Genetically-Directed, Cell Type-Specific Sparse Labeling for the Analysis of Neuronal Morphology

Background: In mammals, genetically-directed cell labeling technologies have not yet been applied to the morphologic analysis of neurons with very large and complex arbors, an application that requires extremely sparse labeling and that is only rendered practical by limiting the labeled population to one or a few predetermined neuronal subtypes. Methods and Findings: In the present study we have addressed this application by using CreER technology to noninvasively label very small numbers of neurons so that their morphologies can be fully visualized. Four lines of IRES-CreER knock-in mice were constructed to permit labeling selectively in cholinergic or catecholaminergic neurons [choline acetyltransferase (ChAT)-IRES-CreER or tyrosine hydroxylase (TH)-IRES-CreER], predominantly in projection neurons [neurofilament light chain (NFL)-IRES-CreER], or broadly in neurons and some glia [vesicle-associated membrane protein2 (VAMP2)-IRES-CreER]. When crossed to the Z/AP reporter and exposed to 4-hydroxytamoxifen in the early postnatal period, the number of neurons expressing the human placental alkaline phosphatase reporter can be reproducibly lowered to fewer than 50 per brain. Sparse Cre-mediated recombination in ChAT-IRES-CreER;Z/AP mice shows the full axonal and dendritic arbors of individual forebrain cholinergic neurons, the first time that the complete morphologies of these very large neurons have been revealed in any species. Conclusions: Sparse genetically-directed, cell type-specific neuronal labeling with IRES-creER lines should prove useful fo

GABA Expression and Regulation by Sensory Experience in the Developing Visual System

The developing retinotectal system of the Xenopus laevis tadpole is a model of choice for studying visual experience-dependent circuit maturation in the intact animal. The neurotransmitter gamma-aminobutyric acid (GABA) has been shown to play a critical role in the formation of sensory circuits in this preparation, however a comprehensive neuroanatomical study of GABAergic cell distribution in the developing tadpole has not been conducted. We report a detailed description of the spatial expression of GABA immunoreactivity in the Xenopus laevis tadpole brain at two key developmental stages: stage 40/42 around the onset of retinotectal innervation and stage 47 when the retinotectal circuit supports visually-guided behavior. During this period, GABAergic neurons within specific brain structures appeared to redistribute from clusters of neuronal somata to a sparser, more uniform distribution. Furthermore, we found that GABA levels were regulated by recent sensory experience. Both ELISA measurements of GABA concentration and quantitative analysis of GABA immunoreactivity in tissue sections from the optic tectum show that GABA increased in response to a 4 hr period of enhanced visual stimulation in stage 47 tadpoles. These observations reveal a remarkable degree of adaptability of GABAergic neurons in the developing brain, consistent with their key contributions to circuit development and function

Microinjection and micropipette-controlled phagocytosis methods for neutrophils

The ability to microinject substances into the cytosol of living neutrophils opens the possibility of manipulating the chemistry within the cell and also of monitoring changes using indicators which otherwise cannot be introduced into the cell. However, neutrophils cannot be microinjected by the conventional glass pipette insertion method. Here we outline two techniques which work well with neutrophils, namely, SLAM (Simple Lipid-Assisted Microinjection) and electromicroinjection. As these methods utilize micropipettes, we also include a simple method which uses a micropipette to deliver a phagocytic stimulus to a specific cell at a defined time, enable detailed study of the phagocytic process from particle contact to particle internalization

Integrated Electrowetting Nanoinjector for Single Cell Transfection

Single cell transfection techniques are essential to understand the heterogeneity between cells. We have developed an integrated electrowetting nanoinjector (INENI) to transfect single cells. The high transfection efficiency, controlled dosage delivery and ease of INENI fabrication promote the widespread application of the INENI in cell transfection assays.</p

Targeted single-cell electroporation of mammalian neurons in vivo.

In order to link our knowledge of single neurons with theories of network function, it has been a long-standing goal to manipulate the activity and gene expression of identified subsets of mammalian neurons within the intact brain in vivo. This protocol describes a method for delivering plasmid DNA into single identified mammalian neurons in vivo, by combining two-photon imaging with single-cell electroporation. Surgery, mounting of a chronic recording chamber and targeted electroporation of identified neurons can be performed within 1-2 h. Stable transgene expression can reliably be induced with high success rates both in single neurons as well as in small, spatially defined networks of neurons in the cerebral cortex of rodents