Major Signaling Pathways in Migrating Neuroblasts

Neuronal migration is a key process in the developing and adult brain. Numerous factors act on intracellular cascades of migrating neurons and regulate the final position of neurons. One robust migration route persists postnatally – the rostral migratory stream (RMS). To identify genes that govern neuronal migration in this unique structure, we isolated RMS neuroblasts by making use of transgenic mice that express EGFP in this cell population and performed microarray analysis on RNA. We compared gene expression patterns of neuroblasts obtained from two sites of the RMS, one closer to the site of origin, the subventricular zone, and one closer to the site of the final destination, the olfactory bulb (OB). We identified more than 400 upregulated genes, many of which were not known to be involved in migration. These genes were grouped into functional networks by bioinformatics analysis. Selecting a specific upregulated intracellular network, the cytoskeleton pathway, we confirmed by functional in vitro and in vivo analysis that the identified genes of this network affected RMS neuroblast migration. Based on the validity of this approach, we chose four new networks and tested by functional in vivo analysis their involvement in neuroblast migration. Thus, knockdown of Calm1, Gria1 (GluA1) and Camk4 (calmodulin-signaling network), Hdac2 and Hsbp1 (Akt1-DNA transcription network), Vav3 and Ppm1a (growth factor signaling network) affected neuroblast migration to the OB

Localization of Retinal Ca2+/Calmodulin-Dependent Kinase II-β (CaMKII-β) at Bipolar Cell Gap Junctions and Cross-Reactivity of a Monoclonal Anti-CaMKII-β Antibody With Connexin36

Neuronal gap junctions formed by connexin36 (Cx36) and chemical synapses share striking similarities in terms of plasticity. Ca2+/calmodulin-dependent protein kinase II (CaMKII), an enzyme known to induce memory formation at chemical synapses, has recently been described to potentiate electrical coupling in the retina and several other brain areas via phosphorylation of Cx36. The contribution of individual CaMKII isoforms to this process, however, remains unknown. We recently identified CaMKII-β at electrical synapses in the mouse retina. Now, we set out to identify cell types containing Cx36 gap junctions that also express CaMKII-β. To ensure precise description, we first tested the specificity of two commercially available antibodies on CaMKII-β-deficient retinas. We found that a polyclonal antibody was highly specific for CaMKII-β. However, a monoclonal antibody (CB-β-1) recognized CaMKII-β but also cross-reacted with the C-terminal tail of Cx36, making localization analyses with this antibody inaccurate. Using the polyclonal antibody, we identified strong CaMKII-β expression in bipolar cell terminals that were secretagogin- and HCN1-positive and thus represent terminals of type 5 bipolar cells. In these terminals, a small fraction of CaMKII-β also colocalized with Cx36. A similar pattern was observed in putative type 6 bipolar cells although there, CaMKII expression seemed less pronounced. Next, we tested whether CaMKII-β influenced the Cx36 expression in bipolar cell terminals by quantifying the number and size of Cx36-immunoreactive puncta in CaMKII-β-deficient retinas. However, we found no significant differences between the genotypes, indicating that CaMKII-β is not necessary for the formation and maintenance of Cx36-containing gap junctions in the retina. In addition, in wild-type retinas, we observed frequent association of Cx36 and CaMKII-β with synaptic ribbons, i.e., chemical synapses, in bipolar cell terminals. This arrangement resembled the composition of mixed synapses found for example in Mauthner cells, in which electrical coupling is regulated by glutamatergic activity. Taken together, our data imply that CaMKII-β may fulfill several functions in bipolar cell terminals, regulating both Cx36-containing gap junctions and ribbon synapses and potentially also mediating cross-talk between these two types of bipolar cell outputs

Gene Expression Analysis of In Vivo Fluorescent Cells

BACKGROUND: The analysis of gene expression for tissue homogenates is of limited value because of the considerable cell heterogeneity in tissues. However, several methods are available to isolate a cell type of interest from a complex tissue, the most reliable one being Laser Microdissection (LMD). Cells may be distinguished by their morphology or by specific antigens, but the obligatory staining often results in RNA degradation. Alternatively, particular cell types can be detected in vivo by expression of fluorescent proteins from cell type-specific promoters. METHODOLOGY/PRINCIPAL FINDINGS: We developed a technique for fixing in vivo fluorescence in brain cells and isolating them by LMD followed by an optimized RNA isolation procedure. RNA isolated from these cells was of equal quality as from unfixed frozen tissue, with clear 28S and 18S rRNA bands of a mass ratio of approximately 2ratio1. We confirmed the specificity of the amplified RNA from the microdissected fluorescent cells as well as its usefulness and reproducibility for microarray hybridization and quantitative real-time PCR (qRT-PCR). CONCLUSIONS/SIGNIFICANCE: Our technique guarantees the isolation of sufficient high quality RNA obtained from specific cell populations of the brain expressing soluble fluorescent marker, which is a critical prerequisite for subsequent gene expression studies by microarray analysis or qRT-PCR

AMPA, kainate and NMDA ionotropic glutamate receptor expression: an in situ hybridization atlas

Most rat neocortical neurons have Ca2+-impermeable AMPA receptors containing GluR-A/B, heteromeric receptors will be least numerous in layer IV cells (Fig. 11) (discussed by Conti et al., 1994a). Most GABAergic interneurons have Ca2+-permeable AMPA receptors made from GluR-A/D subunits; these receptors will have fast kinetics and high single-channel conductance

The distribution of 13 GABAA receptor subunit mRNAs in the rat brain - I. Telencephalon, diencephalon, mesencephalon

The expression patterns of 13 GABAA receptor subunit encoding genes (alpha 1-alpha 6, beta 1-beta 3, gamma 1-gamma 3, delta) were determined in adult rat brain by in situ hybridization. Each mRNA displayed a unique distribution, ranging from ubiquitous (alpha 1 mRNA) to narrowly confined (alpha 6 mRNA was present only in cerebellar granule cells). Some neuronal populations coexpressed large numbers of subunit mRNAs, whereas in others only a few GABAA receptor-specific mRNAs were found. Neocortex, hippocampus, and caudate-putamen displayed complex expression patterns, and these areas probably contain a large diversity of GABAA receptors. In many areas, a consistent coexpression was observed for alpha 1 and beta 2 mRNAs, which often colocalized with gamma 2 mRNA. The alpha 1 beta 2 combination was abundant in olfactory bulb, globus pallidus, inferior colliculus, substantia nigra pars reticulata, globus pallidus, zona incerta, subthalamic nucleus, medial septum, and cerebellum. Colocalization was also apparent for the alpha 2 and beta 3 mRNAs, and these predominated in areas such as amygdala and hypothalamus. The alpha 3 mRNA occurred in layers V and VI of neocortex and in the reticular thalamic nucleus. In much of the forebrain, with the exception of hippocampal pyramidal cells, the alpha 4 and delta transcripts appeared to codistribute. In thalamic nuclei, the only abundant GABAA receptor mRNAs were those of alpha 1, alpha 4, beta 2, and delta. In the medial geniculate thalamic nucleus, alpha 1, alpha 4, beta 2, delta, and gamma 3 mRNAs were the principal GABAA receptor transcripts. The alpha 5 and beta 1 mRNAs generally colocalized and may encode predominantly hippocampal forms of the GABAA receptor. These anatomical observations support the hypothesis that alpha 1 beta 2 gamma 2 receptors are responsible for benzodiazepine I (BZ I) binding, whereas receptors containing alpha 2, alpha 3, and alpha 5 contribute to subtypes of the BZ II site. Based on significant mismatches between alpha 4/delta and gamma mRNAs, we suggest that in vivo, the alpha 4 subunit contributes to GABAA receptors that lack BZ modulation