Single-Cell Reconstruction of Oxytocinergic Neurons Reveals Separate Hypophysiotropic and Encephalotropic Subtypes in Larval Zebrafish

Oxytocin regulates a diverse set of processes including stress, analgesia, metabolism, and social behavior. How such diverse functions are mediated by a single hormonal system is not well understood. Different functions of oxytocin could be mediated by distinct cell groups, yet it is currently unknown whether different oxytocinergic cell types exist that specifically mediate peripheral neuroendocrine or various central neuromodulatory processes via dedicated pathways. Using the Brainbow technique to map the morphology and projections of individual oxytocinergic cells in the larval zebrafish brain, we report here the existence of two main types of oxytocinergic cells: those that innervate the pituitary and those that innervate diverse brain regions. Similar to the situation in the adult rat and the adult midshipman, but in contrast to the situation in the adult trout, these two cell types are mutually exclusive and can be distinguished based on morphological and anatomical criteria. Further, our results reveal that complex oxytocinergic innervation patterns are already established in the larval zebrafish brain

Quantitative comparison of DNA detection by GFP-lac repressor tagging, fluorescence in situ hybridization and immunostaining

<p>Abstract</p> <p>Background</p> <p>GFP-fusion proteins and immunostaining are methods broadly applied to investigate the three-dimensional organization of cells and cell nuclei, the latter often studied in addition by fluorescence in situ hybridization (FISH). Direct comparisons of these detection methods are scarce, however.</p> <p>Results</p> <p>We provide a quantitative comparison of all three approaches. We make use of a cell line that contains a transgene array of lac operator repeats which are detected by GFP-lac repressor fusion proteins. Thus we can detect the same structure in individual cells by GFP fluorescence, by antibodies against GFP and by FISH with a probe against the transgene array. Anti-GFP antibody detection was repeated after FISH. Our results show that while all four signals obtained from a transgene array generally showed qualitative and quantitative similarity, they also differed in details.</p> <p>Conclusion</p> <p>Each of the tested methods revealed particular strengths and weaknesses, which should be considered when interpreting respective experimental results. Despite the required denaturation step, FISH signals in structurally preserved cells show a surprising similarity to signals generated before denaturation.</p

The Protein Dendrite Arborization and Synapse Maturation 1 (Dasm-1) Is Dispensable for Dendrite Arborization▿ †

The development of a highly branched dendritic tree is essential for the establishment of functional neuronal connections. The evolutionarily conserved immunoglobulin superfamily member, the protein dendrite arborization and synapse maturation 1 (Dasm-1) is thought to play a critical role in dendrite formation of dissociated hippocampal neurons. RNA interference-mediated Dasm-1 knockdown was previously shown to impair dendrite, but not axonal, outgrowth and branching (S. H. Shi, D. N. Cox, D. Wang, L. Y. Jan, and Y. N. Jan, Proc. Natl. Acad. Sci. USA 101:13341-13345, 2004). Here, we report the generation and analysis of Dasm-1 null mice. We find that genetic ablation of Dasm-1 does not interfere with hippocampal dendrite growth and branching in vitro and in vivo. Moreover, the absence of Dasm-1 does not affect the modulation of dendritic outgrowth induced by brain-derived neurotrophic factor. Importantly, the previously observed impairment in dendrite growth after Dasm-1 knockdown is also observed when the Dasm-1 knockdown is performed in cultured hippocampal neurons from Dasm-1 null mice. These findings indicate that the dendrite arborization phenotype was caused by off-target effects and that Dasm-1 is dispensable for hippocampal dendrite arborization

Comparison of GFP, immunostaining and FISH signals without RNAse digestion, allowing for detection of RNA from the transgene arrays by the FISH probe

<p><b>Copyright information:</b></p><p>Taken from "Quantitative comparison of DNA detection by GFP-lac repressor tagging, fluorescence in situ hybridization and immunostaining"</p><p>http://www.biomedcentral.com/1472-6750/7/92</p><p>BMC Biotechnology 2007;7():92-92.</p><p>Published online 20 Dec 2007</p><p>PMCID:PMC2254608.</p><p></p> For panel labeling see Figure 2. Projections of deconvolved images are shown. Note the larger volume of FISH signals compared to other signals. In this experiment, due to the large differences in signal appearance, pre- and post-FISH signals could not be subjected to automated image processing. Therefore, for this experiment only, post-FISH images were matched to the orientation of pre-FISH images manually

Dual color FISH with two different plasmids detecting the same transgene array

<p><b>Copyright information:</b></p><p>Taken from "Quantitative comparison of DNA detection by GFP-lac repressor tagging, fluorescence in situ hybridization and immunostaining"</p><p>http://www.biomedcentral.com/1472-6750/7/92</p><p>BMC Biotechnology 2007;7():92-92.</p><p>Published online 20 Dec 2007</p><p>PMCID:PMC2254608.</p><p></p> This 50 Mbp transgene array is composed of multiple transgene copies as well as host DNA. pPALZ8.8 (green and center, labeled with FITC) was originally used to generate the transgene array and thus completely covers transgenes while pPS8.8 (red, bottom, labeled with Cy5) detects only the lacO sequence which comprises one sixth of the complete transgene length. CC values are given in percent. They were calculated in 3D, thus they do not necessarily reflect similarity in the projections of deconvolved image stacks shown here. Scale bar: 2 μm

Comparison of GFP, immunostaining and FISH signals from the same nuclear structure

<p><b>Copyright information:</b></p><p>Taken from "Quantitative comparison of DNA detection by GFP-lac repressor tagging, fluorescence in situ hybridization and immunostaining"</p><p>http://www.biomedcentral.com/1472-6750/7/92</p><p>BMC Biotechnology 2007;7():92-92.</p><p>Published online 20 Dec 2007</p><p>PMCID:PMC2254608.</p><p></p> Upper rows show projections of deconvolved image stacks, lower rows those from non-deconvolved images. As indicated at the top of the panel, color overlays on the left are from GFP (green) and antibody signals before FISH (preAB, red, Cy5), color overlays on the right from antibody signals after FISH (postAB, red, Cy5) and FISH signals (green, FITC). Indicated CC values are based on pair-wise comparisons of 3D image stacks, thus they do not necessarily reflect similarity in the projections. Lines on the side of CC values indicate which signals were compared. Shown are examples for high to average CC values (top and center) and low CC values