This work was supported by the Deutsche Forschungsgemeinschaft (SFB 655) and the European Union (Zf-Health).

1. Preparation of the Injection Mixture
<ol>
	<li>Use vivo morpholinos as cells internalize them more efficiently than normal morpholino molecules. See the manufacturer's information for details (Table 1: Reagents and Materials).</li>
	<li>Use a fluorescent tracking dye (e.g. CellTracker Red CMTPX, Invitrogen) to visualize the accuracy of the injection. This dye is metabolized in the cell and becomes fluorescent only in the cell after the uptake. Therefore, this step is crucial to determine the cells that are efficiently targeted by the cerebroventricular microinjection.</li>
	<li>Prepare phosphate-buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 10mM Na2HPO4, 2 mM KH2PO4, pH: 7.4) for diluting morpholino solution. If a dilution of the morpholino solution is required, always use PBS.</li>
	<li>Prepare 10 &mu;l of the injection mix by adding 9 &mu;l of the morpholino solution and 1 &mu;l of the cell tracker dye (diluted or undiluted. The stock concentration of the morpholino solution is 500 &mu;M. Our previous results 22 indicate that several different doses of the morpholino - ranging from 50 &mu;M to 500 &mu;M - result in different levels of knockdown from an efficient knockdown to hypomorphic phenotypes). 1 &mu;l of solution is enough for injection into one zebrafish brain.</li>
	<li>Mix well and store at room temperature until the injection.</li>
</ol>
2. Preparation of the Injection Apparatus
<ol>
	<li>Prepare the glass injection capillaries using a needle puller. Use the following parameters: Heating cycle value: 463, pulling cycle value: 230, velocity: 1.5 sec, time: 75 msec.</li>
	<li>Turn on the pressure source and adjust the pressure settings to 50 psi or 3.5 bars.</li>
	<li>Adjust the microinjector settings as follows: hold pressure 20 psi, eject pressure 10 psi, period value of 2.5 and 100 msec range of gating.</li>
	<li>Turn on the ring illuminator and locate the ring over the microscope stage.</li>
	<li>Place the capillary holder to an appropriate position next to the microscope.</li>
	<li>Insert the glass capillary into the holder. Adjust the injection angle to 45 degrees.</li>
	<li>Snap off the tip of the glass capillary using a fine-end forceps and calibrate the pressure output from the orifice. This must be tested by immersing the tip of the needle into the fish water and giving a continuous pressure pulse. The air bubbles arising should be only one row, which is an indication of optimum pressure/orifice size. Please see the video for demonstration of this calibration.</li>
	<li>Load the glass capillary with the injection solution without trapping air bubbles.</li>
	<li>Apply pressure to remove the air between the tip of the glass capillary and the loaded injection solution.</li>
</ol>
3. Anesthesia
<ol>
	<li>Prepare the stock solution of the anesthetics (0.1% MESAB - ethyl-m-aminobenzoate methanesulphonate).</li>
	<li>Remove desired number of fish from their tanks into plastic mouse cages or other transport container with enough amount of water.</li>
	<li>Prepare the anaesthetization solution in a small plastic box by mixing 200 ml of fish water with 5 ml of anesthetics stock solution. Final concentration of the anesthetics: 0.0025% (v/v).</li>
	<li>Half-fill a plastic Petri dish with anesthetics. Use this dish for injections.</li>
	<li>Incubate the fish in the anesthetics until the fish subdues.</li>
</ol>
4. Incision and Cerebroventricular Microinjection
<ol>
	<li>Remove the fish from the box using a fish net and place it into the Petri dish.</li>
	<li>Gently hold the fish with the forceps and orient the head in a way that it tilts slightly downwards. This will facilitate the incision.</li>
	<li>Generate a small slit on the skull using a 30 gauge barbed-end needle. Use only the tip of the needle and do not penetrate more than enough into the brain as this will cause damage to the brain tissue and will manifest as bleeding.</li>
	<li>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. Avoid touching the brain tissue as this creates tissue damage and also prevents dispersion of the injected solution.</li>
	<li>Inject the solution. The liquid disperses immediately after injection.</li>
</ol>
5. Recovery and Analyzing the Injection Accuracy
<ol>
	<li>Remove the fish from the injection setup and place into a plastic box with fresh fish water.</li>
	<li>Allow recovery.</li>
	<li>Re-anaesthetize the fish with 0.0025% MESAB briefly and check the fish under the fluorescence microscope. A thorough dispersion of red fluorescence is an indication of widespread distribution of the injected liquid.</li>
	<li>Put the fish back to the plastic box.</li>
	<li>Connect the plastic box to the circulation system to oxygenate the fish and maintain for longer periods of time.</li>
	<li>Use the fish for desired purposes such as behavioral analyses or sacrifice the animals at desired time points after the injection to prepare tissue specimens for further analyses (histological stainings, immunohistochemistry, etc.). The following references give examples of how to analyze the adult zebrafish brain: 9,11,17-20,24-27. The quickest time to observe an efficient knockdown (more than 50% knockdown) was 12 hr in our studies 22.</li>
</ol>
6. Suggested Scientific Controls
<ol>
	<li>Use a mismatch morpholino molecule that is not supposed to cause any phenotype as a negative control. Use either a standard morpholino molecule or specifically design morpholino controls for every gene of interest. Additionally, use the solvent (in this case PBS) as a further negative control to assess the non-specific effects or toxicity of the mismatch/control morpholino injections.</li>
	<li>Use PCNA morpholinos described previously 22 as positive controls. This morpholino causes efficient knockdown in the adult zebrafish brain (75-95% knockdown efficiency within 1 day of injection) 22.</li>
</ol>

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Gene Tools, LLC has sponsored additional open access publication fees for this article.

The method we describe here allows easy and rapid administration of morpholinos into the adult zebrafish brain. We have demonstrated that our injection method efficiently blocks gene expression in the ventricular cells and results in functional consequences in the neurogenesis response.
There are important points to be cautious about while executing the cerebroventricular microinjection. For instance, the effect of the morpholino molecules depends on the concentration used. This concentration has to be determined by the end-user. We recommend starting with the stock solution (500 &mu;M) and performing serial dilutions, and ideally to pre-test by injection into embryos using standard embryo injection protocols 28-30. Previously, we obtained different levels of knockdown efficiency with concentrations of morpholinos ranging from 50 &mu;M to 500 &mu;M 22. Second, the orifice of the glass capillary should not be large as this will lead to extensive liquid influx into the brain after the injection. Similarly, the opening must not be too narrow as this will prevent adequate injection. One can determine the optimum orifice-pressure combination by pumping air into a Petri dish with water. The bubbles arising should be in a single row but not in multiple rows. We demonstrate this in the video. Third, incubating the fish in the anesthetics is critical. The fish should not be kept longer than 2 min in the anesthetics. This will hamper the recovery rate after the injection. Fourth, the location of the incision is critical for thoroughly dispersing the injected liquid. The ventricular region over the optic tectum is larger above the midline and gets narrower laterally. Therefore, the experimenter should generate the slit in the skull close to the midline and just caudal to the skull plate covering the telencephalic region.
One of the advantages of the cerebroventricular injection (CVMI) method is its rapidness. CVMI is a quick method for assaying gene function. This feature is important and useful when compared to generation of transgenic lines for functional studies, which generally take several months. Additionally, CVMI leads to uniform distribution of the injected liquid and therefore provides a relatively thorough manipulation of gene activity, when compared to focal injections or electroporation. With CVMI, multiple genes can be knocked down simultaneously by preparing injection mixes containing multiple morpholinos oligonucleotide. CVMI can be used to inject different concentrations of a given morpholino oligonucleotides, and therefore can be used for analyzing hypomorphic phenotypes. Finally, this injection paradigm does not cause toxicity or compromise the survival of the animals.
The CVMI technique might be expanded for other type of studies such as injection of modulatory peptides, drugs, plasmid DNA or modulatory RNA molecules or other substances that might affect the physiology of the cells. Assaying combination of molecules and performing dose response analyses are possible using our method, allowing studying hypomorphic phenotypes. With these properties, CVMI proves to be a quick and easy assay for expression studies in the adult zebrafish brain, and opens up rapid screening and functional analyses.
The adult zebrafish brain can constitutively produce new neurons along the whole rostrocaudal axis and it can also regenerate after traumatic injuries. This is in stark contrast to mammalian brains with limited neurogenesis and if at all, rather poor regenerative capacity. Such widespread stem cell activity and recuperation ability makes zebrafish a useful model organism for understanding the molecular programs required for central nervous system regeneration, which are currently largely unknown. Therefore, investigating the molecular basis of the regenerative aptitude of zebrafish brain is an interesting realm of research that might explain the fundamental difference how fish and mammalian brains react after an injury, and also endow avenues for regenerative medical therapies in humans. In order to understand the molecular infrastructure of vertebrate brain plasticity and regeneration, glial cells serve as an important research area as they are the neurogenic progenitors 3,8,20. Thus, using CVMI technique to alter gene function in the radial glial cells of the zebrafish brain is instrumental in elucidating how fish brain can couple progenitor activity to efficient adult neurogenesis and regenerative response. We have recently shown the involvement and requirement of several factors and signaling pathways in the regenerative neurogenesis response of the adult zebrafish brain 24,26,27, and these studies were made possible by the use of CVMI method. Overall, the knowledge we gain from zebrafish brain could be harnessed to impose regenerative ability to the mammalian glial cells that react to injuries and will hence help designing clinical therapies for human neurological disorders and acute injuries.

Adult neurogenesis is a common trait to vertebrates, however its degree of prevalence and efficiency varies with the phylogeny 1,2,3,4,5,6. For instance, adult mammalian brains contain stem cell regions largely restricted to the forebrain 7,8, while the teleost zebrafish contains sixteen different stem cell domains and associated neurogenic zones in its entire brain 4,5,9,10. This variation between mammals and zebrafish might reflect a disparity in the mechanisms of stem cell maintenance and the neurogenic capacity of the progenitor cells. The lifelong competence of zebrafish to produce neurons in the brain could also pose clinical ramifications as the molecular mechanisms employed by zebrafish brain could be harnessed for therapeutic applications to tackle the neurodegenerative disorders in humans.
Several stem cells zones of the adult zebrafish brain have been analyzed and it has been shown that the cells lining the ventricular surface of those regions serve as progenitor cells 9,11-20. Detailed analyses in the zebrafish telencephalon, for example, identified the radial glial cells, which delineate the ventricular surface of this brain region to be neurogenic progenitors 19, 20. This holds true for other regions such as the cerebellum and the optic tectum, where ventricularly-located neuroepithelial cells provide the neurogenic input 16,21. To this end, understanding the molecular mechanisms governing the widespread neurogenic competency in adult zebrafish brain requires manipulation of gene expression in the progenitor cells.
Various methods were previously described for modulating gene function in zebrafish. These include generation of conditional transgenic lines expressing desired variants of a single protein, plasmid-based focal injections coupled to electroporation or chemical treatments. We previously designed a method for administering various substances including morpholino oligonucleotides or proteins into the adult zebrafish brain using cerebroventricular microinjection as a quick and efficient way of modulating gene function in the ventricular cells of the adult zebrafish forebrain 22. In our studies, vivo morpholinos were used because of their chemistry involving the morpholino molecule covalently linked to arginine rich delivery peptides, which enable efficient delivery to the cells of interest without need for extra permeabilization such as electroporation 23. This would allow a thorough targeting of the desired tissue after injection, just like what we observed in adult zebrafish telencephalon 22. Use of vivo morpholinos are therefore superior to already existing methods of tissue targeting such as electroporation or focal injection of DNA molecules.
Here, we demonstrate visually how we perform this operation and provide a step-by-step protocol. We start with explaining preparation of the injection mixture and the fish to be injected, and proceed by demonstrating how the injection apparatus is set up. We provide a description of the cerebroventricular injection method, which involves generating an incision in the skull and injection of the morpholinos using microinjector. We also elaborate on the critical points one needs to be cautious on during the whole procedure by stating them as optimization or troubleshooting guides.

<table border="1">
	<tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagent/Material</td>
 </tr>
 <tr>
 <td>CellTracker Red CMTPX</td>
 <td>Life Technologies, Invitrogen</td>
 <td>C34552</td>
 <td>Use at 1:100 dilution for measuring the injection accuracy</td>
 </tr>
 <tr>
 <td>MESAB (ethyl-m-aminobenzoate methanesulphonate)</td>
 <td>Sigma-Aldrich</td>
 <td>A5040</td>
 <td>For anesthetizing the fish</td>
 </tr>
 <tr>
 <td>Petri dishes</td>
 <td>Sarstedt</td>
 <td>821,472</td>
 <td>For handling the fish during injection and imaging</td>
 </tr>
 <tr>
 <td>Phosphate-buffered saline</td>
 <td>Life Technologies, GIBCO</td>
 <td>10010-056</td>
 <td>As sterile dilution medium</td>
 </tr>
 <tr>
 <td>Vivo morpholinos</td>
 <td>Gene Tools Inc.</td>
 <td>Customized</td>
 <td>More info on vivo morpholinos: <a href="http://www.gene-tools.com" target="_blank">www.gene-tools.com</a></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Barbed-end needle</td>
 <td>Becton-Dickinson</td>
 <td>305178</td>
 <td>To generate the incision in the skull</td>
 </tr>
 <tr>
 <td>Dissecting microscope</td>
 <td>Olympus, Leica, Zeiss </td>
 <td>Varies with the manufacturer</td>
 <td>Part of the whole injection setup</td>
 </tr>
 <tr>
 <td>Dumont Tweezers</td>
 <td>World Precision Instruments</td>
 <td>501985</td>
 <td>To snap off the tip of the glass capillary</td>
 </tr>
 <tr>
 <td>Fluorescence camera</td>
 <td>Zeiss, Nikon, Leica</td>
 <td>Varies with the manufacturer</td>
 <td>To visualize the fluorescence after injection</td>
 </tr>
 <tr>
 <td>Gillies Dissecting Forceps</td>
 <td>World Precision Instruments</td>
 <td>501265</td>
 <td>To hold the fish in position</td>
 </tr>
 <tr>
 <td>Glass injection capillaries</td>
 <td>World Precision Instruments </td>
 <td>TWF10 </td>
 <td>For microinjection</td>
 </tr>
 <tr>
 <td>PicoNozzle kit (microinjector holder)</td>
 <td>World Precision Instruments</td>
 <td>5430-12</td>
 <td>For microinjection</td>
 </tr>
 <tr>
 <td>Pneumatic PicoPump (microinjector)</td>
 <td>World Precision Instruments</td>
 <td>SYS-PV820 </td>
 <td>For microinjection</td>
 </tr>
 <tr>
 <td>Ring illuminator; Ring Light Guide</td>
 <td>Parkland Scientific</td>
 <td>ILL-RLG </td>
 <td>For illuminating the specimen</td>
 </tr>
	</tbody>
 </table>

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.

Cerebroventricular microinjection leads to widespread distribution of the injected solution
Using the workflow and technical scheme of the cerebroventricular microinjection depicted in Figure 1, we administered morpholino oligonucleotides into the adult zebrafish brain. An accurate injection protocol leads to dispersion of the injected liquid throughout the brain and efficient targeting of the cells close to the ventricular surface (Figure 2A). Injections where the glass capillary impales the brain tissue will have a dense fluorescent signal at the point of injection (Figure 2B). If the injected amount is not enough, weak fluorescent signal will be observed (Figure 2C).
Cerebroventricular microinjection can be used for knocking down gene expression at the ventricular region of the adult zebrafish brain
We have shown that vivo morpholinos can penetrate a few cell diameters inside the ventricular surface and hence can efficiently target the ventricular cells (Figures 3A, 3B). Morpholino injection can knockdown gene expression as it will alleviate the production of the corresponding protein (an example is PCNA as shown in Figures 3C, 3D). We have previously demonstrated that knocking down genes using this technique leads to functional consequences during the adult neurogenesis (Figures 3E, 3F) or regeneration response (Figures 3G, 3H). We demonstrated that the efficient knockdown range (more than 50% knockdown) is attained approximately 12 hr after injection of the morpholinos 22. We also observed that efficient knockdown period is until approximately 5 days after injection for the PCNA protein 22. More than 70% knockdown was seen until 3 days after the injection 22. The knockdown curves are dependent on the level of expression of the endogenous proteins and the efficiency of the morpholino molecules; and has to be determined for every gene by the experimenter. In our studies, we did not see that the CVMI procedure compromises the survival of the animal, probably because the administration of the morpholino molecules is local and does not cause a systemic toxicity.
<img alt="Figure 1" fo:content-width="6in" fo:src="/files/ftp_upload/50415/50415fig1highres.jpg" src="/files/ftp_upload/50415/50415fig1.jpg" /> 
Figure 1. Schematic representation of the cerebroventricular injection technique. (A) The injection is performed from the dorsal of the adult zebrafish (1) at the level of optic tectum (ot, 2). An incision is made over the optic tectal skull plate with a 30-gauge needle (3). Using glass capillaries, the morpholino molecules are injected into the ventricular fluid (4). A thorough dispersion is achieved instantaneously (5). (B) The canula and its barbed-end is shown in high magnification. (C) The orientation of the needle before the incision. (D) The incision is circled and outlined by dashed lines. (E) The injection capillary is located as shown and injection is performed. All panels were adapted from ref. 22. <a href="https://www.jove.com/files/ftp_upload/50415/50415fig1large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 2" src="/files/ftp_upload/50415/50415fig2.jpg" /> 
Figure 2. Comparison of injection efficiency and accuracy. (A) A good injection leads to targeting of the cells close to the ventricular surface. (B) If the glass capillary is inserted into the brain tissue, this will generate a centrally located fluorescence signal. (C) If the amount of injection is low, this will lead to weaker fluorescence signal.
<img alt="Figure 3" fo:content-width="5.5in" fo:src="/files/ftp_upload/50415/50415fig3highres.jpg" src="/files/ftp_upload/50415/50415fig3.jpg" /> 
Figure 3. Morpholino penetration, efficiency of gene knockdowns and functional consequences. (A) GFP immunostaining on the telencephalic cross sections of control morpholino-injected Tg(H2A:GFP) transgenic line showing GFP-positive nuclei. (B) GFP immunostaining on the telencephalic cross sections of gfp antisense morpholino-injected Tg(H2A:GFP) transgenic line showing GFP-positive nuclei. Note the difference in the GFP-signal at the ventricular region. v: ventricle. (C) PCNA immunostaining on control morpholino-injected brains showing widespread distribution of PCNA-positive cells. (D) PCNA immunostaining on pcna antisense morpholino-injected brains showing significantly reduced amounts of PCNA-positive cells, showing that CVMI can knockdown endogenous genes. (E) BrdU and HuC/D immunostaining on control morpholino-injected brains to determine newborn neurons after a BrdU pulse-chase experiment described before 22. (F) BrdU and HuC/D immunostaining on pcna antisense morpholino-injected brains to determine newborn neurons after a BrdU pulse-chase experiment described before 22. (G) Knocking-down PCNA leads to significant reduction in generation of newborn neurons, indicating that CVMI can be used to knockdown endogenous genes, which give rise to functional consequences. All panels were adapted from ref. 22 <a href="https://www.jove.com/files/ftp_upload/50415/50415fig3large.jpg" target="_blank">Click here to view larger figure</a>.

Watch this Scientific Journal Video about Micromanipulation of Gene Expression in the Adult Zebrafish Brain Using Cerebroventricular Microinjection of Morpholino Oligonucleotides at JoVE.com

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

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 ...

micromanipulation-gene-expression-adult-zebrafish-brain-using

Research

JoVE Journal

Neuroscience

19.2K Görüntüleme.  Cluster of Excellence (CRTD) and Biotechnology Center (BIOTEC) of the Technische Universität Dresden. Bu yazıda, morfolino antisens oligonükleotidler kullanılarak yetişkin zebrabalıkları telencephalon ventriküler hücrelerde gen ifadesinin manipülasyonu için bir yöntem ortaya koymaktadır. Biz, yetişkin omurgalı beynin fonksiyonel çalışmalar için kullanılan bir hızlı ve verimli protokolü olarak bu yöntem sunuyoruz.

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

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Optimized Analysis of DNA Methylation and Gene Expression from Small, Anatomically-defined Areas of the Brain

Exposure to diet, drugs and early life adversity during sensitive windows of life 1,2 can lead to lasting changes in gene expression that contribute to the display of physiological and behavioural phenotypes. Such environmental programming is likely to increase the susceptibility to metabolic, cardiovascular and mental diseases 3,4.
DNA methylation and histone modifications are considered key processes in the mediation of the gene-environment dialogue and appear also to underlay environmental programming 5. In mammals, DNA methylation typically comprises the covalent addition of a methyl group at the 5-position of cytosine within the context of CpG dinucleotides.
CpG methylation occurs in a highly tissue- and cell-specific manner making it a challenge to study discrete, small regions of the brain where cellular heterogeneity is high and tissue quantity limited. Moreover, because gene expression and methylation are closely linked events, increased value can be gained by comparing both parameters in the same sample.
Here, a step-by-step protocol (Figure 1) for the investigation of epigenetic programming in the brain is presented using the 'maternal separation' paradigm of early life adversity for illustrative purposes. The protocol describes the preparation of micropunches from differentially-aged mouse brains from which DNA and RNA can be simultaneously isolated, thus allowing DNA methylation and gene expression analyses in the same sample.

A streamlined workflow to study DNA methylation and gene expression changes upon early-life stress is shown. Starting from maternal separation of newborn mice and isolation of discrete brain tissues, we represent a protocol to simultaneously isolate DNA and RNA from brain tissue punches for subsequent bisulfite sequencing and RT-PCR analysis.

Exposure to diet, drugs and early life adversity during sensitive windows of life 1,2 can lead to lasting changes in gene expression that contribute to ...

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 ...

Analysis of Gene Expression Changes in the Rat Hippocampus After Deep Brain Stimulation of the Anterior Thalamic Nucleus

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective treatment for several movement disorders that have failed to respond to medication. Recent progress in the field of DBS surgery has begun to extend the application of this surgical technique to other conditions as diverse as morbid obesity, depression and obsessive compulsive disorder. Despite these expanding indications, little is known about the underlying physiological mechanisms that facilitate the beneficial effects of DBS surgery. One approach to this question is to perform gene expression analysis in neurons that receive the electrical stimulation. Previous studies have shown that neurogenesis in the rat dentate gyrus is elicited in DBS targeting of the anterior nucleus of the thalamus1. DBS surgery targeting the ATN is used widely for treatment refractory epilepsy. It is thus of much interest for us to explore the transcriptional changes induced by electrically stimulating the ATN. In this manuscript, we describe our methodologies for stereotactically-guided DBS surgery targeting the ATN in adult male Wistar rats. We also discuss the subsequent steps for tissue dissection, RNA isolation, cDNA preparation and quantitative RT-PCR for measuring gene expression changes. This method could be applied and modified for stimulating the basal ganglia and other regions of the brain commonly clinically targeted. The gene expression study described here assumes a candidate target gene approach for discovering molecular players that could be directing the mechanism for DBS.

The mechanism underlying the therapeutic effects of Deep Brain Stimulation (DBS) surgery needs investigation. The methods presented in this manuscript describe an experimental approach to examine the cellular events triggered by DBS by analyzing the gene expression profile of candidate genes that can facilitate neurogenesis post DBS surgery.

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective ...

Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant cell type in the brain, neurons are the most widely studied of these cell types given their direct role in impacting behaviors. Other cell types in the brain also impact neuronal function and behavior via the signaling molecules they produce. Neuroscientists must understand the interactions between the cell types in the brain to better understand how these interactions impact neural function and disease. To date, the most common method of analyzing protein or gene expression utilizes the homogenization of whole tissue samples, usually with blood, and without regard for cell type. This approach is an informative approach for examining general changes in gene or protein expression that may influence neural function and behavior; however, this method of analysis does not lend itself to a greater understanding of cell-type-specific gene expression and the effect of cell-to-cell communication on neural function. Analysis of behavioral epigenetics has been an area of growing focus which examines how modifications of the deoxyribonucleic acid (DNA) structure impact long-term gene expression and behavior; however, this information may only be relevant if analyzed in a cell-type-specific manner given the differential lineage and thus epigenetic markers that may be present on certain genes of individual neural cell types. The Fluorescence Activated Cell Sorting (FACS) technique described below provides a simple and effective way to isolate individual neural cells for the subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA. This technique can also be modified to isolate more specific neural cell types in the brain for subsequent cell-type-specific analysis.

The goal of this protocol is to use the fluorescence activated cell sorting (FACS) technique to sort specific types of neural cells for subsequent analysis of cell-type-specific gene expression, epigenetic markers, and or protein expression.

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant ...

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.

Modeling Amyloid-&amp;#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 ...

In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters

This technique combines optical fiber mediated fluorescence recordings with the precise delivery of recombinant adeno-associated virus based gene reporters. This new and easy to use in vivo fluorescence monitoring system was developed to record the transcriptional rhythm of the clock gene, Cry1, in the suprachiasmatic nucleus (SCN) of freely moving mice. To do so, a Cry1 transcription fluorescence reporter was designed and packaged into Adeno-associated virus. Purified, concentrated virus was injected into the mouse SCN followed by the insertion of an optic fiber, which was then fixed onto the surface of the brain. The animals were returned to their home cages and allowed a 1-month post-operative recovery period to ensure sufficient reporter expression. Fluorescence was then recorded in freely moving mice via an in vivo monitoring system that was constructed at our institution. For the in vivo recording system, a 488 nm laser was coupled with a 1 &#215; 4 beam-splitter that divided the light into four laser excitation outputs of equal power. This setup enabled us to record from four animals simultaneously. Each of the emitted fluorescence signals was collected via a photomultiplier tube and a data acquisition card. In contrast to the previous bioluminescence in vivo circadian clock recording technique, this fluorescence in vivo recording system allowed the recording of circadian clock gene expression during the light cycle.

In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters

This newly developed fluorescence-based technology enables long-term monitoring of the transcription of circadian clock genes in the suprachiasmatic nucleus (SCN) of freely moving mice in real-time and at a high temporal resolution.

This technique combines optical fiber mediated fluorescence recordings with the precise delivery of recombinant adeno-associated virus based gene ...

Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study of neuroscience as cellular function is determined by the composition and activity of cellular proteins. In this article, we describe how immunocytochemistry can be combined with near-infrared high-resolution scanning to provide a semi-quantitative measure of protein expression in distinct brain regions. This technique can be used for single or double protein expression in the same brain region. Measuring proteins in this fashion can be used to obtain a relative change in protein expression with an experimental manipulation, molecular signature of learning and memory, activity in molecular pathways, and neural activity in multiple brain regions. Using the correct proteins and statistical analysis, functional connectivity among brain regions can be determined as well. Given the ease of implementing immunocytochemistry in a laboratory, using immunocytochemistry with near-infrared high-resolution scanning can expand the ability of the neuroscientist to examine neurobiological processes at a systems level.

Here, we present a protocol that uses near-infrared dyes in conjunction with immunohistochemistry and high-resolution scanning to assay proteins in brain regions.

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study ...

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 ...