to sever neuronal processes or ablate cells in vivo.

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron ...

Micromanipulation of Gene Expression in the Adult Zebrafish Brain Using Cerebroventricular Microinjection of Morpholino Oligonucleotides

Manipulation of gene expression in tissues is required to perform functional studies. In this paper, we demonstrate the cerebroventricular microinjection (CVMI) technique as a means to modulate gene expression in the adult zebrafish brain. By using CVMI, substances can be administered into the cerebroventricular fluid and be thoroughly distributed along the rostrocaudal axis of the brain. We particularly focus on the use of antisense morpholino oligonucleotides, which are potent tools for knocking down gene expression in vivo. In our method, when applied, morpholino molecules are taken up by the cells lining the ventricular surface. These cells include the radial glial cells, which act as neurogenic progenitors. Therefore, knocking down gene expression in the radial glial cells is of utmost importance to analyze the widespread neurogenesis response in zebrafish, and also would provide insight into how vertebrates could sustain adult neurogenesis response. Such an understanding would also help the efforts for clinical applications in human neurodegenerative disorders and central nervous system regeneration. Thus, we present the cerebroventricular microinjection method as a quick and efficient way to alter gene expression and neurogenesis response in the adult zebrafish forebrain. We also provide troubleshooting tips and other useful information on how to carry out the CVMI procedure.

In this article, we demonstrate a method for manipulation of gene expression in the ventricular cells of the adult zebrafish telencephalon using antisense morpholino oligonucleotides. We present this method as an efficient and quick protocol that can be used for functional studies in the adult vertebrate brain.

Manipulation of gene expression in tissues is  required to perform functional studies. In this paper, we demonstrate the  cerebroventricular ...

Electrophysiological Recording in the Brain of Intact Adult Zebrafish

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram recordings. This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments, allowing recording of neural activity. Immobilization of the adult requires a mechanism to deliver dissolved oxygen to the gills in lieu of buccal and opercular movement. With our technique, animals are immobilized and perfused with habitat water to fulfill this requirement. A craniotomy is performed under tricaine methanesulfonate (MS-222; tricaine) anesthesia to provide access to the brain. The primary electrode is then positioned within the craniotomy window to record extracellular brain activity. Through the use of a multitube perfusion system, a variety of pharmacological compounds can be administered to the adult fish and any alterations in the neural activity can be observed. The methodology not only allows for observations to be made regarding changes in neurological activity, but it also allows for comparisons to be made between larval and adult zebrafish. This gives researchers the ability to identify the alterations in neurological activity due to the introduction of various compounds at different life stages.

This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments to allow recordings and manipulation of neural activity in an intact animal.

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram ...

Anatomically Inspired Three-dimensional Micro-tissue Engineered Neural Networks for Nervous System Reconstruction, Modulation, and Modeling

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory environment and the limited capacity for neurogenesis. We are developing&#160;a strategy to simultaneously address&#160;neuronal and axonal pathway loss within the damaged CNS. This manuscript presents the fabrication protocol for micro-tissue engineered neural networks (micro-TENNs), implantable constructs consisting of neurons and aligned axonal tracts spanning the extracellular matrix (ECM) lumen of a preformed hydrogel cylinder hundreds of microns in diameter that may extend centimeters in length. Neuronal aggregates are delimited to the extremes of the three-dimensional encasement and are spanned by axonal projections. Micro-TENNs are uniquely poised as a strategy for CNS reconstruction, emulating aspects of brain connectome cytoarchitecture and potentially providing means for network replacement. The neuronal aggregates may synapse with host tissue to form new functional relays to restore and/or modulate missing or damaged circuitry. These constructs may also act as pro-regenerative&#160;&#34;living scaffolds&#34; capable of exploiting developmental mechanisms for cell migration and axonal pathfinding, providing synergistic structural and soluble cues based on the state of regeneration. Micro-TENNs are fabricated by pouring liquid hydrogel into a cylindrical mold containing a longitudinally centered needle. Once the hydrogel has gelled, the needle is removed, leaving a hollow micro-column. An ECM solution is added to the lumen to provide an environment suitable for neuronal adhesion and axonal outgrowth. Dissociated neurons are mechanically aggregated for precise seeding within one or both ends of the micro-column. This methodology reliably produces self-contained miniature constructs with long-projecting axonal tracts that may recapitulate features of brain neuroanatomy. Synaptic immunolabeling and genetically encoded calcium indicators suggest that micro-TENNs possess extensive synaptic distribution and intrinsic electrical activity. Consequently, micro-TENNs represent a promising strategy for targeted neurosurgical reconstruction of brain pathways and may also be applied as biofidelic models to study neurobiological phenomena in vitro.

This manuscript details the fabrication of micro-tissue engineered neural networks: three-dimensional micron-sized constructs comprised of long aligned axonal tracts spanning aggregated neuronal population(s) encased in a tubular hydrogel. These living scaffolds can serve as functional relays to reconstruct or modulate neural circuitry or as biofidelic test-beds mimicking gray-white matter neuroanatomy.

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory ...

Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is important to isolate these cells without changing their gene expression profile. The zebrafish model provides a large number of transgenic fish lines in which specific cell types are labelled; for example neurons in the NBT:DsRed line or macrophages/microglia in the mpeg1:eGFP line. Furthermore, antibodies have been developed to stain specific cells, such as microglia with the 4C4 antibody.
Here, we describe the isolation of neurons, macrophages and microglia from larval zebrafish brains. Central to this protocol is the avoidance of an enzymatic tissue digestion at 37 &#176;C, which could modify cellular profiles. Instead a mechanical system of tissue homogenization at 4 &#176;C is used. This protocol entails homogenization of brains into cell suspension, their immuno-staining and the isolation of neurons, macrophages and microglia by FACS. Afterwards, we extracted RNA from those cells and evaluated their quality/quantity. We managed to obtain RNA of high quality (RNA Integrity Number (RIN) &#62; 7) to perform qPCR on macrophages/microglia and neurons, and transcriptomic analysis on microglia. This approach enables a better characterization of these cells, as well as a clearer understanding about their role in development and pathologies.

We present a protocol to isolate neurons, macrophages and microglia from larval zebrafish brains under physiological and pathological conditions. Upon isolation, RNA is extracted from these cells to analyze their gene expression profile. This protocol allows for the collection of high-quality RNA for performing downstream analysis like qPCR and transcriptomics.

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is ...

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from parallel fibers (PF) to Purkinje cells (PC) is considered the basis for motor learning, and deficiencies of both LTD and motor learning are observed in various gene-manipulated animals. Common motor learning sets, such as adaptation of the optokinetic reflex (OKR), the vestibular-ocular reflex (VOR), and rotarod test were used for evaluation of motor learning ability. However, results obtained from the GluA2-carboxy terminus modified knock-in mice demonstrated normal adaptation of the VOR and the OKR, despite lacking PF-LTD. In that report, induction of LTD was only attempted using one type of stimulation protocol at room temperature. Thus, conditions to induce cerebellar LTD were explored in the same knock-in mutants using various protocols at near physiological temperature. Finally, we found stimulation protocols, by which LTD could be induced in these gene-manipulated mice. In this study, a set of protocols are proposed to evaluate LTD-induction, which will more accurately allow examination of the causal relationship between LTD and motor learning. In conclusion, experimental conditions are crucial when evaluating LTD in gene-manipulated mice.

In some gene-manipulated animals, using a single protocol may fail to induce LTD in cerebellar Purkinje cells, and there may be a discrepancy between LTD and motor learning. Multiple protocols are necessary to assess LTD-induction in gene-manipulated animals. Standard protocols are shown.

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...

Visualizing Synaptic Degeneration in Adult Drosophila in Association with Neurodegeneration

Drosophila serves as a useful model for assessing synaptic structure and function associated with neurodegenerative diseases. While much work has focused on neuromuscular junctions (NMJs) in Drosophila larvae, assessing synaptic integrity in adult Drosophila has received much less attention. Here we provide a straightforward method for dissection of the dorsal longitudinal muscles (DLMs), which are required for flight ability. In addition to flight as a behavioral readout, this dissection allows for the both DLM synapses and muscle tissue to be amenable to structural analysis using fluorescently labeled antibodies for synaptic markers or proteins of interest. This protocol allows for the evaluation of the structural integrity of synapses in adult Drosophila during aging to model the progressive, age-dependent nature of most neurodegenerative diseases.

Visualizing Synaptic Degeneration in Adult Drosophila in Association with Neurodegeneration

The goal of this procedure is to dissect the dorsal longitudinal muscle (DLM) tissue to assess the structural integrity of DLM neuromuscular junctions (NMJs) in neurodegenerative disease models using Drosophila melanogaster.

Drosophila serves as a useful model for assessing synaptic structure and function associated with neurodegenerative diseases. While much work has focused ...

A Scalable Model to Study the Effects of Blunt-Force Injury in Adult Zebrafish

Blunt-force traumatic brain injuries (TBI) are the most common form of head trauma, which spans a range of severities and results in complex and heterogenous secondary effects. While there is no mechanism to replace or regenerate the lost neurons following a TBI in humans, zebrafish possess the ability to regenerate neurons throughout their body, including the brain. To examine the breadth of pathologies exhibited in zebrafish following a blunt-force TBI and to study the mechanisms underlying the subsequent neuronal regenerative response, we modified the commonly used rodent Marmarou weight drop for the use in adult zebrafish. Our simple blunt-force TBI model is scalable, inducing a mild, moderate, or severe TBI, and recapitulates many of the phenotypes observed following human TBI, such as contact- and post-traumatic seizures, edema, subdural and intracerebral hematomas, and cognitive impairments, each displayed in an injury severity-dependent manner. TBI sequelae, which begin to appear within minutes of the injury, subside and return to near undamaged control levels within 7 days post-injury. The regenerative process begins as early as 48 hours post-injury (hpi), with the peak cell proliferation observed by 60 hpi. Thus, our zebrafish blunt-force TBI model produces characteristic primary and secondary injury TBI pathologies similar to human TBI, which allows for investigating disease onset and progression, along with the mechanisms of neuronal regeneration that is unique to zebrafish.

We modified the Marmarou weight drop model for adult zebrafish to examine a breadth of pathologies following blunt-force traumatic brain injury (TBI) and the mechanisms underlying subsequent neuronal regeneration. This blunt-force TBI model is scalable, induces a mild, moderate, or severe TBI, and recapitulates injury heterogeneity observed in human TBI.

Blunt-force traumatic brain injuries (TBI) are the most common form of head trauma, which spans a range of severities and results in complex and ...

Shuttle Box Assay as an Associative Learning Tool for Cognitive Assessment in Learning and Memory Studies using Adult Zebrafish

Cognitive deficits, including impaired learning and memory, are a primary symptom of various developmental and age-related neurodegenerative diseases and traumatic brain injury (TBI). Zebrafish are an important neuroscience model due to their transparency during development and robust regenerative capabilities following neurotrauma. While various cognitive tests exist in zebrafish, most of the cognitive assessments that are rapid examine non-associative learning. At the same time, associative-learning assays often require multiple days or weeks. Here, we describe a rapid associative-learning test that utilizes an adverse stimulus (electric shock) and requires minimal preparation time. The shuttle box assay, presented here, is simple, ideal for novice investigators, and requires minimal equipment. We demonstrate that, following TBI, this shuttle box test reproducibly assesses cognitive deficit and recovery from young to old zebrafish. Additionally, the assay is adaptable to examine either immediate or delayed memory. We demonstrate that both a single TBI and repeated TBI events negatively affect learning and immediate memory but not delayed memory. We, therefore, conclude that the shuttle box assay reproducibly tracks the progression and recovery of cognitive impairment.

Learning and memory are potent metrics in studying either developmental, disease-dependent, or environmentally induced cognitive impairments. Most cognitive assessments require specialized equipment and extensive time commitments. However, the shuttle box assay is an associative learning tool that utilizes a conventional gel box for rapid and reliable assessment of adult zebrafish cognition.

Cognitive deficits, including impaired learning and memory, are a primary symptom of various developmental and age-related neurodegenerative diseases and ...

Determination of Mitochondrial Respiration and Glycolysis in Ex Vivo Retinal Tissue Samples

Mitochondrial respiration is a critical energy-generating pathway in all cells, especially retinal photoreceptors that possess a highly active metabolism. In addition, photoreceptors also exhibit high aerobic glycolysis like cancer cells. Precise measurements of these metabolic activities can provide valuable insights into cellular homeostasis under physiological conditions and in disease states. High throughput microplate-based assays have been developed to measure mitochondrial respiration and various metabolic activities in live cells. However, a vast majority of these are developed for cultured cells and have not been optimized for intact tissue samples and for application ex vivo. Described here is a detailed step-by-step protocol, using microplate-based fluorescence technology, to directly measure oxygen consumption rate (OCR) as an indicator of mitochondrial respiration, as well as extracellular acidification rate (ECAR) as an indicator of glycolysis, in intact ex vivo retinal tissue. This method has been used to successfully assess metabolic activities in adult mouse retina and demonstrate its application in investigating cellular mechanisms of aging and disease.

Determination of Mitochondrial Respiration and Glycolysis in Ex Vivo Retinal Tissue Samples

Described here is a detailed protocol for performing mitochondrial stress assay and glycolytic rate assay in ex vivo retinal tissue samples using a commercial bioanalyzer.

Mitochondrial respiration is a critical energy-generating pathway in all cells, especially retinal photoreceptors that possess a highly active metabolism. ...