Double labeling of neurons by retrograde axonal tracing and non-radioactive in situ hybridization in the CNS of adult zebrafish

Lieberoth, Bettina C; Becker, Catherina G.; Becker, Thomas

doi:10.1023/b:mics.0000006848.57869.4c

Cited by 16 publications

(11 citation statements)

References 14 publications

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“…Digoxigenin (DIG)-labeled RNA sense and antisense probes for mouse CRP1 (NM_007791, full length) were generated using the Megascript system (Ambion) according to the manufacturer's protocol, and in situ hybridization was performed as described previously (Becker et al, 1998;Lieberoth et al, 2003). In brief, 20 m-thick sagittal brain sections were incubated with 0.1N HCl for 10 min, followed by three washes in PBS, pH 7.4, and digested for 10 min with 10 g/ml proteinase-K (Roche) at room temperature, followed by fixation in 4% paraformaldehyde after two washes with glycine (2 mg/ml) in PBS, pH 7.4.…”

Section: Methodsmentioning

confidence: 99%

CRP1, a Protein Localized in Filopodia of Growth Cones, Is Involved in Dendritic Growth

Ma¹,

Greenwood²,

Schachner³

2011

J. Neurosci.

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The cysteine-rich protein (CRP) family is a subgroup of LIM domain proteins. CRP1, which cross-links actin filaments to make actin bundles, is the only CRP family member expressed in the CNS with little known about its function in nerve cells. Here, we report that CRP1 colocalizes with actin in the filopodia of growth cones in cultured rat hippocampal neurons. Knockdown of CRP1 expression by short hairpin RNA interference results in inhibition of filopodia formation and dendritic growth in neurons. Overexpression of CRP1 increases filopodia formation and neurite branching, which require its actin-bundling activity. Expression of CRP1 with a constitutively active form of Cdc42, a GTPase involved in filopodia formation, increases filopodia formation in COS-7 cells, suggesting cooperation between the two proteins. Moreover, we demonstrate that neuronal activity upregulates CRP1 expression in hippocampal neurons via Ca 2ϩ influx after depolarization. Ca 2ϩ /calmodulin-dependent protein kinase IV (CaMKIV) and cAMP response element binding protein mediate the Ca 2ϩ -induced upregulation of CRP1 expression. Furthermore, CRP1 is required for the dendritic growth induced by Ca 2ϩ influx or CaMKIV. Together, these data are the first to demonstrate a role for CRP1 in dendritic growth.

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Section: Methodsmentioning

confidence: 99%

CRP1, a Protein Localized in Filopodia of Growth Cones, Is Involved in Dendritic Growth

Ma¹,

Greenwood²,

Schachner³

2011

J. Neurosci.

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“…The method for nonradioactive in situ hybridization on 50-lm vibrating blade microtome sections (Lieberoth et al, 2003) and the plasmids to generate probes for th2 (Filippi et al, 2010) have been described. Solutions were made up with RNAse-free water.…”

Section: In Situ Hybridizationmentioning

confidence: 99%

Plasticity of tyrosine hydroxylase and serotonergic systems in the regenerating spinal cord of adult zebrafish

Kuscha

Barreiro‐Iglesias

Becker

et al. 2012

J of Comparative Neurology

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Monoaminergic innervation of the spinal cord has important modulatory functions for locomotion. Here we performed a quantitative study to determine the plastic changes of tyrosine hydroxylase-positive (TH1(+); mainly dopaminergic), and serotonergic (5-HT(+)) terminals and cells during successful spinal cord regeneration in adult zebrafish. TH1(+) innervation in the spinal cord is derived from the brain. After spinal cord transection, TH1(+) immunoreactivity is completely lost from the caudal spinal cord. Terminal varicosities increase in density rostral to the lesion site compared with unlesioned controls and are re-established in the caudal spinal cord at 6 weeks post lesion. Interestingly, axons mostly fail to re-innervate more caudal levels of the spinal cord even after prolonged survival times. However, densities of terminal varicosities correlate with recovery of swimming behavior, which is completely lost again after re-lesion of the spinal cord. Similar observations were made for terminals derived from descending 5-HT(+) axons from the brain. In addition, spinal 5-HT(+) neurons were newly generated after a lesion and transiently increased in number up to fivefold, which depended in part on hedgehog signaling. Overall, TH1(+) and 5-HT(+) innervation is massively altered in the successfully regenerated spinal cord of adult zebrafish. Despite these changes in TH and 5-HT systems, a remarkable recovery of swimming capability is achieved, suggesting significant plasticity of the adult spinal network during regeneration.

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“…We used standard methods (Thisse et al, 1993) to label mRNA in whole-zebrafish embryos. In situ hybridization in adult specimens was performed as previously described by Lieberoth et al (2003). Subsequent immunohistochemistry was performed as above.…”

Section: In Situ Hybridizationmentioning

confidence: 99%

Claudin k is specifically expressed in cells that form myelin during development of the nervous system and regeneration of the optic nerve in adult zebrafish

Münzel

Schaefer

Obirei

et al. 2011

Glia

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The zebrafish has become an important model organism to study myelination during development and after a lesion of the adult central nervous system (CNS). Here, we identify Claudin k as a myelin-associated protein in zebrafish and determine its localization during development and adult optic nerve regeneration. We find Claudin k in subcellular compartments consistent with location in autotypic tight junctions of oligodendrocytes and myelinating Schwann cells. Expression starts in the hindbrain at 2 days (mRNA) and 3 days (protein) postfertilization and is maintained in adults. A newly generated claudin k:green fluorescent protein (GFP) reporter line allowed us to characterize oligodendrocytes in the adult retina that express Claudin k and olig2, but not P0 and uniquely only form loose wraps of membrane around axons. After a crush of the adult optic nerve, Claudin k protein levels were first reduced and then recovered within 4 weeks postlesion, concomitant with optic nerve myelin de- and regeneration. During optic nerve regeneration, oligodendrocytes, many of which were newly generated, repopulated the lesion site and exhibited increasing morphological complexity over time. Thus, Claudin k is a novel myelin-associated protein expressed by oligodendrocytes and Schwann cells from early stages of wrapping and myelin formation in zebrafish development and adult regeneration, suggesting important functions of the gene for myelin formation and maintenance. Our Claudin k antibodies and claudin k:GFP reporter line represent excellent ways to visualize oligodendrocyte and Schwann cell differentiation in vivo.

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Double labeling of neurons by retrograde axonal tracing and non-radioactive in situ hybridization in the CNS of adult zebrafish

Cited by 16 publications

References 14 publications

CRP1, a Protein Localized in Filopodia of Growth Cones, Is Involved in Dendritic Growth

CRP1, a Protein Localized in Filopodia of Growth Cones, Is Involved in Dendritic Growth

Plasticity of tyrosine hydroxylase and serotonergic systems in the regenerating spinal cord of adult zebrafish

Claudin k is specifically expressed in cells that form myelin during development of the nervous system and regeneration of the optic nerve in adult zebrafish

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