Back-propagating action potentials (bAPs) are involved in associative synaptic plasticity and the modulation of dendritic excitability. We have used high-speed confocal and two-photon imaging to measure calcium and voltage signals associated with action potential propagation into oblique branches of CA1 pyramidal neurons in adult hippocampal slices. The spatial profile of the bAP-associated Ca(2+) influx was biphasic, with an initial increase in the proximity of the branch point followed by a progressive decrease. Voltage imaging in the branches showed that bAP amplitude was initially constant and then steadily declined with distance from the soma. To determine the role of transient K(+) channels in this profile, we used external Ba(2+) (150 microm) as a channel blocker, after characterizing its effect on A-type K(+) channels in the apical trunk. Bath application of Ba(2+) significantly reduced the A-type K(+) current in outside-out patches and nearly eliminated the distance-dependent decrease in bAP amplitude and its associated Ca(2+) signal. Finally, small amplitude bAPs at more distal oblique branch locations could be boosted by simultaneous branch depolarization, such that the paired Ca(2+) signal became nearly the same for proximal and distal oblique dendrites. These data suggest that dendritic K(+) channels regulate the amplitude of bAPs to create a dendritic Ca(2+) signal whose magnitude is inversely related to the electrotonic distance from the soma when bAPs are not associated with a significant amount of localized synaptic input. This distance-dependent Ca(2+) signal from bAPs, however, can be amplified and a strong associative signal is produced once the proper correlation between synaptic activation and AP output is achieved. We hypothesize that these two signals may be involved in the regulation of the expression and activity of dendritic voltage- and ligand-gated ion channels.

Staining the mRNA of a gene via in situ hybridization (ISH) during the development of a D. melanogaster embryo delivers the detailed spatio-temporal pattern of expression of the gene. Many biological problems such as the detection of co-expressed genes, co-regulated genes, and transcription factor binding motifs rely heavily on the analyses of these image patterns. The increasing availability of ISH image data motivates the development of automated computational approaches to the analysis of gene expression patterns.

Gene expression patterns obtained by in situ mRNA hybridization provide important information about different genes during Drosophila embryogenesis. So far, annotations of these images are done by manually assigning a subset of anatomy ontology terms to an image. This time-consuming process depends heavily on the consistency of experts.

Automatic segmentation of nuclei in 3D microscopy images is essential for many biological studies including high throughput analysis of gene expression level, morphology, and phenotypes in single cell level. The complexity and variability of the microscopy images present many difficulties to the traditional image segmentation methods. In this paper, we present a new method based on 3D watershed algorithm to segment such images. By using both the intensity information of the image and the geometry information of the appropriately detected foreground mask, our method is robust to intensity fluctuation within nuclei and at the same time sensitive to the intensity and geometrical cues between nuclei. Besides, the method can automatically correct potential segmentation errors by using several post-processing steps. We tested this algorithm on the 3D confocal images of C.elegans, an organism that has been widely used in biological studies. Our results show that the algorithm can segment nuclei in high accuracy despite the non-uniform background, tightly clustered nuclei with different sizes and shapes, fluctuated intensities, and hollow-shaped staining patterns in the images.

Aldolases are enzymes with potential applications in biosynthesis, depending on their activity, specificity and stability. In the present study, the genomes of Sulfolobus species were screened for aldolases. Two new KDGA [2-keto-3-deoxygluconate (2-oxo-3-deoxygluconate) aldolases] from Sulfolobus acidocaldarius and Sulfolobus tokodaii were identified, overexpressed in Escherichia coli and characterized. Both enzymes were found to have biochemical properties similar to the previously characterized S. solfataricus KDGA, including the condensation of pyruvate and either D,L-glyceraldehyde or D,L-glyceraldehyde 3-phosphate. The crystal structure of S. acidocaldarius KDGA revealed the presence of a novel phosphate-binding motif that allows the formation of multiple hydrogen-bonding interactions with the acceptor substrate, and enables high activity with glyceraldehyde 3-phosphate. Activity analyses with unnatural substrates revealed that these three KDGAs readily accept aldehydes with two to four carbon atoms, and that even aldoses with five carbon atoms are accepted to some extent. Water-mediated interactions permit binding of substrates in multiple conformations in the spacious hydrophilic binding site, and correlate with the observed broad substrate specificity.

Multi-neuronal recordings with Ca2+ indicator dyes usually relate [Ca2+]i to action potentials (APs) assuming a stereotypical dependency between the two. However, [Ca2+]i affects and is affected by numerous complex mechanisms that differ from cell type to cell type, from cell compartment to cell compartment. Moreover, [Ca2+]i depends on the specific way a cell is activated. Here we investigate, by combining calcium imaging and on-cell patch clamp recordings, the relationship between APs (spiking) and somatic [Ca2+]i in mitral and granule cells of the olfactory bulb in Xenopus laevis tadpoles. Both cell types exhibit ongoing and odour-modulated [Ca2+]i dynamics. In mitral cells, the occurrence of APs in both spontaneous and odour-evoked situations correlates tightly to step-like [Ca2+]i increases. Moreover, odorant-induced suppression of spontaneous firing couples to a decrease in [Ca2+]i. In contrast, granule cells show a substantial number of uncorrelated events such as increases in [Ca2+]i without APs occurring or APs without any effect upon [Ca2+]i. The correlation between spiking and [Ca2+]i is low, possibly due to somatic NMDAR-mediated and subthreshold voltage-activated Ca2+ entries, and thus does not allow a reliable prediction of APs based on calcium imaging. Taken together, our results demonstrate that the relationship between somatic [Ca2+]i and APs can be cell type specific. Taking [Ca2+]i dynamics as an indicator for spiking activity is thus only reliable if the correlation has been established in the system of interest. When [Ca2+]i and APs are precisely correlated, fast calcium imaging is an extremely valuable tool for determining spatiotemporal patterns of APs in neuronal population.

The functions of cortical areas depend on their inputs and outputs, but the detailed circuits made by long-range projections are unknown. We show that the light-gated channel channelrhodopsin-2 (ChR2) is delivered to axons in pyramidal neurons in vivo. In brain slices from ChR2-expressing mice, photostimulation of ChR2-positive axons can be transduced reliably into single action potentials. Combining photostimulation with whole-cell recordings of synaptic currents makes it possible to map circuits between presynaptic neurons, defined by ChR2 expression, and postsynaptic neurons, defined by targeted patching. We applied this technique, ChR2-assisted circuit mapping (CRACM), to map long-range callosal projections from layer (L) 2/3 of the somatosensory cortex. L2/3 axons connect with neurons in L5, L2/3 and L6, but not L4, in both ipsilateral and contralateral cortex. In both hemispheres the L2/3-to-L5 projection is stronger than the L2/3-to-L2/3 projection. Our results suggest that laminar specificity may be identical for local and long-range cortical projections.

Recent advances in single-neuron biophysics have enhanced our understanding of information processing on the cellular level, but how the detailed properties of individual neurons give rise to large-scale behavior remains unclear. Here, we present a model of the hippocampal network based on observed biophysical properties of hippocampal and entorhinal cortical neurons. We assembled our model to simulate spatial alternation, a task that requires memory of the previous path through the environment for correct selection of the current path to a reward site. The convergence of inputs from entorhinal cortex and hippocampal region CA3 onto CA1 pyramidal cells make them potentially important for integrating information about place and temporal context on the network level. Our model shows how place and temporal context information might be combined in CA1 pyramidal neurons to give rise to splitter cells, which fire selectively based on a combination of place and temporal context. The model leads to a number of experimentally testable predictions that may lead to a better understanding of the biophysical basis of information processing in the hippocampus.

Methods for the selective and reproducible expression of genetically encoded tools in targeted subsets of cells are required to facilitate studies of neuronal development, connectivity, and function in living animals. In the absence of techniques for synthesizing promoters that target defined cell groups, current methods exploit the regulatory elements of endogenous genes to achieve specificity of transgene expression. However, single promoters often have expression patterns too broad to pinpoint the functional roles of specific neurons. In this review, we describe emerging combinatorial techniques that make transgene expression contingent not upon a single promoter, but upon two or more promoters. Although only a few such techniques are currently available, recent developments promise rapid growth in this area in the coming years.

The pea aphid, Acyrthosiphon pisum, exhibits several environmentally cued polyphenisms, in which discrete, alternative phenotypes are produced. At low-density, parthenogenetic females produce unwinged female progeny, but at high-density females produce progeny that develop with wings. These alternative phenotypes represent a solution to the competing demands of dispersal and reproduction. Males also develop as either winged or unwinged, but these alternatives are determined by a genetic polymorphism. Winged and unwinged males are morphologically less distinct from each other than winged and unwinged females, possibly because males experience fewer trade-offs between dispersal and reproduction. To assess whether shared physiological differences mirror the shared morphological differences that characterize the wing polyphenism and polymorphism, we used a cDNA microarray representing an estimated 10% of the coding genome (1734 genes) to examine differential transcript accumulation between winged and unwinged females and males. We identified several transcripts that differentially accumulate between winged and unwinged morphs in both sexes, the majority of which are involved in energy production. Unexpectedly, the extent of differential transcript accumulation between winged and unwinged morphs was greater for adult males than for adult females. Together, these results suggest not only that similar physiological differences underlie the polyphenism and polymorphism, but that male morphs, like females, are subject to trade-offs between reproduction and dispersal that are reflected in levels of transcript accumulation and possibly genome-wide patterns of gene regulation. These data also provide a baseline for future studies of the molecular and physiological basis of life-history trade-offs.