Decoding temporally encoded sensory input by cortical oscillations and thalamic phase comparators

The temporally encoded information obtained by vibrissal touch could be decoded “passively,” involving only input-driven elements, or “actively,” utilizing intrinsically driven oscillators. A previous study suggested that the trigeminal somatosensory system of rats does not obey the bottom-up order of activation predicted by passive decoding. Thus, we have tested whether this system obeys the predictions of active decoding. We have studied cortical single units in the somatosensory cortices of anesthetized rats and guinea pigs and found that about a quarter of them exhibit clear spontaneous oscillations, many of them around whisking frequencies (≈10 Hz). The frequencies of these oscillations could be controlled locally by glutamate. These oscillations could be forced to track the frequency of induced rhythmic whisker movements at a stable, frequency-dependent, phase difference. During these stimulations, the response intensities of multiunits at the thalamic recipient layers of the cortex decreased, and their latencies increased, with increasing input frequency. These observations are consistent with thalamocortical loops implementing phase-locked loops, circuits that are most efficient in decoding temporally encoded information like that obtained by active vibrissal touch. According to this model, and consistent with our results, populations of thalamic “relay” neurons function as phase “comparators” that compare cortical timing expectations with the actual input timing and represent the difference by their population output rate.

Synchronization between prefrontal and posterior association cortex during human working memory

We measured coherence between the electroencephalogram at different scalp sites while human subjects performed delayed response tasks. The tasks required the retention of either verbalizable strings of characters or abstract line drawings. In both types of tasks, a significant enhancement in coherence in the θ range (4–7 Hz) was found between prefrontal and posterior electrodes during 4-s retention intervals. During 6-s perception intervals, far fewer increases in θ coherence were found. Also in other frequency bands, coherence increased; however, the patterns of enhancement made a relevance for working memory processes seem unlikely. Our results suggest that working memory involves synchronization between prefrontal and posterior association cortex by phase-locked, low frequency (4–7 Hz) brain activity.

Blind separation of auditory event-related brain responses into independent components

Averaged event-related potential (ERP) data recorded from the human scalp reveal electroencephalographic (EEG) activity that is reliably time-locked and phase-locked to experimental events. We report here the application of a method based on information theory that decomposes one or more ERPs recorded at multiple scalp sensors into a sum of components with fixed scalp distributions and sparsely activated, maximally independent time courses. Independent component analysis (ICA) decomposes ERP data into a number of components equal to the number of sensors. The derived components have distinct but not necessarily orthogonal scalp projections. Unlike dipole-fitting methods, the algorithm does not model the locations of their generators in the head. Unlike methods that remove second-order correlations, such as principal component analysis (PCA), ICA also minimizes higher-order dependencies. Applied to detected—and undetected—target ERPs from an auditory vigilance experiment, the algorithm derived ten components that decomposed each of the major response peaks into one or more ICA components with relatively simple scalp distributions. Three of these components were active only when the subject detected the targets, three other components only when the target went undetected, and one in both cases. Three additional components accounted for the steady-state brain response to a 39-Hz background click train. Major features of the decomposition proved robust across sessions and changes in sensor number and placement. This method of ERP analysis can be used to compare responses from multiple stimuli, task conditions, and subject states.

A new mechanism of sound generation in songbirds

Our current understanding of the sound-generating mechanism in the songbird vocal organ, the syrinx, is based on indirect evidence and theoretical treatments. The classical avian model of sound production postulates that the medial tympaniform membranes (MTM) are the principal sound generators. We tested the role of the MTM in sound generation and studied the songbird syrinx more directly by filming it endoscopically. After we surgically incapacitated the MTM as a vibratory source, zebra finches and cardinals were not only able to vocalize, but sang nearly normal song. This result shows clearly that the MTM are not the principal sound source. The endoscopic images of the intact songbird syrinx during spontaneous and brain stimulation-induced vocalizations illustrate the dynamics of syringeal reconfiguration before phonation and suggest a different model for sound production. Phonation is initiated by rostrad movement and stretching of the syrinx. At the same time, the syrinx is closed through movement of two soft tissue masses, the medial and lateral labia, into the bronchial lumen. Sound production always is accompanied by vibratory motions of both labia, indicating that these vibrations may be the sound source. However, because of the low temporal resolution of the imaging system, the frequency and phase of labial vibrations could not be assessed in relation to that of the generated sound. Nevertheless, in contrast to the previous model, these observations show that both labia contribute to aperture control and strongly suggest that they play an important role as principal sound generators.

A family of phase-variable restriction enzymes with differing specificities generated by high-frequency gene rearrangements

The hsd genes of Mycoplasma pulmonis encode restriction and modification enzymes exhibiting a high degree of sequence similarity to the type I enzymes of enteric bacteria. The S subunits of type I systems dictate the DNA sequence specificity of the holoenzyme and are required for both the restriction and the modification reactions. The M. pulmonis chromosome has two hsd loci, both of which contain two hsdS genes each and are complex, site-specific DNA inversion systems. Embedded within the coding region of each hsdS gene are a minimum of three sites at which DNA inversions occur to generate extensive amino acid sequence variations in the predicted S subunits. We show that the polymorphic hsdS genes produced by gene rearrangement encode a family of functional S subunits with differing DNA sequence specificities. In addition to creating polymorphisms in hsdS sequences, DNA inversions regulate the phase-variable production of restriction activity because the other genes required for restriction activity (hsdR and hsdM) are expressed only from loci that are oriented appropriately in the chromosome relative to the hsd promoter. These data cast doubt on the prevailing paradigms that restriction systems are either selfish or function to confer protection from invasion by foreign DNA.

Adaptive evolution of a candidate gene for aging in Drosophila

Examination of the phenotypic effects of specific mutations has been extensively used to identify candidate genes affecting traits of interest. However, such analyses do not reveal anything about the evolutionary forces acting at these loci, or whether standing allelic variation contributes to phenotypic variance in natural populations. The Drosophila gene methuselah (mth) has been proposed as having major effects on organismal stress response and longevity phenotype. Here, we examine patterns of polymorphism and divergence at mth in population level samples of Drosophila melanogaster, D. simulans, and D. yakuba. Mth has experienced an unusually high level of adaptive amino acid divergence concentrated in the intra- and extracellular loop domains of the receptor protein, suggesting the historical action of positive selection on those regions of the molecule that modulate signal transduction. Further analysis of single nucleotide polymorphisms (SNPs) in D. melanogaster provided evidence for contemporary and spatially variable selection at the mth locus. In ten surveyed populations, the most common mth haplotype exhibited a 40% cline in frequency that coincided with population level differences in multiple life-history traits including lifespan. This clinal pattern was not associated with any particular SNP in the coding region, indicating that selection is operating at a closely linked site that may be involved in gene expression. Together, these consistently nonneutral patterns of inter- and intraspecific variation suggest adaptive evolution of a signal transduction pathway that may modulate lifespan in nature.

High-frequency, spin-label EPR of nonaxial lipid ordering and motion in cholesterol-containing membranes

The EPR spectra of spin-labeled lipid chains in fully hydrated bilayer membranes of dimyristoyl phosphatidylcholine containing 40 mol % of cholesterol have been studied in the liquid-ordered phase at a microwave radiation frequency of 94 GHz. At such high field strengths, the spectra should be optimally sensitive to lateral chain ordering that is expected in the formation of in-plane domains. The high-field EPR spectra from random dispersions of the cholesterol-containing membranes display very little axial averaging of the nitroxide g-tensor anisotropy for lipids spin labeled toward the carboxyl end of the sn-2 chain (down to the 8-C atom). For these positions of labeling, anisotropic 14N-hyperfine splittings are resolved in the gzz and gyy regions of the nonaxial EPR spectra. For positions of labeling further down the lipid chain, toward the terminal methyl group, the axial averaging of the spectral features systematically increases and is complete at the 14-C atom position. Concomitantly, the time-averaged 〈Azz〉 element of the 14N-hyperfine tensor decreases, indicating that the axial rotation at the terminal methyl end of the chains arises from correlated torsional motions about the bonds of the chain backbone, the dynamics of which also give rise to a differential line broadening of the 14N-hyperfine manifolds in the gzz region of the spectrum. These results provide an indication of the way in which lateral ordering of lipid chains in membranes is induced by cholesterol.

Neural mechanisms underlying binocular fusion and stereopsis: Position vs. phase

The visual system utilizes binocular disparity to discriminate the relative depth of objects in space. Since the striate cortex is the first site along the central visual pathways at which signals from the left and right eyes converge onto a single neuron, encoding of binocular disparity is thought to begin in this region. There are two possible mechanisms for encoding binocular disparity through simple cells in the striate cortex: a difference in receptive field (RF) position between the two eyes (RF position disparity) and a difference in RF profile between the two eyes (RF phase disparity). Although there have been studies supporting each of the two encoding mechanisms, both mechanisms have not been examined in a single study. Therefore, the relative roles of the two mechanisms have not been determined. To address this issue, we have mapped left and right eye RFs of simple cells in the cat’s striate cortex using binary m-sequence noise, and then we have estimated RF position and phase disparities. We find that RF position disparities are generally limited to small values that are not sufficient to encode large binocular disparities. In contrast, RF phase disparities cover a wide range of binocular disparities and exhibit dependencies on orientation and spatial frequency in a manner expected for a mechanism that encodes binocular disparity. These results indicate that binocular disparity is mainly encoded through RF phase disparity. However, RF position disparity may play a significant role for cells with high spatial frequency selectivity, which are constrained to small RF phase disparities.

Rapid down-regulation of mammalian Period genes during behavioral resetting of the circadian clock

The pervasive role of circadian clocks in regulating physiology and behavior is widely recognized. Their adaptive value is their ability to be entrained by environmental cues such that the internal circadian phase is a reliable predictor of solar time. In mammals, both light and nonphotic behavioral cues can entrain the principal oscillator of the hypothalamic suprachiasmatic nuclei (SCN). However, although light can advance or delay the clock during circadian night, behavioral events trigger phase advances during the subjective day, when the clock is insensitive to light. The recent identification of Period (Per) genes in mammals, homologues of dperiod, which encodes a core element of the circadian clockwork in Drosophila, now provides the opportunity to explain circadian timing and entrainment at a molecular level. In mice, expression of mPer1 and mPer2 in the SCN is rhythmic and acutely up-regulated by light. Moreover, the temporal relations between mRNA and protein cycles are consistent with a clock based on a transcriptional/translational feedback loop. Here we describe circadian oscillations of Per1 and Per2 in the SCN of the Syrian hamster, showing that PER1 protein and mRNA cycles again behave in a manner consistent with a negative-feedback oscillator. Furthermore, we demonstrate that nonphotic resetting has the opposite effect to light: acutely down-regulating these genes. Their sensitivity to nonphotic resetting cues supports their proposed role as core elements of the circadian oscillator. Moreover, this study provides an explanation at the molecular level for the contrasting but convergent effects of photic and nonphotic cues on the clock.

Effects of Genome Position and the DNA Damage Checkpoint on the Structure and Frequency of sod2 Gene Amplification in Fission Yeast

The Schizosaccharomyces pombe sod2 gene, located near the telomere on the long arm of chromosome I, encodes a Na+ (or Li+)/H+ antiporter. Amplification of sod2 has previously been shown to confer resistance to LiCl. We analyzed 20 independent LiCl-resistant strains and found that the only observed mechanism of resistance is amplification of sod2. The amplicons are linear, extrachromosomal elements either 225 or 180 kb long, containing both sod2 and telomere sequences. To determine whether proximity to a telomere is necessary for sod2 amplification, a strain was constructed in which the gene was moved to the middle of the same chromosomal arm. Selection of LiCl-resistant strains in this genetic background also yielded amplifications of sod2, but in this case the amplified DNA was exclusively chromosomal. Thus, proximity to a telomere is not a prerequisite for gene amplification in S. pombe but does affect the mechanism. Relative to wild-type cells, mutants with defects in the DNA damage aspect of the rad checkpoint control pathway had an increased frequency of sod2 amplification, whereas mutants defective in the S-phase completion checkpoint did not. Two models for generating the amplified DNA are presented.

Mechanical bases of frequency tuning and neural excitation at the base of the cochlea: Comparison of basilar-membrane vibrations and auditory-nerve-fiber responses in chinchilla

We review the mechanical origin of auditory-nerve excitation, focusing on comparisons of the magnitudes and phases of basilar-membrane (BM) vibrations and auditory-nerve fiber responses to tones at a basal site of the chinchilla cochlea with characteristic frequency ≈ 9 kHz located 3.5 mm from the oval window. At this location, characteristic frequency thresholds of fibers with high spontaneous activity correspond to magnitudes of BM displacement or velocity in the order of 1 nm or 50 μm/s. Over a wide range of stimulus frequencies, neural thresholds are not determined solely by BM displacement but rather by a function of both displacement and velocity. Near-threshold, auditory-nerve responses to low-frequency tones are synchronous with peak BM velocity toward scala tympani but at 80–90 dB sound pressure level (in decibels relative to 20 microPascals) and at 100–110 dB sound pressure level responses undergo two large phase shifts approaching 180°. These drastic phase changes have no counterparts in BM vibrations. Thus, although at threshold levels the encoding of BM vibrations into spike trains appears to involve only relatively minor signal transformations, the polarity of auditory-nerve responses does not conform with traditional views of how BM vibrations are transmitted to the inner hair cells. The response polarity at threshold levels, as well as the intensity-dependent phase changes, apparently reflect micromechanical interactions between the organ of Corti, the tectorial membrane and the subtectorial fluid, and/or electrical and synaptic processes at the inner hair cells.

Cellular mechanisms for resolving phase ambiguity in the owl's inferior colliculus

Both mammals and birds use the interaural time difference (ITD) for localization of sound in the horizontal plane. They may localize either real or phantom sound sources, when the signal consists of a narrow frequency band. This ambiguity does not occur with broadband signals. A plot of impulse rates or amplitude of excitatory postsynaptic potentials against ITDs (ITD curve) consists of peaks and troughs. In the external nucleus (ICX) of the owl's inferior colliculus, ITD curves show multiple peaks when the signal is narrow-band, such as tones. Of these peaks, one occurs at ITDi, which is independent of frequency, and others at ITDi ± T, where T is the tonal period. The ITD curve of the same neuron shows a large peak (main peak) at ITDi and no or small peaks (side peaks) at ITDi ± T, when the signal is broadband. ITD curves for postsynaptic potentials indicate that ICX neurons integrate the results of binaural cross-correlation in different frequency bands. However, the difference between the main and side peaks is small. ICX neurons further enhance this difference in the process of converting membrane potentials to impulse rates. Inhibition also appears to augment the difference between the main and side peaks.

Rad52 forms DNA repair and recombination centers during S phase

Maintenance of genomic integrity and stable transmission of genetic information depend on a number of DNA repair processes. Failure to faithfully perform these processes can result in genetic alterations and subsequent development of cancer and other genetic diseases. In the eukaryote Saccharomyces cerevisiae, homologous recombination is the major pathway for repairing DNA double-strand breaks. The key role played by Rad52 in this pathway has been attributed to its ability to seek out and mediate annealing of homologous DNA strands. In this study, we find that S. cerevisiae Rad52 fused to green fluorescent protein (GFP) is fully functional in DNA repair and recombination. After induction of DNA double-strand breaks by γ-irradiation, meiosis, or the HO endonuclease, Rad52-GFP relocalizes from a diffuse nuclear distribution to distinct foci. Interestingly, Rad52 foci are formed almost exclusively during the S phase of mitotic cells, consistent with coordination between recombinational repair and DNA replication. This notion is further strengthened by the dramatic increase in the frequency of Rad52 focus formation observed in a pol12-100 replication mutant and a mec1 DNA damage checkpoint mutant. Furthermore, our data indicate that each Rad52 focus represents a center of recombinational repair capable of processing multiple DNA lesions.

Stationary-phase mutation in the bacterial chromosome: Recombination protein and DNA polymerase IV dependence

Several microbial systems have been shown to yield advantageous mutations in slowly growing or nongrowing cultures. In one assay system, the stationary-phase mutation mechanism differs from growth-dependent mutation, demonstrating that the two are different processes. This system assays reversion of a lac frameshift allele on an F′ plasmid in Escherichia coli. The stationary-phase mutation mechanism at lac requires recombination proteins of the RecBCD double-strand-break repair system and the inducible error-prone DNA polymerase IV, and the mutations are mostly −1 deletions in small mononucleotide repeats. This mutation mechanism is proposed to occur by DNA polymerase errors made during replication primed by recombinational double-strand-break repair. It has been suggested that this mechanism is confined to the F plasmid. However, the cells that acquire the adaptive mutations show hypermutation of unrelated chromosomal genes, suggesting that chromosomal sites also might experience recombination protein-dependent stationary-phase mutation. Here we test directly whether the stationary-phase mutations in the bacterial chromosome also occur via a recombination protein- and pol IV-dependent mechanism. We describe an assay for chromosomal mutation in cells carrying the F′ lac. We show that the chromosomal mutation is recombination protein- and pol IV-dependent and also is associated with general hypermutation. The data indicate that, at least in these male cells, recombination protein-dependent stationary-phase mutation is a mechanism of general inducible genetic change capable of affecting genes in the bacterial chromosome.

Adaptive mutations in Escherichia coli as a model for the multiple mutational origins of tumors.

The cells in most tumors are found to carry multiple mutations; however, based upon mutation rates determined by fluctuation tests, the frequency of such multiple mutations should be so low that tumors are never detected within human populations. Fluctuation tests, which determine the cell-division-dependent mutation rate per cell generation in growing cells, may not be appropriate for estimating mutation rates in nondividing or very slowly dividing cells. Recent studies of time-dependent, "adaptive" mutations in nondividing populations of microorganisms suggest that similar measurements may be more appropriate to understanding the mutation origins of tumors. Here I use the ebgR and ebgA genes of Escherichia coli to measure adaptive mutation rates where multiple mutations are required for rapid growth. Mutations in either ebgA or ebgR allow very slow growth on lactulose (4-O-beta-D-galactosyl-D-fructose), with doubling times of 3.2 and 17.3 days, respectively. However, when both mutations are present, cells can grow rapidly with doubling times of 2.7 hr. I show that during prolonged (28-day) selection for growth on lactulose, the number of lactulose-utilizing mutants that accumulate is 40,000 times greater than can be accounted for on the basis of mutation rates measured by fluctuation tests, but is entirely consistent with the time-dependent adaptive mutation rates measured under the same conditions of prolonged selection.