Distribution of bursting neurons in the CA1 region and the subiculum of the rat hippocampus.

We performed patch-clamp recordings from morphologically identified and anatomically mapped pyramidal neurons of the ventral hippocampus to test the hypothesis that bursting neurons are distributed on a gradient from the CA2/CA1 border (proximal) through the subiculum (distal), with more bursting observed at distal locations. We find that the well-defined morphological boundaries between the hippocampal subregions CA1 and subiculum do not correspond to abrupt changes in electrophysiological properties. Rather, we observed that the percentage of bursting neurons is linearly correlated with position in the proximal-distal axis across the CA1 and the subiculum, the percentages of bursting neurons being 10% near the CA1-CA2 border, 24% at the CA1-subiculum border, and higher than 50% in the distal subiculum. The distribution of bursting neurons was paralleled by a gradient in afterdepolarization (ADP) amplitude. We also tested the hypothesis that there was an association between bursting and two previously described morphologically distinct groups of pyramidal neurons (twin and single apical dendrites) in the CA1 region. We found no difference in output mode between single and twin apical dendrite morphologies, which was consistent with the observation that the two morphologies were equally distributed across the transverse axis of the CA1 region. Taken together with the known organization of connections from CA3 to CA1 and CA1 to subiculum, our results indicate that bursting neurons are most likely to be connected to regular spiking neurons and vice versa.

Isolation of rat telencephalic neural explants to assay Shh GABAergic interneuron differentiation-inducing activity.

Multiple assays for Shh activity using cell lines, primary cultures, and explanted tissue have been described. We first described the use of E11.5 rat dorsal telencephalic explants to assay Shh ventralizing and differentiation-inducing activity in Kohtz et al. (1). Using this assay, we subsequently showed that N-lipid modification is critical for Shh activity in the telencephalon (2). In vivo assays for lipid-modified Shh support the results of our E11.5 telencephalic neural explant assay (2). More recently, the method of isolating telencephaic explants was improved by an intraocular grid, increasing both its accuracy and reproducibility (3). Shh induces the expression of the following ventral telencephalic markers: MASH-1, the Dlx's, and Islet 1/2. Therefore, this assay for Shh induction of GABAergic interneurons defines a competent, but naïve region within the E11.5 dorsal telencephalon, allowing the study of GABAergic interneuron induction and differentiation from an unspecified progenitor population.

The Evf-2 noncoding RNA is transcribed from the Dlx-5/6 ultraconserved region and functions as a Dlx-2 transcriptional coactivator.

The identification of ultraconserved noncoding sequences in vertebrates has been associated with developmental regulators and DNA-binding proteins. One of the first of these was identified in the intergenic region between the Dlx-5 and Dlx-6 genes, members of the Dlx/dll homeodomain-containing protein family. In previous experiments, we showed that Sonic hedgehog treatment of forebrain neural explants results in the activation of Dlx-2 and the novel noncoding RNA (ncRNA), Evf-1. In this report, we show that the Dlx-5/6 ultraconserved region is transcribed to generate an alternatively spliced form of Evf-1, the ncRNA Evf-2. Evf-2 specifically cooperates with Dlx-2 to increase the transcriptional activity of the Dlx-5/6 enhancer in a target and homeodomain-specific manner. A stable complex containing the Evf-2 ncRNA and the Dlx-2 protein forms in vivo, suggesting that the Evf-2 ncRNA activates transcriptional activity by directly influencing Dlx-2 activity. These experiments identify a novel mechanism whereby transcription is controlled by the cooperative actions of an ncRNA and a homeodomain protein. The possibility that a subset of vertebrate ultraconserved regions may function at both the DNA and RNA level to control key developmental regulators may explain why ultraconserved sequences exhibit 90% or more conservation even after 450 million years of vertebrate evolution.