Although cellular mitochondrial DNA (mtDNA) copy number varies widely among cell lines and tissues, little is known about the mechanism of mtDNA copy number control. Most nascent replication strands from the leading, heavy-strand origin (O(H)) are prematurely terminated, defining the 3’ boundary of the displacement loop (D-loop). We have depleted mouse LA9 cell mtDNA to approximately 20% of normal levels by treating with 2’,3’-dideoxycytidine (ddC) and subsequently allowed recovery to normal levels of mtDNA. A quantitative ligation-mediated PCR assay was used to determine the levels of both terminated and extended nascent O(H) strands during mtDNA depletion and repopulation. Depleting mtDNA leads to a release of replication termination until mtDNA copy number approaches a normal level. Detectable total nascent strands per mtDNA genome remain below normal. Therefore, it is likely that the level of replication termination plays a significant role in copy number regulation in this system. However, termination of D-loop strand synthesis is persistent, indicating formation of the D-loop structure has a purpose that is required under conditions of rapid recovery of depleted mtDNA.

Hydrogen peroxide (H(2)O(2)) is the major reactive oxygen species (ROS) produced in sperm. High concentrations of H(2)O(2) in sperm induce nuclear DNA fragmentation and lipid peroxidation and result in cell death. The respiratory chain of the mitochondrion is one of the most productive ROS generating systems in sperm, and thus the destruction of ROS in mitochondria is critical for the cell. It was recently reported that H(2)O(2) generated by the respiratory chain of the mitochondrion can be efficiently destroyed by the cytochrome c-mediated electron-leak pathway where the electron of ferrocytochrome c migrates directly to H(2)O(2) instead of to cytochrome c oxidase. In our studies, we found that mouse testis-specific cytochrome c (T-Cc) can catalyze the reduction of H(2)O(2) three times faster than its counterpart in somatic cells (S-Cc) and that the T-Cc heme has the greater resistance to being degraded by H(2)O(2). Together, these findings strongly imply that T-Cc can protect sperm from the damages caused by H(2)O(2). Moreover, the apoptotic activity of T-Cc is three to five times greater than that of S-Cc in a well established apoptosis measurement system using Xenopus egg extract. The dramatically stronger apoptotic activity of T-Cc might be important for the suicide of male germ cells, considered a physiological mechanism that regulates the number of sperm produced and eliminates those with damaged DNA. Thus, it is very likely that T-Cc has evolved to guarantee the biological integrity of sperm produced in mammalian testis.

Multiple studies have investigated the mechanisms of aggressive behavior in Drosophila; however, little is known about the effects of chronic fighting experience. Here, we investigated if repeated fighting encounters would induce an internal state that could affect the expression of subsequent behavior. We trained wild-type males to become winners or losers by repeatedly pairing them with hypoaggressive or hyperaggressive opponents, respectively. As described previously, we observed that chronic losers tend to lose subsequent fights, while chronic winners tend to win them. Olfactory conditioning experiments showed that winning is perceived as rewarding, while losing is perceived as aversive. Moreover, the effect of chronic fighting experience generalized to other behaviors, such as gap-crossing and courtship. We propose that in response to repeatedly winning or losing aggressive encounters, male flies form an internal state that displays persistence and generalization; fight outcomes can also have positive or negative valence. Furthermore, we show that the activities of the PPL1-γ1pedc dopaminergic neuron and the MBON-γ1pedc>α/β mushroom body output neuron are required for aversion to an olfactory cue associated with losing fights.

The established strand-displacement model for mammalian mitochondrial DNA (mtDNA) replication has recently been questioned in light of new data using two-dimensional (2D) agarose gel electrophoresis. It has been proposed that a synchronous, strand-coupled mode of replication occurs in tissues, thereby casting doubt on the general validity of the "orthodox," or strand-displacement model. We have examined mtDNA replicative intermediates from mouse liver using atomic force microscopy and 2D agarose gel electrophoresis in order to resolve this issue. The data provide evidence for only the orthodox, strand-displacement mode of replication and reveal the presence of additional, alternative origins of lagging light-strand mtDNA synthesis. The conditions used for 2D agarose gel analysis are favorable for branch migration of asymmetrically replicating nascent strands. These data reconcile the original displacement mode of replication with the data obtained from 2D gel analyses.

Mammalian mitochondrial DNA maintains a novel displacement-loop region containing the major sites of transcriptional initiation and the origin of heavy strand DNA replication. Because the exact map positions of the 5’ termini of nascent mouse displacement-loop strands are known, it is possible to examine directly a potential relationship between replication priming and transcription. Analyses of in vivo nucleic acids complementary to the displacement-loop region reveal two species with identical 5’ ends at map position 16 183. One is entirely RNA and the other is RNA covalently linked to DNA. In the latter the transition from RNA to DNA is sharp, occurring near or within a series of previously identified conserved sequences 74-163 nucleotides downstream from the transcriptional initiation site. These data suggest that the initial events in replication priming and transcription are the same and that the decision to synthesize DNA or RNA is a downstream event under the control of short, conserved displacement-loop template sequences.

BACKGROUND: In a series of landmark papers, Kyriacou, Hall, and colleagues reported that the average inter-pulse interval of Drosophila melanogaster male courtship song varies rhythmically (KH cycles), that the period gene controls this rhythm, and that evolution of the period gene determines species differences in the rhythm's frequency. Several groups failed to recover KH cycles, but this may have resulted from differences in recording chamber size.

RESULTS: Here, using recording chambers of the same dimensions as used by Kyriacou and Hall, I found no compelling evidence for KH cycles at any frequency. By replicating the data analysis procedures employed by Kyriacou and Hall, I found that two factors--data binned into 10-second intervals and short recordings--imposed non-significant periodicity in the frequency range reported for KH cycles. Randomized data showed similar patterns.

CONCLUSIONS: All of the results related to KH cycles are likely to be artifacts of binning data from short songs. Reported genotypic differences in KH cycles cannot be explained by this artifact and may have resulted from the use of small sample sizes and/or from the exclusion of samples that did not exhibit song rhythms.

Genetically encoded calcium indicators (GECIs), based on recombinant fluorescent proteins, have been engineered to observe calcium transients in living cells and organisms. Through observation of calcium, these indicators also report neural activity. We review progress in GECI construction and application, particularly toward in vivo monitoring of sparse action potentials (APs). We summarize the extrinsic and intrinsic factors that influence GECI performance. A simple model of GECI response to AP firing demonstrates the relative significance of these factors. We recommend a standardized protocol for evaluating GECIs in a physiologically relevant context. A potential method of simultaneous optical control and recording of neuronal circuits is presented.

Animals exhibit a behavioral response to novel sensory stimuli about which they have no prior knowledge. We have examined the neural and behavioral correlates of novelty and familiarity in the olfactory system of Drosophila. Novel odors elicit strong activity in output neurons (MBONs) of the α'3 compartment of the mushroom body that is rapidly suppressed upon repeated exposure to the same odor. This transition in neural activity upon familiarization requires odor-evoked activity in the dopaminergic neuron innervating this compartment. Moreover, exposure of a fly to novel odors evokes an alerting response that can also be elicited by optogenetic activation of α'3 MBONs. Silencing these MBONs eliminates the alerting behavior. These data suggest that the α'3 compartment plays a causal role in the behavioral response to novel and familiar stimuli as a consequence of dopamine-mediated plasticity at the Kenyon cell-MBONα'3 synapse.