Cerebroventricular Microinjection (CVMI) into Adult Zebrafish Brain Is an Efficient Misexpression Method for Forebrain Ventricular Cells
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{"title"=>"Cerebroventricular microinjection (CVMI) into adult zebrafish brain is an efficient misexpression method for forebrain ventricular cells", "type"=>"journal", "authors"=>[{"first_name"=>"Caghan", "last_name"=>"Kizil", "scopus_author_id"=>"25723356500"}, {"first_name"=>"Michael", "last_name"=>"Brand", "scopus_author_id"=>"54894031500"}], "year"=>2011, "source"=>"PLoS ONE", "identifiers"=>{"issn"=>"19326203", "scopus"=>"2-s2.0-80455156263", "sgr"=>"80455156263", "pui"=>"362874137", "isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)", "pmid"=>"22076157", "doi"=>"10.1371/journal.pone.0027395"}, "id"=>"3d33da9b-8583-30ad-8b40-8ca6ffb17888", "abstract"=>"The teleost fish Danio rerio (zebrafish) has a remarkable ability to generate newborn neurons in its brain at adult stages of its lifespan-a process called adult neurogenesis. This ability relies on proliferating ventricular progenitors and is in striking contrast to mammalian brains that have rather restricted capacity for adult neurogenesis. Therefore, investigating the zebrafish brain can help not only to elucidate the molecular mechanisms of widespread adult neurogenesis in a vertebrate species, but also to design therapies in humans with what we learn from this teleost. Yet, understanding the cellular behavior and molecular programs underlying different biological processes in the adult zebrafish brain requires techniques that allow manipulation of gene function. As a complementary method to the currently used misexpression techniques in zebrafish, such as transgenic approaches or electroporation-based delivery of DNA, we devised a cerebroventricular microinjection (CVMI)-assisted knockdown protocol that relies on vivo morpholino oligonucleotides, which do not require electroporation for cellular uptake. This rapid method allows uniform and efficient knockdown of genes in the ventricular cells of the zebrafish brain, which contain the neurogenic progenitors. We also provide data on the use of CVMI for growth factor administration to the brain--in our case FGF8, which modulates the proliferation rate of the ventricular cells. In this paper, we describe the CVMI method and discuss its potential uses in zebrafish.", "link"=>"http://www.mendeley.com/research/cerebroventricular-microinjection-cvmi-adult-zebrafish-brain-efficient-misexpression-method-forebrai", "reader_count"=>66, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>5, "Librarian"=>2, "Researcher"=>14, "Student > Doctoral Student"=>1, "Student > Ph. D. Student"=>16, "Student > Postgraduate"=>3, "Student > Master"=>11, "Other"=>2, "Student > Bachelor"=>10, "Professor"=>2}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>5, "Librarian"=>2, "Researcher"=>14, "Student > Doctoral Student"=>1, "Student > Ph. D. Student"=>16, "Student > Postgraduate"=>3, "Student > Master"=>11, "Other"=>2, "Student > Bachelor"=>10, "Professor"=>2}, "reader_count_by_subject_area"=>{"Biochemistry, Genetics and Molecular Biology"=>7, "Agricultural and Biological Sciences"=>48, "Medicine and Dentistry"=>7, "Neuroscience"=>4}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>7}, "Neuroscience"=>{"Neuroscience"=>4}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>48}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>7}}, "reader_count_by_country"=>{"Argentina"=>1, "United States"=>1, "Japan"=>1, "Brazil"=>1, "Macau"=>1, "Portugal"=>2, "Germany"=>3}, "group_count"=>2}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/716539"], "description"=>"<p>(A) Schematic representation of the neurogenesis assay after PCNA knock-down. Following morpholino injection (control and PCNA-antisense), BrdU is given between 12 and 36 hours. Brains were analyzed at 7 days post injection (dpi) for BrdU (marker for proliferated cells), HuC (neuronal marker) and DAPI as a nuclear counterstain. (B) BrdU immunohistochemistry on the dorsal region of telencephalon from control morpholino-injected brain. (C) Co-staining for HuC and BrdU in control morpholino-injected dorsal telencephalon. (D) BrdU immunohistochemistry on the dorsal region of telencephalon from PCNA morpholino-injected brain. (E) Co-staining for HuC and BrdU in PCNA morpholino-injected dorsal telencephalon. (F) Graph depicts the average number of newborn neurons (HuC-BrdU double-positive cells) in control and PCNA morpholino-injected brains. Scale bars 50 µm. N = 4 adult fish.</p>", "links"=>[], "tags"=>["pcna", "neurogenesis"], "article_id"=>386904, "categories"=>["Physiology", "Biochemistry", "Neuroscience", "Cell Biology"], "users"=>["Caghan Kizil", "Michael Brand"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0027395.g005", "stats"=>{"downloads"=>3, "page_views"=>12, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Knocking_down_PCNA_reduces_neurogenesis_as_a_functional_consequence_/386904", "title"=>"Knocking-down PCNA reduces neurogenesis as a functional consequence.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 12:02:23"}
  • {"files"=>["https://ndownloader.figshare.com/files/716250"], "description"=>"<p>(A) GFP immunohistochemistry (IHC) on control morpholino-injected (Ctrl-MO) brain sections. (B) DAPI colocalization of A. (C) GFP immunohistochemistry (IHC) on GFP antisense morpholino-injected (GFP-MO) brain sections. (D) DAPI colocalization of C. Red arrows exemplify fully knocked-down cells. (E) 3D surface plot of A. x-axis: distance from the ventricle; y-axis: ventral to dorsal orientation; z-axis: relative level of fluorescence intensity. (F) 3D surface plot of C. x-axis: distance from the ventricle; y-axis: ventral to dorsal orientation; z-axis: relative level of fluorescence intensity. White arrow shows the gap, which indicates the knocked down cells. (G) Intensity quantification plot depicting relative fluorescence intensity versus distance from the ventricle in Ctrl-MO (black lines) and GFP-MO (red lines) injections. Black asterisk indicates the ventricle. Blue bars delineate the borders where GFP-MO injection leads to overt reduction in GFP fluorescence intensity. (H) Penetration prediction graph. The ratio of fluorescence intensity values between Ctrl-MO (FI<sub>Ctrl-MO</sub>) and GFP-MO (FI<sub>GFP-MO</sub>) versus cell diameters away from the ventricle is sketched. The trendline indicates the extent of knockdown in relation to cell diameters. Scale bars 20 µm.</p>", "links"=>[], "tags"=>["morpholino", "penetration", "knockdown"], "article_id"=>386610, "categories"=>["Physiology", "Biochemistry", "Neuroscience", "Cell Biology"], "users"=>["Caghan Kizil", "Michael Brand"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0027395.g002", "stats"=>{"downloads"=>1, "page_views"=>9, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Analysis_of_morpholino_penetration_and_efficient_knockdown_range_/386610", "title"=>"Analysis of morpholino penetration and efficient knockdown range.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 12:00:47"}
  • {"files"=>["https://ndownloader.figshare.com/files/716713"], "description"=>"<p>(A) Pairwise sequence alignment between human FGF8 (NP_149355) and zebrafish Fgf8 (NP_571356) showing 86.4% similarity between two proteins. Red boxes indicate the residues required for ligand-receptor interaction and they are completely conserved between human and zebrafish. Asterisks indicate identical residues; semicolons indicate conservative substitutions. (B) PCNA immunohistochemistry (IHC) on the telencephalon of BSA-injected brains at 1 dpi. (C) DAPI counterstaining on A. (C') High magnification of dorsal telencephalon. (D) PCNA immunohistochemistry (IHC) on the telencephalon of FGF8-injected brains at 1 dpi. (E) DAPI counterstaining on C. (E') High magnification of dorsal telencephalon. (F) Graph depicts the average number of PCNA-positive cells per section. Scale bars 50 µm. N = 3 adult fish for each injection.</p>", "links"=>[], "tags"=>["fgf8", "increases", "ventricular"], "article_id"=>387075, "categories"=>["Physiology", "Biochemistry", "Neuroscience", "Cell Biology"], "users"=>["Caghan Kizil", "Michael Brand"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0027395.g007", "stats"=>{"downloads"=>0, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_CVMI_of_FGF8_increases_ventricular_cell_proliferation_/387075", "title"=>"CVMI of FGF8 increases ventricular cell proliferation.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 12:03:25"}
  • {"files"=>["https://ndownloader.figshare.com/files/716067"], "description"=>"<p>(A) CVMI is performed at the dorsal surface of the head (1) and it targets, in this example, the forebrain that is rostral to the optic tectum (2). For injection, an incision is made into the skull over the optic tectum using a barbed-end canula (3). Through this slit, liquid is injected using a glass capillary (4). Injected liquid disperses rostrally (5). (B) The canula used for incision. (C) The incision on an adult fish. Dorsal view. (D) The incision site marked by dashed lines. (E) Injection with the glass capillary (*) (dotted lines mark the outline). (F) Dorsal view of an uninjected adult zebrafish head in red fluorescence channel. (G) Dorsal view of a CMTPX-injected adult zebrafish head in red fluorescence channel. (H) Cross section through the telencephalon. Ventricular cells are labelled with CMTPX. (H') Higher magnification of the box in H. (I) Cross section through the midbrain. Ventricular cells are labelled with CMTPX. (I') Higher magnification of the box in I. (J) Injection apparatus. J1: halogen light source with ring illuminator. J2: vacuum pump for microinjection. J3: pressurized air source. J4: dissecting microscope. J5: injection holder, needle and tubing. Scale bars: 500 µm A–G, 100 µm H-I'. ot: optic tectum, tel: telencephalon, ob: olfactory bulb, c: cerebellum, med: medulla, v: ventricle.</p>", "links"=>[], "tags"=>["cerebroventricular", "microinjection", "paradigm"], "article_id"=>386424, "categories"=>["Physiology", "Biochemistry", "Neuroscience", "Cell Biology"], "users"=>["Caghan Kizil", "Michael Brand"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0027395.g001", "stats"=>{"downloads"=>7, "page_views"=>78, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Overview_of_cerebroventricular_microinjection_CVMI_paradigm_and_its_target_regions_/386424", "title"=>"Overview of cerebroventricular microinjection (CVMI) paradigm and its target regions.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 11:59:48"}
  • {"files"=>["https://ndownloader.figshare.com/files/716619"], "description"=>"<p>(A) The dose-response analyses were performed on dorsal regions of telencephalic sections. PCNA immunohistochemistry coupled to DAPI nuclear counterstaining on 500 µM control morpholino-injected (B), 500 µM PCNA morpholino-injected (C), 250 µM PCNA morpholino-injected (D), 100 µM PCNA morpholino-injected (E), 50 µM PCNA morpholino-injected (F) brains. (G) Graph depicts the relative amount of PCNA-positive cells in dorsal telencephalon after every dose in control and PCNA morpholino-injected brains. Scale bars 50 µm. N = 3 adult fish for every dose.</p>", "links"=>[], "tags"=>["knock-down", "cvmi"], "article_id"=>386988, "categories"=>["Physiology", "Biochemistry", "Neuroscience", "Cell Biology"], "users"=>["Caghan Kizil", "Michael Brand"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0027395.g006", "stats"=>{"downloads"=>1, "page_views"=>14, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Morpholino_mediated_gene_knock_down_using_CVMI_is_dose_dependent_/386988", "title"=>"Morpholino-mediated gene knock-down using CVMI is dose-dependent.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 12:02:54"}
  • {"files"=>["https://ndownloader.figshare.com/files/716439"], "description"=>"<p>(A) PCNA immunohistochemistry (IHC) on rostral telencephalon of control morpholino-injected (ctrl-MO) brains. (B) DAPI counterstaining on A. (C) High magnification image of medial ventricular region of B. (D) PCNA IHC on rostral telencephalon of PCNA morpholino-injected (PCNA-MO) brains. (E) DAPI counterstaining on D. (F) High magnification image of medial ventricular region of E. (G) PCNA IHC on rostral telencephalon of ctrl-MO brains. (H) DAPI counterstaining on G. (I) High magnification image of medial ventricular region of H. (J) PCNA IHC on telencephalon of PCNA-MO brains on a more caudal level. (K) DAPI counterstaining on J. (L) High magnification image of medial ventricular region of K. (M) PCNA IHC on rostral optic tectum of ctrl-MO brains. (N) DAPI counterstaining on M. (O) High magnification image of dorsal region of N. (P) PCNA IHC on rostral optic tectum of PCNA-MO brains. (Q) DAPI counterstaining on P. (R) High magnification image of dorsal region of Q. (S) Graph depicts the average number of PCNA-positive cells in PCNA antisense morpholino-injected brains (green line) over a time course relative to control morpholino-injected brains (red line). Scale bars 50 µm. hpi: hours post injection, dpi: days post injection. N = 3 adult fish for each time point.</p>", "links"=>[], "tags"=>["pcna", "endogenous"], "article_id"=>386797, "categories"=>["Physiology", "Biochemistry", "Neuroscience", "Cell Biology"], "users"=>["Caghan Kizil", "Michael Brand"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0027395.g004", "stats"=>{"downloads"=>1, "page_views"=>7, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Knocking_down_PCNA_as_an_endogenous_gene_using_CVMI_/386797", "title"=>"Knocking-down PCNA as an endogenous gene using CVMI.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 12:01:48"}
  • {"files"=>["https://ndownloader.figshare.com/files/716340"], "description"=>"<p>(A) Fluorescence reporter activity of <i>Tg(her4.1:mCherry)</i> transgenic line in the radial glial cells (red arrows along the ventricular surface) of the rostral telencephalon. (A') Higher magnification of the dorsomedial region of A. (B) Fluorescence reporter activity in rostral telencephalon injected with translation-blocking vivo morpholinos for mCherry transgene. (B') Higher magnification of the dorsomedial region of B, indicating the significant reduction of reporter activity. (C) Fluorescence reporter activity of <i>Tg(her4.1:mCherry)</i> transgenic line in the radial glial cells (red arrows along the ventricular surface) of the caudal telencephalon. (C') Higher magnification of the medial region of C. (D) Fluorescence reporter activity in caudal telencephalon injected with translation-blocking vivo morpholinos for mCherry transgene. (D') Higher magnification of the medial region of D, indicating the significant reduction of reporter activity. (E) Graph depicts the average mCherry fluorescence intensity in mCherry antisense morpholino-injected brains (green line) over a time course relative to control morpholino-injected brains (red line). Scale bars 50 µm. v: ventricle, tel: telencephalon, hpi: hours post injection, dpi: days post injection. N = 6 adult fish for each time point.</p>", "links"=>[], "tags"=>["knockdown", "cvmi"], "article_id"=>386703, "categories"=>["Physiology", "Biochemistry", "Neuroscience", "Cell Biology"], "users"=>["Caghan Kizil", "Michael Brand"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0027395.g003", "stats"=>{"downloads"=>1, "page_views"=>22, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Morpholino_mediated_gene_knockdown_using_CVMI_in_Tg_her4_1_mCherry_reporter_line_/386703", "title"=>"Morpholino-mediated gene knockdown using CVMI in <i>Tg(her4.1:mCherry)</i> reporter line.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 12:01:18"}

PMC Usage Stats | Further Information

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  • {"unique-ip"=>"9", "full-text"=>"5", "pdf"=>"3", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"3", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2014", "month"=>"7"}
  • {"unique-ip"=>"14", "full-text"=>"9", "pdf"=>"4", "abstract"=>"1", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"9", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2014", "month"=>"8"}
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