It is known that point mutations and rearrangements (deletions and duplications) of mammalian mitochondrial DNA (mtDNA) can result in mitochondrial dysfunction and human disease. Very little attention has been paid to mtDNA circular dimers (a complex form consisting of two genomes joined head-to-tail) despite their close association with human neoplasia. MtDNA dimers are frequently found in human leukemia, but the clinical relevance of their presence remains unknown. To begin to investigate the role of circular dimer mtDNA in the tumorigenic phenotype, we have created isogenic cell lines containing monomer and dimer mitochondrial genomes and compared the respective nuclear mRNA expression using Affymetrix gene array analysis. Surprisingly, a large number of nuclear gene changes were observed, with one of the largest category of genes being associated with remodeling of the cell surface and extracellular matrix. Since cell growth, migration, apoptosis, and many other cellular processes are influenced by signals initiating from the cell surface, the changes associated with the presence of mtDNA dimers could lead to significant alterations in tumorigenic potential and/or progression.

Color and motion are used by many species to identify salient objects. They are processed largely independently, but color contributes to motion processing in humans, for example, enabling moving colored objects to be detected when their luminance matches the background. Here, we demonstrate an unexpected, additional contribution of color to motion vision in Drosophila. We show that behavioral ON-motion responses are more sensitive to UV than for OFF-motion, and we identify cellular pathways connecting UV-sensitive R7 photoreceptors to ON and OFF-motion-sensitive T4 and T5 cells, using neurogenetics and calcium imaging. Remarkably, this contribution of color circuitry to motion vision enhances the detection of approaching UV discs, but not green discs with the same chromatic contrast, and we show how this could generalize for systems with ON- and OFF-motion pathways. Our results provide a computational and circuit basis for how color enhances motion vision to favor the detection of saliently colored objects.

Color and motion are used by many species to identify salient objects. They are processed largely independently, but color contributes to motion processing in humans, for example, enabling moving colored objects to be detected when their luminance matches the background. Here, we demonstrate an unexpected, additional contribution of color to motion vision in Drosophila. We show that behavioral ON-motion responses are more sensitive to UV than for OFF-motion, and we identify cellular pathways connecting UV-sensitive R7 photoreceptors to ON and OFF-motion-sensitive T4 and T5 cells, using neurogenetics and calcium imaging. Remarkably, this contribution of color circuitry to motion vision enhances the detection of approaching UV discs, but not green discs with the same chromatic contrast, and we show how this could generalize for systems with ON- and OFF-motion pathways. Our results provide a computational and circuit basis for how color enhances motion vision to favor the detection of saliently colored objects.

MHR3 is an ecdysone-inducible transcription factor whose expression in both Manduca sexta epidermis and the Manduca GV1 cell line is induced by 20-hydroxyecdysone (20E) in vitro. There are four putative ecdysone response elements (EcRE) in the 2.6-kb flanking region of the MHR3 promoter. The most proximal, EcRE1, is necessary for activation of the promoter by 20E in the GV1 cells because the mutation of EcRE1 caused the loss of responsiveness to 20E. Previous studies showed that EcR-B1/USP-1 bound only to EcRE1 and high levels of this complex increased the 20E-induced activation, whereas the presence of high USP-2 prevented this increased activation. When we expressed EcR-A alone or in combination with USP-1 under the control of Autographa californica baculovirus promoter (pIE1hr), the activation of the 2.6-kb promoter by 20E was reduced by about 50%. Moreover, when EcR-A was expressed together with both EcR-B1 and USP-1, it reduced the normal activation caused by EcR-B1 and USP-1 by 50%. Gel mobility shift assays showed no binding of EcR-A/USP-1 to EcRE1. The presence of EcR-A, however, reduced the binding of EcR-B1/USP-1 by about 50%. These findings suggest that EcR-A competes with EcR-B1 for binding of USP-1, leading to a decline in activity of the promoter. In addition, E75A, another ecdysone-induced transcription factor, and MHR3 itself suppressed MHR3 promoter activity by binding to the monomeric response element (MRE2). Therefore, MHR3 can be down-regulated both by itself and by E75A.

Trace fear conditioning is characterized by a stimulus-free trace interval (TI) between the conditioned stimulus (CS) and the unconditioned stimulus (US), which requires an array of brain structures to support the formation and storage of associative memory. The entorhinal cortex (EC) has been proposed to provide essential neural code for resolving temporal discontinuity in conjunction with the hippocampus. However, how the CS and TI are encoded at the neuronal level in the EC is not clear. In Exp. 1, we tested the effect of bilateral pre-training electrolytic lesions of EC on trace vs. delay fear conditioning using rats as subjects. We found that the lesions impaired the acquisition of trace but not delay fear conditioning confirming that EC is a critical brain area for trace fear memory formation. In Exp. 2, single-unit activities from EC were recorded during the pre-training baseline and post-training retention sessions following trace or delay conditioning. The recording results showed that a significant proportion of the EC neurons modulated their firing during TI after the trace conditioning, but not after the delay fear conditioning. Further analysis revealed that the majority of modulated units decreased the firing rate during the TI or the CS. Taken together, these results suggest that EC critically contributes to trace fear conditioning by modulating neuronal activity during the TI to facilitate the association between the CS and US across a temporal gap.

Ca2+ signals associated with action potentials (APs) and metabotropic glutamate receptor (mGluR) activation exert distinct influences on neuronal activity and synaptic plasticity. However, it is not clear how these two types of Ca2+ signals are differentially regulated by neurotransmitter inputs in a single neuron. We investigated this issue in dopaminergic neurons of the ventral midbrain using brain slices. Intracellular Ca2+ was assessed by measuring Ca2+-sensitive K+ currents or imaging the fluorescence of Ca2+ indicator dyes. Tonic activation of metabotropic neurotransmitter receptors (mGluRs, alpha1 adrenergic receptors, and muscarinic acetylcholine receptors), attained by superfusion of agonists or weak, sustained (approximately 1 s) synaptic stimulation, augmented AP-induced Ca2+ transients. In contrast, Ca2+ signals elicited by strong, transient (50-200 ms) activation of mGluRs with aspartate iontophoresis were suppressed by superfusion of agonists. These opposing effects on Ca2+ signals were both mediated by an increase in intracellular inositol 1,4,5-trisphosphate (IP3) levels, because they were blocked by heparin, an IP3 receptor antagonist, and reproduced by photolytic application of IP3. Evoking APs repetitively at low frequency (2 Hz) caused inactivation of IP3 receptors and abolished IP3 facilitation of single AP-induced Ca2+ signals, whereas facilitation of Ca2+ signals triggered by bursts of APs (five at 20 Hz) was attenuated by less than half. We further obtained evidence suggesting that the psychostimulant amphetamine may augment burst-induced Ca2+ signals via both depression of basal firing and production of IP3. We propose that intracellular IP3 tone provides a mechanism to selectively amplify burst-induced Ca2+ signals in dopaminergic neurons.

The central complex is a prominent structure in the Drosophila brain. Visual learning experiments in the flight simulator, with flies with genetically altered brains, revealed that two groups of horizontal neurons in one of its substructures, the fan-shaped body, were required for Drosophila visual pattern memory. However, little is known about the role of other components of the central complex for visual pattern memory. Here we show that a small set of neurons in the ellipsoid body, which is another substructure of the central complex and connected to the fan-shaped body, is also required for visual pattern memory. Localized expression of rutabaga adenylyl cyclase in either the fan-shaped body or the ellipsoid body is sufficient to rescue the memory defect of the rut(2080) mutant. We then performed RNA interference of rutabaga in either structure and found that they both were required for visual pattern memory. Additionally, we tested the above rescued flies under several visual pattern parameters, such as size, contour orientation, and vertical compactness, and revealed differential roles of the fan-shaped body and the ellipsoid body for visual pattern memory. Our study defines a complex neural circuit in the central complex for Drosophila visual pattern memory.

This study provides a new perspective on the long-standing problem of the nature of the decapod crustacean blood-brain interface. Previous studies of crustacean blood-brain interface permeability have relied on invasive histological, immunohistochemical and electrophysiological techniques, indicating a leaky non-selective blood-brain barrier. The present investigation involves the use of magnetic resonance imaging (MRI), a method for non-invasive longitudinal tracking of tracers in real-time. Differential uptake rates of two molecularly distinct MRI contrast agents, namely manganese (Mn(II)) and Magnevist® (Gd-DTPA), were observed and quantified in the crayfish, Cherax destructor. Contrast agents were injected into the pericardium and uptake was observed with longitudinal MRI for approximately 14.5 h. Mn(II) was taken up quickly into neural tissue (within 6.5 min), whereas Gd-DTPA was not taken up into neural tissue and was instead restricted to the intracerebral vasculature or excreted into nearby sinuses. Our results provide evidence for a charge-selective intracerebral blood-brain interface in the crustacean nervous system, a structural characteristic once considered too complex for a lower-order arthropod.

Modern applications in the life sciences are frequently based on in vivo imaging of biological specimens, a domain for which light microscopy approaches are typically best suited. Often, quantitative information must be obtained from large multicellular organisms at the cellular or even subcellular level and with a good temporal resolution. However, this usually requires a combination of conflicting features: high imaging speed, low photobleaching and low phototoxicity in the specimen, good three-dimensional (3D) resolution, an excellent signal-to-noise ratio, and multiple-view imaging capability. The latter feature refers to the capability of recording a specimen along multiple directions, which is crucial for the imaging of large specimens with strong light-scattering or light-absorbing tissue properties. An imaging technique that fulfills these requirements is essential for many key applications: For example, studying fast cellular processes over long periods of time, imaging entire embryos throughout development, or reconstructing the formation of morphological defects in mutants. Here, we discuss digital scanned laser light sheet fluorescence microscopy (DSLM) as a novel tool for quantitative in vivo imaging in the post-genomic era and show how this emerging technique relates to the currently most widely applied 3D microscopy techniques in biology: confocal fluorescence microscopy and two-photon microscopy.