Axonal Patterns and Targets of dA1 Interneurons in the Chick Hindbrain

Kohl, Ayelet; Hadas, Yoav; Klar, Avihu; Sela-Donenfeld, Dalit

doi:10.1523/jneurosci.4231-11.2012

Cited by 27 publications

(56 citation statements)

References 113 publications

(160 reference statements)

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“…This protocol was recently used to uncover the axonal patterns and projection sites of dA1 subgroup of interneurons in the chick hindbrain 10 .…”

Section: Representative Resultsmentioning

confidence: 99%

“…To specifically label these axons, an enhancer element (Atoh1), that has previously been characterized as specific for spinal dI1 neurons 8,12,13 , was confirmed to be expressed in hindbrain dA1 cells 10 . The element was cloned upstream to Cre recombinase and co-electroporated at E2.75 along with a Cre dependent cytoplasmic GFP reporter plasmid (pCAGG-LoxP-Stop-LoxP-cGFP; Figure 1BI).…”

Section: Representative Resultsmentioning

confidence: 99%

“…A reporter Cre-conditional-myristoylated-GFP (mGFP) cassette, cloned between the two PB arms (PB-CAG-LoxP-STOP-LoxP-mGFP-PB), was electroporated at E2.75 along with the Atoh1::Cre enhancer and the transposase vector (CAG::PBase). In this strategy, the electroporated Creconditional mGFP is integrated into the chick chromosomes and consequently the STOP cassette is removed only in dA1 neurons, enabling prolonged expression of GFP in dA1 cells (Figure 1BII; 10 ). The myristoylated form of the GFP tethers it to the membrane such that it is localized along the axonal membrane rather than being diluted in the cytoplasm.…”

Section: Representative Resultsmentioning

confidence: 99%

“…In addition, molecular identities of subgroups of neurons that projected into the reported funiculi could not be revealed. Our combined method of electroporation and sustained genetic tracing provides the unique ability to follow the development of selected hindbrain neurons from their specification stages to axonal navigation and projection targets 10 . Notably, our approach was previously applied to trace the axonal trajectories of spinal cord dorsal interneurons for shorter periods of time 8,9 .…”

Section: Discussionmentioning

confidence: 99%

“…Based on this method, we labeled the dorsal-most subgroup of hindbrain interneurons (dA1/Atoh1 + cells) and revealed two contralateral ascending axonal projection patterns, each derives from a different AP location and elongates in a distinct funiculus. dA1 axons were found to project and form synapses in the auditory nuclei, midbrain and in multiple layers of the cerebellum 10 .…”

Section: Introductionmentioning

confidence: 99%

See 4 more Smart Citations

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Kohl

Hadas

Klar

et al. 2013

JoVE

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Electroporation of the chick embryonic neural tube has many advantages such as being quick and efficient for the expression of foreign genes into neuronal cells. In this manuscript we provide a method that demonstrates uniquely how to electroporate DNA into the avian hindbrain at E2.75 in order to specifically label a subset of neuronal progenitors, and how to follow their axonal projections and synaptic targets at much advanced stages of development, up to E14.5. We have utilized novel genetic tools including specific enhancer elements, Cre/Loxbased plasmids and the PiggyBac-mediated DNA transposition system to drive GFP expression in a subtype of hindbrain cells (the dorsal most subgroup of interneurons, dA1). Axonal trajectories and targets of dA1 axons are followed at early and late embryonic stages at various brainstem regions. This strategy contributes advanced techniques for targeting cells of interest in the embryonic hindbrain and for tracing circuit formation at multiple stages of development. Video LinkThe video component of this article can be found at

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“…This protocol was recently used to uncover the axonal patterns and projection sites of dA1 subgroup of interneurons in the chick hindbrain 10 .…”

Section: Representative Resultsmentioning

confidence: 99%

Section: Representative Resultsmentioning

confidence: 99%

Section: Representative Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Kohl

Hadas

Klar

et al. 2013

JoVE

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Axon guidance at the spinal cord midline—A live imaging perspective

2021

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During neural circuit formation, axons navigate several choice points to reach their final target. At each one of these intermediate targets, growth cones need to switch responsiveness from attraction to repulsion in order to move on. Molecular mechanisms that allow for the precise timing of surface expression of a new set of receptors that support the switch in responsiveness are difficult to study in vivo. Mostly, mechanisms are inferred from the observation of snapshots of many different growth cones analyzed in different preparations of tissue harvested at distinct time points. However, to really understand the behavior of growth cones at choice points, a single growth cone should be followed arriving at and leaving the intermediate target. Existing ex vivo preparations, like cultures of an “open‐book” preparation of the spinal cord have been successfully used to study floor plate entry and exit, but artifacts prevent the analysis of growth cone behavior at the floor plate exit site. Here, we describe a novel spinal cord preparation that allows for live imaging of individual axons during navigation in their intact environment. When comparing growth cone behavior in our ex vivo system with snapshots from in vivo navigation, we do not see any differences. The possibility to observe the dynamics of single growth cones navigating their intermediate target allows for measuring growth speed, changes in morphology, or aberrant behavior, like stalling and wrong turning. Moreover, observation of the intermediate target—the floor plate—revealed its active participation and interaction with commissural axons during midline crossing.

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Specific labeling of climbing fibers shows early synaptic interactions with immature Purkinje cells in the prenatal cerebellum

Kita

Tanaka

Murakami

2014

Developmental Neurobiology

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During development, growing axons must locate target cells to form synapses. This is not easy, since target cells are also growing and even actively migrating. In some brain regions, such axons have been reported to wait for the timing when target cells become mature, without invading their target region. However, in the cerebellum climbing fibers (CFs), major afferent axons, arrive near their target neurons, Purkinje cells, when the neurons are still actively migrating. We, therefore, examined whether synaptic contacts are established at such early stages. To specifically label CFs, we introduced by in utero electroporation a mixture of genes encoding for Ptf1a-enhancer-driven Cre recombinase and Cre-dependent fluorescent protein into the mouse hindbrain at embryonic day (E) 10.5 and observed them during development. The earliest stages at which labeled CFs were observed in the cerebellar primordium were E15.5-E16.5. These fibers were fasciculated in the dorsal region and entered the cerebellar primordium. Some fibers defasciculated and reached the caudal region. At E17.5 and E18.5, fasciculated fibers were also found in the mantle region, and some grew toward the surface of the primordium to penetrate a mass of Purkinje cells. Interestingly, as early as E16.5, labeled fibers were found to run in close apposition to Purkinje cell dendrites and to express a presynaptic marker. These observations suggest that CFs form synapses with Purkinje cells as soon as the fibers enter the cerebellum.

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Axonal Patterns and Targets of dA1 Interneurons in the Chick Hindbrain

Cited by 27 publications

References 113 publications

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Axon guidance at the spinal cord midline—A live imaging perspective

Specific labeling of climbing fibers shows early synaptic interactions with immature Purkinje cells in the prenatal cerebellum

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