Spontaneous EEG oscillations reveal periodic sampling of visual attention

An important effect of sustained attention is the facilitation of perception. Although the term “sustained” suggests that this beneficial effect endures continuously as long as something is attended, we present electrophysiological evidence that perception at attended locations is actually modulated periodically. Subjects detected brief light flashes that were presented peripherally at locations that were either attended or unattended. We analyzed the correlation between detection performance for attended and unattended stimuli and the phase of ongoing EEG oscillations, which relate to subsecond fluctuations of neuronal excitability. Although on average, detection performance was improved by attention—indicated by reduced detection thresholds at attended locations—we found that detection performance for attended stimuli actually fluctuated over time along with the phase of spontaneous oscillations in the θ (≈7 Hz) frequency band just before stimulus onset. This fluctuation was absent for unattended stimuli. This pattern of results suggests that “sustained” attention in fact exerts its facilitative effect on perception in a periodic fashion.Our senses are constantly confronted with a flow of information that exceeds the limits of our sensory systems. One mechanism that limits this input to a manageable amount is selective attention—the ability to enhance processing of a particular object or location. The investigation of attention has been central to our understanding of cognition. Here we present evidence for a largely unexplored feature of selective attention: its periodic rather than continuous operation.The most prominent effect of attention is the facilitation of perception—selected information is processed more efficiently (1, 2) or faster (3). One example of this facilitation is enhanced contrast sensitivity at attended locations (4, 5). Two types of selective attention are usually distinguished: transient automatic attention and sustained voluntary attention (6, 7). Transient attention is captured automatically by sudden salient events, whereas sustained attention can be controlled at will. Although the term “sustained” suggests that the facilitative effect of attention endures continuously as long as attention is deployed at a particular location, it is conceivable that attention operates in a periodic rather than a continuous fashion (8, 9). This periodicity of attention is implicit in many visual search tasks in which attention is assumed to switch successively between the different display elements (10–12). On the basis of modeling of human psychometric functions in a signal detection task, VanRullen et al. (8) recently proposed that attention could sample information at a rate of approximately seven items per second—no matter whether attention was focused on multiple targets or on just a single item. A similar attention-driven periodic sampling mechanism (albeit at a different frequency of ≈13 Hz) has been suggested to account for illusory motion reversals in the continuous wagon-wheel illusion (9, 13, 14).Periodic processes are not only found in psychophysical data; they are very prominent in electrophysiological recordings. Here, oscillations correspond to periodic fluctuations of the local electrical field and the intrinsic excitability of neuronal populations (15–21). Of course, these electrical neural oscillations could provide the physiological basis of periodic perceptual and attentional sampling phenomena (22). Recently, two studies have demonstrated that perceptual performance fluctuates between favorable and less-favorable periods along with the phase of 7–10 Hz oscillations of the human EEG (23, 24).In the present study we investigated how this connection between ongoing EEG oscillations and perception is modulated by selective attention to reveal possible fluctuations of “sustained” attention. Human observers detected luminance targets that were presented at threshold either at attended or unattended locations. In keeping with the idea that “sustained” attention in fact operates in a periodic fashion, we found that detection performance was correlated with the phase of ongoing EEG oscillations around 7 Hz, but only for stimuli presented at attended locations.     

Optogenetic activation of septal cholinergic neurons suppresses sharp wave ripples and enhances theta oscillations in the hippocampus

Theta oscillations in the limbic system depend on the integrity of the medial septum. The different populations of medial septal neurons (cholinergic and GABAergic) are assumed to affect different aspects of theta oscillations. Using optogenetic stimulation of cholinergic neurons in ChAT-Cre mice, we investigated their effects on hippocampal local field potentials in both anesthetized and behaving mice. Cholinergic stimulation completely blocked sharp wave ripples and strongly suppressed the power of both slow oscillations (0.5–2 Hz in anesthetized, 0.5–4 Hz in behaving animals) and supratheta (6–10 Hz in anesthetized, 10–25 Hz in behaving animals) bands. The same stimulation robustly increased both the power and coherence of theta oscillations (2–6 Hz) in urethane-anesthetized mice. In behaving mice, cholinergic stimulation was less effective in the theta (4–10 Hz) band yet it also increased the ratio of theta/slow oscillation and theta coherence. The effects on gamma oscillations largely mirrored those of theta. These findings show that medial septal cholinergic activation can both enhance theta rhythm and suppress peri-theta frequency bands, allowing theta oscillations to dominate.Subcortical neuromodulators play a critical role in shifting states of the brain (1, 2). State changes can occur both during sleep and in the waking animal and are instrumental in affecting local circuit computation that supports various functions, including attention, learning, memory, and action (3–5). The septo-hippocampal cholinergic system has been hypothesized to play a critical role in setting network states in the limbic system (4, 6). ACh can affect both short- and long-term plasticity of synaptic connections and provide favorable conditions for encoding information (7–9). These plastic states are associated with hippocampal theta oscillations (10). High theta states are characterized by increased release of ACh that varies in a task-dependent manner on the time scale of seconds (11–13). In contrast, reduced cholinergic activity allows effective spread of excitation in the recurrent CA3 network, giving rise to synchronous sharp wave ripples (SPW-R) (14–16).Inactivation of the medial septum (MS)/diagonal band of Broca abolishes theta oscillations in the hippocampus and entorhinal cortex (17) and results in severe learning deficit (18, 19). Similarly, selective toxin lesion of septal cholinergic neurons produces a several-fold decrease of theta power but not its frequency (20). The phase of the local field potentials (LFP) theta oscillations shifts from the septal to the temporal pole and in the CA3–CA1 axis by ∼180° (21, 22). Thus, at each point in time neurons residing at different locations of the three-dimensional structure of the hippocampus spike at different theta phases yet are bound together by the global theta signal. These numerous sources of theta generators are believed to be coordinated by the reciprocal connections between the septum and hippocampus (23), but the nature of this spatial–temporal coordination is not well understood (24). Both cholinergic and GABAergic neurons, and a small fraction of VGlut2 immunoreactive neurons (25), are believed to play a critical role in such global coordination (26, 27). Although GABAergic neurons of the MS were demonstrated to be entrained at theta frequency, identified cholinergic neurons did not show theta-related discharge pattern (28, 29). Additionally, both GABAergic and cholinergic neurons are affected by the feedback long-range hippocampo-septal inhibitory connections (30).Early studies, performed in anesthetized animals, already suggested a critical role for the cholinergic septo-hippocampal projection in the generation of theta oscillations (6). Indeed, the low-frequency theta present under urethane anesthesia can be fully abolished by antimuscarinic drugs (31). In contrast, atropine or scopolamine fail to abolish theta oscillations during waking exploration (31, 32), although they affect the theta waveform and its amplitude-phase depth profile in the hippocampus (33). Although these previous works are compatible with the hypothesis that the role of septal cholinergic projections is mainly permissive and affects theta power without modulating its frequency (20, 26, 28), direct evidence is missing. The role of septal cholinergic neurons on gamma oscillation and SPW-R is even less understood (14). To address these issues, we used optogenetic activation of septal cholinergic input and examined its impact on hippocampal theta, peri-theta bands, gamma, and ripple oscillations in both anesthetized and freely moving mice.     

Minimal model for collective kinetochore–microtubule dynamics

Chromosome segregation during cell division depends on interactions of kinetochores with dynamic microtubules (MTs). In many eukaryotes, each kinetochore binds multiple MTs, but the collective behavior of these coupled MTs is not well understood. We present a minimal model for collective kinetochore–MT dynamics, based on in vitro measurements of individual MTs and their dependence on force and kinetochore phosphorylation by Aurora B kinase. For a system of multiple MTs connected to the same kinetochore, the force–velocity relation has a bistable regime with two possible steady-state velocities: rapid shortening or slow growth. Bistability, combined with the difference between the growing and shrinking speeds, leads to center-of-mass and breathing oscillations in bioriented sister kinetochore pairs. Kinetochore phosphorylation shifts the bistable region to higher tensions, so that only the rapidly shortening state is stable at low tension. Thus, phosphorylation leads to error correction for kinetochores that are not under tension. We challenged the model with new experiments, using chemically induced dimerization to enhance Aurora B activity at metaphase kinetochores. The model suggests that the experimentally observed disordering of the metaphase plate occurs because phosphorylation increases kinetochore speeds by biasing MTs to shrink. Our minimal model qualitatively captures certain characteristic features of kinetochore dynamics, illustrates how biochemical signals such as phosphorylation may regulate the dynamics, and provides a theoretical framework for understanding other factors that control the dynamics in vivo.Microtubule (MT) dynamics are critical for cell division. Plus ends of spindle MTs interact with kinetochores, protein complexes that assemble at the centromere of each chromosome, and these dynamic MTs exert forces to move chromosomes. Individual MTs are “dynamically unstable,” spontaneously switching between a polymerizing state and a depolymerizing state (1) with growth, shortening, and switching rates that are regulated by the forces exerted at the MT tips (2–6). For many eukaryotes, however, multiple MTs are connected to each kinetochore, giving rise to collective MT behavior that is not well understood and can be entirely different from the behavior of individual MTs. Here, we develop a model of collective MT dynamics based on the measured force-dependent dynamics of individual MTs.Accurate chromosome segregation depends on correctly biorienting the kinetochore pairs by attaching sister kinetochores to opposite spindle poles. Properly attached kinetochores undergo center-of-mass (CM) and breathing oscillations that are regulated by collective MT dynamics (7–12). Incorrect attachments—such as syntelic attachment of both kinetochores to the same pole—must be corrected (13–17). Tension may cue this process because bioriented kinetochore pairs are under tension while syntelically attached kinetochores are not (7, 9, 15, 17, 18). Error correction is also mediated by Aurora B kinase phosphorylating MT-binding kinetochore proteins (13–17, 19–21). A consistent theory of metaphase kinetochore–MT dynamics should capture CM and breathing oscillations for correctly attached pairs and elucidate the contributions of tension and phosphorylation to syntelic error correction.Several models suggest that chromosome oscillations result from competition between poleward MT-based pulling and antipoleward “polar ejection” forces (22–24). Another model proposes that oscillations occur via a general mechanobiochemical feedback (25). Models of force-dependent MTs interacting with the same object also exhibit cooperative behavior (5, 26–29). However, these models do not explain error correction dynamics. Thus, the underlying physical mechanisms coordinating metaphase chromosome motions are unclear.We address these issues by developing a minimal model for collective MT dynamics based on in vitro measurements of single MTs interacting dynamically with kinetochore proteins (4, 6, 20, 21). In the model, MT polymerization and rescue are promoted by tension and inhibited by compression, whereas depolymerization and catastrophe are enhanced by compression and reduced by tension. With just these features, we find a robust and versatile mechanism by which force-dependent MTs coupled to the same kinetochore may drive metaphase chromosome motions. The force–velocity relation for a MT bundle is fundamentally different from that of a single dynamically unstable MT, exhibiting bistable behavior. Bistability gives rise to kinetochore oscillations and is shifted by phosphorylation to produce error correction. The model qualitatively predicts kinetochore motions in our experiments in which Aurora B is hyperactivated in bioriented kinetochore pairs. Thus, we find that many characteristics of metaphase kinetochore dynamics emerge simply from the force coupling of many MTs to the same kinetochore, and chemical signals such as phosphorylation can regulate this physical mechanism.     

Degenerate target sites mediate rapid primed CRISPR adaptation

Prokaryotes encode adaptive immune systems, called CRISPR-Cas (clustered regularly interspaced short palindromic repeats–CRISPR associated), to provide resistance against mobile invaders, such as viruses and plasmids. Host immunity is based on incorporation of invader DNA sequences in a memory locus (CRISPR), the formation of guide RNAs from this locus, and the degradation of cognate invader DNA (protospacer). Invaders can escape type I-E CRISPR-Cas immunity in Escherichia coli K12 by making point mutations in the seed region of the protospacer or its adjacent motif (PAM), but hosts quickly restore immunity by integrating new spacers in a positive-feedback process termed “priming.” Here, by using a randomized protospacer and PAM library and high-throughput plasmid loss assays, we provide a systematic analysis of the constraints of both direct interference and subsequent priming in E. coli. We have defined a high-resolution genetic map of direct interference by Cascade and Cas3, which includes five positions of the protospacer at 6-nt intervals that readily tolerate mutations. Importantly, we show that priming is an extremely robust process capable of using degenerate target regions, with up to 13 mutations throughout the PAM and protospacer region. Priming is influenced by the number of mismatches, their position, and is nucleotide dependent. Our findings imply that even outdated spacers containing many mismatches can induce a rapid primed CRISPR response against diversified or related invaders, giving microbes an advantage in the coevolutionary arms race with their invaders.Bacteria and Archaea are regularly exposed to bacteriophages and other mobile genetic elements, such as plasmids. To control the competing effects of horizontal gene transfer, a spectrum of resistance strategies have evolved in prokaryotes (1). One of the most widespread and well-characterized are the CRISPR-Cas (clustered regularly interspaced short palindromic repeats–CRISPR-associated) systems, which provide bacterial “adaptive immunity” (1–8). Simply, CRISPR-Cas functions in three major steps. First, in a process termed “adaptation,” short sequences are derived from the invading element and incorporated into a CRISPR array (9). CRISPR arrays are composed of short repeats that are separated by the foreign-derived sequences, termed “spacers.” Second, CRISPRs are transcribed into a pre-CRISPR RNA (pre-crRNA), which is then processed into short crRNAs, which encompass portions of the repeats and most—or all—of the spacer. Finally, as part of a Cas ribonucleoprotein complex, the crRNAs guide a sequence-specific targeting of complementary nucleic acids (for recent reviews, see refs. 1–7).CRISPR-Cas systems are divided into three major types (I–III) and further categorized into subtypes (e.g., I-A to I-F) (10). The mechanisms of both crRNA generation and interference differ between the types and there are even significant differences between closely related subtypes. However, Cas1 and Cas2 are the only two Cas proteins completely conserved across all CRISPR-Cas systems and they are crucial for adaptation in Escherichia coli (10–12). The acquisition of new spacers is the most poorly understood stage in CRISPR-Cas immunity, mainly hindered by the paucity of robust laboratory assays to monitor this process (reviewed in ref. 9). Streptococcus thermophilus is highly proficient at spacer acquisition and provided much of the early insight into adaptation, showing that new spacers are typically acquired at one end of the CRISPR array from either phages (13–15) or plasmids (16). Recently, spacer acquisition has been detected in a variety of other systems (11, 12, 17–20). Adjacent to the expanding end of the array is the leader region, which harbors the promoter for pre-crRNA expression and sequences important for spacer acquisition (12, 21). Recent studies in E. coli in the type I-E system have shown that spacer acquisition can occur from phages and plasmids either when the Cas1 and Cas2 proteins are overexpressed or if the native cas genes are up-regulated, because of deletion of hns (11, 12, 20–22). The DNA targets (termed “protospacers”) of newly acquired spacers are consistently flanked by protospacer-adjacent motifs (PAMs), with the E. coli type I-E consensus 5′-protospacer-CTT-3′. PAMs were originally identified computationally (23) and were shown to play a role in interference in an early study (14). The importance of PAMs in the recognition and selection of precursor-spacers (prespacers) during adaptation was demonstrated unequivocally using assays that were independent of interference (12, 21). The simple overexpression of Cas1 and Cas2, in the absence of other cas genes, demonstrated these are the only Cas proteins essential for adaptation and are likely to recognize PAMs (12).Adaptation consists of two related stages, termed “naïve” and “primed” (9). Naïve adaptation occurs when a bacterium harboring a CRISPR-Cas system is infected by a new foreign element that it has not previously encountered. Although the acquisition of a new spacer can result in effective protection from the element, point mutations within the protospacer or PAM allow the element to escape CRISPR-Cas targeting (14, 24, 25). This aspect had been viewed as a weakness of CRISPR-Cas interference, but recent studies show that a positive feedback loop—called priming—occurs, which enables one or more new spacers to be acquired (11, 20, 22). Specifically, single mutations within either the PAM or the seed region of the protospacer, although inactive for interference, promote the rapid acquisition of new spacers from the same target (11). Priming is proposed to allow an effective response against viral or plasmid escapees through the incorporation of new spacers. Unlike naïve adaptation, priming is more complex, and in type I-E systems requires Cas1, Cas2, crRNA, the targeting complex termed Cascade [CRISPR-associated complex for antiviral defence, composed of Cse1, Cse2, Cas7, Cas5, and Cas6e (26–28)] and the Cas3 nuclease/helicase (11). Interestingly, the vast majority of spacers acquired through priming are derived from the same DNA strand as the original priming spacer (11, 20, 22). In addition, priming in E. coli was abolished by two mutations in the protospacer and PAM regions (11).In this study, we generated a mutagenic variant library of a protospacer and PAM region and used both individual high-throughput plasmid-loss assays and next-generation sequencing to determine the limits of both direct interference and indirect interference through priming. Our results demonstrate that direct interference tolerates mutations mostly at very specific positions in the protospacer, whereas priming tolerates extensive mutation of the PAM and protospacer regions. The results have wide evolutionary consequences for primed acquisition and could explain the retention of multiple “older” spacers in CRISPR arrays.     

From the Cover: Why the proportion of transmission during early-stage HIV infection does not predict the long-term impact of treatment on HIV incidence

Antiretroviral therapy (ART) reduces the infectiousness of HIV-infected persons, but only after testing, linkage to care, and successful viral suppression. Thus, a large proportion of HIV transmission during a period of high infectiousness in the first few months after infection (“early transmission”) is perceived as a threat to the impact of HIV “treatment-as-prevention” strategies. We created a mathematical model of a heterosexual HIV epidemic to investigate how the proportion of early transmission affects the impact of ART on reducing HIV incidence. The model includes stages of HIV infection, flexible sexual mixing, and changes in risk behavior over the epidemic. The model was calibrated to HIV prevalence data from South Africa using a Bayesian framework. Immediately after ART was introduced, more early transmission was associated with a smaller reduction in HIV incidence rate—consistent with the concern that a large amount of early transmission reduces the impact of treatment on incidence. However, the proportion of early transmission was not strongly related to the long-term reduction in incidence. This was because more early transmission resulted in a shorter generation time, in which case lower values for the basic reproductive number (R₀) are consistent with observed epidemic growth, and R₀ was negatively correlated with long-term intervention impact. The fraction of early transmission depends on biological factors, behavioral patterns, and epidemic stage and alone does not predict long-term intervention impacts. However, early transmission may be an important determinant in the outcome of short-term trials and evaluation of programs.Recent studies have confirmed that effective antiretroviral therapy (ART) reduces the transmission of HIV among stable heterosexual couples (1–3). This finding has generated interest in understanding the population-level impact of HIV treatment on reducing the rate of new HIV infections in generalized epidemic settings (4). Research, including mathematical modeling (5–10), implementation research (11), and major randomized controlled trials (12–14), are focused on how ART provision might be expanded strategically to maximize its public health benefits (15, 16).One concern is that if a large fraction of HIV transmission occurs shortly after a person becomes infected, before the person can be diagnosed and initiated on ART, this will limit the potential impact of HIV treatment on reducing HIV incidence (9, 17, 18). Data suggest that persons are more infectious during a short period of “early infection” after becoming infected with HIV (19–22), although there is debate about the extent, duration, and determinants of elevated infectiousness (18, 23). The amount of transmission that occurs also will depend on patterns of sexual behavior and sexual networks (17, 24–27). There have been estimates for the contribution of early infection to transmission from mathematical models (7, 17, 21, 24–26) and phylogenetic analyses (28–31), but these vary widely, from 5% to above 50% (23).In this study, we use a mathematical model to quantify how the proportion of transmission that comes from persons who have been infected recently affects the impact of treatment scale-up on HIV incidence. The model is calibrated to longitudinal HIV prevalence data from South Africa using a Bayesian framework. Thus, the model accounts for not only the early epidemic growth rate highlighted in previous research (5, 9, 18), but also the heterogeneity and sexual behavior change to explain the peak and decline in HIV incidence observed in sub-Saharan African HIV epidemics (32, 33).The model calibration allows uncertainty about factors that determine the amount of early transmission, including the relative infectiousness during early infection, heterogeneity in propensity for sexual risk behavior, assortativity in sexual partner selection, reduction in risk propensity over the life course, and population-wide reductions in risk behavior in response to the epidemic (32, 33). This results in multiple combinations of parameter values that are consistent with the observed epidemic and variation in the amount of early transmission. We simulated the impact of a treatment intervention and report how the proportion of early transmission correlates with the reduction in HIV incidence from the intervention over the short- and long-term.     

Hippocampal molecular mechanisms involved in the enhancement of fear extinction caused by exposure to novelty

Exposure to a novel environment enhances the extinction of contextual fear. This has been explained by tagging of the hippocampal synapses used in extinction, followed by capture of proteins from the synapses that process novelty. The effect is blocked by the inhibition of hippocampal protein synthesis following the novelty or the extinction. Here, we show that it can also be blocked by the postextinction or postnovelty intrahippocampal infusion of the NMDA receptor antagonist 2-amino-5-phosphono pentanoic acid; the inhibitor of calcium/calmodulin-dependent protein kinase II (CaMKII), autocamtide-2–related inhibitory peptide; or the blocker of L-voltage–dependent calcium channels (L-VDCCs), nifedipine. Inhibition of proteasomal protein degradation by β-lactacystin has no effect of its own on extinction or on the influence of novelty thereon but blocks the inhibitory effects of all the other substances except that of rapamycin on extinction, suggesting that their action depends on concomitant synaptic protein turnover. Thus, the tagging-and-capture mechanism through which novelty enhances fear extinction involves more molecular processes than hitherto thought: NMDA receptors, L-VDCCs, CaMKII, and synaptic protein turnover.Frey and Morris (1, 2) and their collaborators (3–7) proposed a mechanism whereby relatively “weak” hippocampal long-term potentiation (LTP) or long-term depression (LTD) lasting only a few minutes can nevertheless “tag” the synapses involved with proteins synthesized ad hoc, so that other plasticity-related proteins (PRPs) produced at other sets of synapses by other LTPs or LTDs can be captured by the tagged synapses and strengthen their activity to “long” LTPs or LTDs lasting hours or days (8). LTDs and LTPs can “cross”-tag each other; that is, LTDs can enhance both LTDs and LTPs, and vice versa (6, 8). Because many learned behaviors rely on hippocampal LTP or LTD (7–9), among them the processing of novelty (9, 10) and the making of extinction (11–13), interactions between consecutive learnings can also be explained by the “tagging-and-capture” hypothesis (9, 10, 13), whose application to behavior became known as “behavioral tagging and capture” (5, 7, 9, 13). Typically, exposure to a novel environment [e.g., a nonanxiogenic 50 × 50 × 40-cm open field (OF) (5, 7, 9, 10, 14)] is interpolated before testing for another task, which becomes enhanced (4–10, 13). The usual reaction to novelty is orienting and exploration (14), followed by habituation of this response (14–16). Habituation is perhaps the simplest form of learning, and it consists of inhibition of the orienting/exploratory response (14, 16).We recently showed that the brief exposure of rats to a novel environment (the OF) within a limited time window enhances the extinction of contextual fear conditioning (CFC) through a mechanism of synaptic tagging and capture (13), which is a previously unidentified example of behavioral tagging of inhibitory learning. Fear extinction is most probably due to LTD in the hippocampus (11, 12), although the possibility that it may also involve LTP is not discarded (13). The enhancement of extinction by novelty probably relies on the habituation to the novel environment, which is also probably due to LTD (15, 16). The enhancement of extinction by the exposure to novelty depends on hippocampal gene expression and ribosomal protein synthesis following extinction training and on both ribosomal and nonribosomal protein synthesis caused by the novel experience (13). Nonribosomal protein synthesis that can be blocked by rapamycin is believed to be dendritic (13, 17), so it would be strategically located for tagging-and-capture processes, but it has not been studied in synaptic tagging to date (3–8) or in other forms of behavioral tagging (7–10). As occurs with the interactions between LTPs and/or LTDs (4), the enhancement of extinction by novelty relies on hippocampal but not amygdalar processes (13).Recent findings indicate that several hippocampal processes related to learning and memory, such as the reconsolidation of spatial learning, are highly dependent on NMDA glutamate receptors, calcium/calmodulin protein kinase II (CaMKII), and long-term voltage channel blockers (L-VDCCs), which, in turn, rely on the proteasomal degradation of proteins (18). Here, we study the effects of an NMDA blocker, 2-amino-5-phosphono pentanoic acid (AP5); the L-VDCC blocker nifedipine (Nife); a CaMKII inhibitor, the autocamtide-2–related inhibitory peptide (AIP); and the irreversible proteasome blocker β-lactacystin (12, 13) on the interaction between novelty and extinction (11). As will be seen, we found that both the setting up of tags by extinction and the presumable production of PRPs by the processing of novelty are dependent on NMDA receptors, CaMKII, and L-VDCCs. This endorses and expands the hypothesis that the novelty–extinction interaction relies on synaptic tagging and capture (13).     

Kinesin processivity is gated by phosphate release

Kinesin-1 is a dimeric motor protein, central to intracellular transport, that steps hand-over-hand toward the microtubule (MT) plus-end, hydrolyzing one ATP molecule per step. Its remarkable processivity is critical for ferrying cargo within the cell: over 100 successive steps are taken, on average, before dissociation from the MT. Despite considerable work, it is not understood which features coordinate, or “gate,” the mechanochemical cycles of the two motor heads. Here, we show that kinesin dissociation occurs subsequent to, or concomitant with, phosphate (P_i) release following ATP hydrolysis. In optical trapping experiments, we found that increasing the steady-state population of the posthydrolysis ADP·P_i state (by adding free P_i) nearly doubled the kinesin run length, whereas reducing either the ATP binding rate or hydrolysis rate had no effect. The data suggest that, during processive movement, tethered-head binding occurs subsequent to hydrolysis, rather than immediately after ATP binding, as commonly suggested. The structural change driving motility, thought to be neck linker docking, is therefore completed only upon hydrolysis, and not ATP binding. Our results offer additional insights into gating mechanisms and suggest revisions to prevailing models of the kinesin reaction cycle.Since its discovery nearly 30 years ago (1), kinesin-1—the founding member of the kinesin protein superfamily—has emerged as an important model system for studying biological motors (2, 3). During “hand-over-hand” stepping, kinesin dimers alternate between a two–heads-bound (2-HB) state, with both heads attached to the microtubule (MT), and a one–head-bound (1-HB) state, where a single head, termed the tethered head, remains free of the MT (4, 5). The catalytic cycles of the two heads are maintained out of phase by a series of gating mechanisms, thereby enabling the dimer to complete, on average, over 100 steps before dissociating from the MT (6–8). A key structural element for this coordination is the neck linker (NL), a ∼14-aa segment that connects each catalytic head to a common stalk (9). In the 1-HB state, nucleotide binding is thought to induce a structural reconfiguration of the NL, immobilizing it against the MT-bound catalytic domain (2, 3, 10–17). This transition, called “NL docking,” is believed to promote unidirectional motility by biasing the position of the tethered head toward the next MT binding site (2, 3, 10–17). The completion of an 8.2-nm step (18) entails the binding of this tethered head to the MT, ATP hydrolysis, and detachment of the trailing head, thereby returning the motor to the ATP-waiting state (2, 3, 10–17). Prevailing models of the kinesin mechanochemical cycle (2, 3, 10, 14, 15, 17), which invoke NL docking upon ATP binding, explain the highly directional nature of kinesin motility and offer a compelling outline of the sequence of events following ATP binding. Nevertheless, these abstractions do not speak directly to the branching transitions that determine whether kinesin dissociates from the MT (off-pathway) or continues its processive reaction cycle (on-pathway). The distance moved by an individual motor before dissociating—the run length—is limited by unbinding from the MT. The propensity for a dimer to unbind involves a competition among multiple, force-dependent transitions in the two heads, which are not readily characterized by traditional structural or bulk biochemical approaches. Here, we implemented high-resolution single-molecule optical trapping techniques to determine transitions in the kinesin cycle that govern processivity.     

Effects of dopamine depletion on LFP oscillations in striatum are task- and learning-dependent and selectively reversed by l-DOPA

A major physiologic sign in Parkinson disease is the occurrence of abnormal oscillations in cortico-basal ganglia circuits, which can be normalized by l-DOPA therapy. Under normal circumstances, oscillatory activity in these circuits is modulated as behaviors are learned and performed, but how dopamine depletion affects such modulation is not yet known. We here induced unilateral dopamine depletion in the sensorimotor striatum of rats and then recorded local field potential (LFP) activity in the dopamine-depleted region and its contralateral correspondent as we trained the rats on a conditional T-maze task. Unexpectedly, the dopamine depletion had little effect on oscillations recorded in the pretask baseline period. Instead, the depletion amplified oscillations across delta (∼3 Hz), theta (∼8 Hz), beta (∼13 Hz), and low-gamma (∼48 Hz) ranges selectively during task performance times when each frequency band was most strongly modulated, and only after extensive training had occurred. High-gamma activity (65–100 Hz), in contrast, was weakened independent of task time or learning stage. The depletion also increased spike-field coupling of fast-spiking interneurons to low-gamma oscillations. l-DOPA therapy normalized all of these effects except those at low gamma. Our findings suggest that the task-related and learning-related dynamics of LFP oscillations are the primary targets of dopamine depletion, resulting in overexpression of behaviorally relevant oscillations. l-DOPA normalizes these dynamics except at low-gamma, linked by spike-field coupling to fast-spiking interneurons, now known to undergo structural changes after dopamine depletion and to lack normalization of spike activity following l-DOPA therapy.Loss of the dopamine-containing innervation of the basal ganglia is a primary pathology in Parkinson disease, resulting, in addition to its behavioral effects, in abnormal local field potential (LFP) oscillations within cortico-basal ganglia circuits (1–4). Clinical evidence suggests that successful therapies for Parkinson disease reduce these abnormal LFP oscillations (3–6), establishing them as a central feature of Parkinson disease. In particular, abnormally strong beta-range oscillations (12–30 Hz) and weakened high-frequency gamma oscillations (>70 Hz) have been found in basal ganglia structures. The “antimovement” beta-band oscillations are reduced by both l-DOPA therapy and by deep brain stimulation (DBS) (3–6). How these observations relate to the proposed network functions of oscillatory neural activity is not yet clear. LFP oscillations have been linked not only to motor control but also to sensory perception, attention, learning, memory formation, and interregional communication (7–11). In Parkinson disease models, abnormal patterns of synchrony have been found in rest and locomotion (12–14), but the effect of dopamine loss on LFP oscillations during complex tasks requiring learning and decision making has not been explored.Here we report that dopamine depletion in the sensorimotor striatum has striking effects both on oscillatory power in multiple frequency ranges and on spike-field synchrony, but that the abnormal patterns of synchronization are behaviorally regulated and are not omnipresent features of the dopamine-depleted state.     

Cortical entrainment to music and its modulation by expertise

Recent studies establish that cortical oscillations track naturalistic speech in a remarkably faithful way. Here, we test whether such neural activity, particularly low-frequency (<8 Hz; delta–theta) oscillations, similarly entrain to music and whether experience modifies such a cortical phenomenon. Music of varying tempi was used to test entrainment at different rates. In three magnetoencephalography experiments, we recorded from nonmusicians, as well as musicians with varying years of experience. Recordings from nonmusicians demonstrate cortical entrainment that tracks musical stimuli over a typical range of tempi, but not at tempi below 1 note per second. Importantly, the observed entrainment correlates with performance on a concurrent pitch-related behavioral task. In contrast, the data from musicians show that entrainment is enhanced by years of musical training, at all presented tempi. This suggests a bidirectional relationship between behavior and cortical entrainment, a phenomenon that has not previously been reported. Additional analyses focus on responses in the beta range (∼15–30 Hz)—often linked to delta activity in the context of temporal predictions. Our findings provide evidence that the role of beta in temporal predictions scales to the complex hierarchical rhythms in natural music and enhances processing of musical content. This study builds on important findings on brainstem plasticity and represents a compelling demonstration that cortical neural entrainment is tightly coupled to both musical training and task performance, further supporting a role for cortical oscillatory activity in music perception and cognition.Cortical oscillations in specific frequency ranges are implicated in many aspects of auditory perception. Principal among these links are “delta–theta phase entrainment” to sounds, hypothesized to parse signals into chunks (<8 Hz) (1–6), “alpha suppression,” correlated with intelligible speech (∼10 Hz) (7, 8), and “beta oscillatory modulation,” argued to reflect the prediction of rhythmic inputs (∼20 Hz) (9–13).In particular, delta–theta tracking is driven by both stimulus acoustics and speech intelligibility (14, 15). Such putative cortical entrainment is, however, not limited to speech but is elicited by stimuli such as FM narrowband noise (16, 17) or click trains (18). Overall, the data suggest that, although cortical entrainment is necessary to support intelligibility of continuous speech—perhaps by parsing the input stream into chunks for subsequent decoding—the reverse does not hold: “intelligibility” is not required to drive entrainment. The larger question of the function of entrainment in general auditory processing remains unsettled. Therefore, we investigate here whether this mechanism extends to a different, salient, ecological stimulus: music.In addition, higher-frequency activity merits scrutiny. Rhythmic stimuli evince beta band activity (15–30 Hz) (10, 19), associated with synchronization across sensorimotor cortical networks (20, 21). Beta power is modulated at the rate of isochronous tones, and its variability reduces with musical training (19). These effects have been noted in behavioral (tapping to the beat) tasks: musicians show less variability, greater accuracy, and improved detection of temporal deviations (22, 23). Furthermore, prestimulus beta plays a role in generating accurate temporal predictions even in pure listening tasks (9). Here, we test whether this mechanism scales to naturalistic and complex rhythmic stimuli such as music.Musical training has wide-ranging effects on the brain, e.g., modulating auditory processing of speech and music (24–27) and inducing structural changes in several regions (28–30). Recent work (31) has shown that musical training induces changes in amplitude and latency to brainstem responses. Building on effects such as these, we expect that increased entrainment and reliability in musicians’ cortical responses may reflect expertise. We explore three hypotheses: (i) like speech, music will trigger cortical neural entrainment processes at frequencies corresponding to the dominant note rate; (ii) musical training enhances cortical entrainment; and (iii) beta rhythms coupled with entrained delta–theta oscillations underpin accuracy in musical processing.This study focuses on a low-level stimulus feature: note rate. This methodological choice allows us to study the rate of the music without delving into the complexities of beat and meter, which can shift nonmonotonically with increasing overall rate (i.e., faster notes are often arranged into larger groupings). As the entrainment mechanism occurs for a wide range of acoustic stimuli, we find it unlikely to be following beat or meter in music: such clear groupings do not exist in other stimulus types. It should, however, be noted that influential recent research has addressed cortical beat or meter entrainment (11, 12, 32–37). In particular, using EEG, selective increases in power at beat and meter frequencies have been observed, even when those frequencies are not dominant in the acoustic spectrum (35, 36). Similarly, Large and Snyder (12) have proposed a framework in which beta and gamma support auditory–motor interactions and encoding of musical beat. Such beat induction has been linked with more frontal regions such as supplementary motor area, premotor cortex, and striatum (33), suggesting higher-order processing of the stimulus that is linked to motor output.In relation to these interesting findings, our goal is to identify a distinct entrainment process and, by hypothesis, a necessary precursor to the higher-order beat entrainment mechanism: the parsing of individual notes within a stream of music. We suggest further that beta activity may reflect such low-level temporal regularity in the generation of temporal predictions for upcoming notes. As such, the metric, notes per second (Materials and Methods), is meant to be associated with this more general process of event chunking—which must occur in all forms of sound stream processing—and should therefore not be confused with beat, meter, and the complex cognitive processes associated with these operations.     

Brain system for mental orientation in space,time, and person

Orientation is a fundamental mental function that processes the relations between the behaving self to space (places), time (events), and person (people). Behavioral and neuroimaging studies have hinted at interrelations between processing of these three domains. To unravel the neurocognitive basis of orientation, we used high-resolution 7T functional MRI as 16 subjects compared their subjective distance to different places, events, or people. Analysis at the individual-subject level revealed cortical activation related to orientation in space, time, and person in a precisely localized set of structures in the precuneus, inferior parietal, and medial frontal cortex. Comparison of orientation domains revealed a consistent order of cortical activity inside the precuneus and inferior parietal lobes, with space orientation activating posterior regions, followed anteriorly by person and then time. Core regions at the precuneus and inferior parietal lobe were activated for multiple orientation domains, suggesting also common processing for orientation across domains. The medial prefrontal cortex showed a posterior activation for time and anterior for person. Finally, the default-mode network, identified in a separate resting-state scan, was active for all orientation domains and overlapped mostly with person-orientation regions. These findings suggest that mental orientation in space, time, and person is managed by a specific brain system with a highly ordered internal organization, closely related to the default-mode network.Orientation in space, time, and person is a fundamental cognitive function and the bedrock of neurological and psychiatric mental status examination (1, 2). Orientation is defined as the “tuning between the subject and the internal representation he forms of the corresponding public reference system”: that is, the external world (1). Although the representation of the external world by means of a cognitive map has been widely investigated (3–5), the way in which the self refers to this map has yet to be understood. Moreover, the behaving self refers not only to spatial landmarks but also to remembered or imagined events, or to people around, yielding a “cognitive mapping” of the time and person domains of the mental world (2, 6–9). However, it is still unknown whether mental orientation in space, time, and person relies on similar or distinct neurocognitive systems.Several lines of research support the idea that similar neurocognitive systems underlie orientation in these three domains. Behavioral studies indicate a common psychological metric for proximity estimations (“cognitive distance”) in space, time, and person (7); for example, manipulation of stimuli’s distance in one orientation domain affects the perceived distance in the other two domains (10, 11). Accordingly, a recent neuroimaging study mapped cognitive distance estimations in the three domains to a single region in the inferior parietal lobe (IPL) (12). However, other neuroimaging studies that investigated processing of places, events, and people separately have found activation in brain regions besides the IPL, including the precuneus and posterior cingulate cortices, medial prefrontal cortex (mPFC), and lateral frontal and temporal lobes (6, 13–29). Notably, these regions constitute a part of the default-mode network (DMN), a system involved in self-referential processes (24, 30–35). These findings suggest a common brain system for orientation across domains, possibly related to the DMN.Clinical observations in patients with disorientation in space, time, and person are less clear: on the one hand, clinical syndromes may involve disorientation in several domains simultaneously, and disorientation disorders in the three domains involve lesions in similar brain regions, usually overlapping with the DMN (1, 2). On the other hand, disorientation may be limited to one specific domain – space, time, or person (2, 36–40). In addition, patients with traumatic brain injury or after electro-convulsive therapy regain their orientation gradually, from personal to spatial and temporal orientation (41, 42) whereas patients with Alzheimer’s disease typically lose orientation in time first, then in place, and then in person, suggesting that partially separate systems underlie orientation in each domain.Here, we investigated the neurocognitive system underlying orientation in space, time, and person and its relation to the DMN. To this aim, we used a mental-orientation task, with individually tailored stimuli in the space (places), time (events), and person (people) domains. To gain high anatomical specificity, we used high-resolution 7-Tesla functional MRI (fMRI). To capitalize on the spatial acuity of the high-resolution fMRI, we applied a strategy of analyzing each subject individually in native space and combined the results to compare activations for the three domains. Finally, we compared our results to the DMN as identified in each individual subject by analysis of resting-state fMRI. We hypothesized that orientation across different domains relies on a shared “core” brain system, in close relation to the DMN, yet orientation in specific domains may involve additional specialized subsystems.     

Southern East Asian origin and coexpansion of Mycobacterium tuberculosis Beijing family with Han Chinese

The Beijing family is the most successful genotype of Mycobacterium tuberculosis and responsible for more than a quarter of the global tuberculosis epidemic. As the predominant genotype in East Asia, the Beijing family has been emerging in various areas of the world and is often associated with disease outbreaks and antibiotic resistance. Revealing the origin and historical dissemination of this strain family is important for understanding its current global success. Here we characterized the global diversity of this family based on whole-genome sequences of 358 Beijing strains. We show that the Beijing strains endemic in East Asia are genetically diverse, whereas the globally emerging strains mostly belong to a more homogenous subtype known as “modern” Beijing. Phylogeographic and coalescent analyses indicate that the Beijing family most likely emerged around 30,000 y ago in southern East Asia, and accompanied the early colonization by modern humans in this area. By combining the genomic data and genotyping result of 1,793 strains from across China, we found the “modern” Beijing sublineage experienced massive expansions in northern China during the Neolithic era and subsequently spread to other regions following the migration of Han Chinese. Our results support a parallel evolution of the Beijing family and modern humans in East Asia. The dominance of the “modern” Beijing sublineage in East Asia and its recent global emergence are most likely driven by its hypervirulence, which might reflect adaption to increased human population densities linked to the agricultural transition in northern China.Tuberculosis has plagued human beings since ancient times and remains a leading cause of global morbidity and mortality. The causative agent of human tuberculosis is the Mycobacterium tuberculosis complex (MTBC), a group of organisms that harbor little genetic diversity compared with other bacteria (1). MTBC most likely originated in Africa, although its age is being debated (2–4). The human-adapted MTBC is highly clonal and is classified into seven main phylogenetic groups, designated lineage 1 through lineage 7 (2). These seven lineages show strong biogeographic associations that have been proposed to result from codiversification with different human populations (2, 5). Lineage 2 that dominates in East Asia is one of the most successful MTBC variants; more than a quarter of the global tuberculosis epidemic is caused by this lineage (6, 7). Lineage 2 contains strains that mostly belong to the so-called Beijing family (8, 9). This strain family has attracted great attentions due to its global emergence in recent decades (6, 7, 10–12), its tendency to cause disease outbreak (13–17), and its association with antibiotic resistance (12, 18). Experimental and clinical evidences suggest a hypervirulent phenotype of Beijing strains (12, 19), and a higher mutation rate compared with other strains (20).According to genotyping data from previous molecular-epidemiology studies, most Beijing strains from widespread geographic areas showed a remarkable degree of genetic similarity (6, 21), suggesting this strain family might have emerged from recent expansions. It was hypothesized that vaccination with Bacille Calmette Guerin (bacillus Calmette–Guérin) that has been widely implemented in East Asian countries might be the force driving the dominance of this strain family in this area (21). Moreover, the global emergence of the Beijing family may have been due to its hypervirulence and association with drug resistance (7, 18). However, there were discrepant results regarding the relative protective effect of bacillus Calmette–Guérin vaccination against Beijing strains from animal infection experiments (19), and many epidemiological studies failed to find any association between bacillus Calmette–Guérin vaccination and Beijing strains (22–25). The link between drug resistance and the Beijing family has primarily been observed in regions where this family has emerged recently (e.g., Cuba, South Africa, countries of the former Soviet Union) but not in East Asian, where the Beijing family has been endemic for a long time (18, 26). Furthermore, more recent studies indicate that the expansion of the Beijing family may have started long before the introduction of vaccination and antibiotic treatment (2, 3, 27).With the increased availability of genotyping data, the Beijing strains were proved more heterogeneous than initially estimated, and several Beijing sublineages have been identified (28–31). However, a full understanding of the genetic diversity of Beijing family is constrained by the low amount of nucleotide variation (8, 32). Whole-genome sequencing provides an ideal tool to study the genetic diversity of MTBC, and new insights into the origin and evolution of MTBC have been gained (2, 4, 20, 33–35). The genomic diversity of Beijing family was initially studied in a most recent study, in which a general East Asian origin and recent expansions of this strain family were suggested (36). However, the details about the origin and primary dissemination of Beijing family remain unclear. Answering of these questions is important to better understand the virulence of this lineage and its global success. Here, we combined whole-genome sequencing of key strains with detailed single nucleotide polymorphism (SNP) typing of a large collection of clinical MTBC strains isolated from across China. Our results strongly support a southern East Asian origin of the MTBC Beijing family and suggest a parallel evolution of this family with modern humans in East Asia during the last 30,000 y.     

Site-specific cation release drives actin filament severing by vertebrate cofilin

Actin polymerization powers the directed motility of eukaryotic cells. Sustained motility requires rapid filament turnover and subunit recycling. The essential regulatory protein cofilin accelerates network remodeling by severing actin filaments and increasing the concentration of ends available for elongation and subunit exchange. Although cofilin effects on actin filament assembly dynamics have been extensively studied, the molecular mechanism of cofilin-induced filament severing is not understood. Here we demonstrate that actin filament severing by vertebrate cofilin is driven by the linked dissociation of a single cation that controls filament structure and mechanical properties. Vertebrate cofilin only weakly severs Saccharomyces cerevisiae actin filaments lacking this “stiffness cation” unless a stiffness cation-binding site is engineered into the actin molecule. Moreover, vertebrate cofilin rescues the viability of a S. cerevisiae cofilin deletion mutant only when the stiffness cation site is simultaneously introduced into actin, demonstrating that filament severing is the essential function of cofilin in cells. This work reveals that site-specific interactions with cations serve a key regulatory function in actin filament fragmentation and dynamics.Actin polymerization powers the directed motility of eukaryotic cells and some pathogenic bacteria (1–3). Actin assembly also plays critical roles in endocytosis, cytokinesis, and establishment of cell polarity. Sustained motility requires filament disassembly and subunit recycling. The essential regulatory protein cofilin severs actin filaments (4–6), which accelerates actin network reorganization by increasing the concentration of filament ends available for subunit exchange (7).Cofilin binding alters the structure and mechanical properties of filaments, which effectively introduces local “defects” that compromise filament integrity and promote severing (5). Filaments with bound cofilin have altered twist (8, 9) and are more compliant in both bending and twisting than bare filaments (10–13). It has been suggested that deformations in filament shape promote fragmentation at or near regions of topological and mechanical discontinuities, such as boundaries between bare and cofilin-decorated segments along partially decorated filaments (5, 12, 14–18).Cations modulate actin filament structure and mechanical properties (19) and cofilin dissociates filament-associated cations (20), leading us to hypothesize that cation-binding interactions regulate filament severing by cofilin. Cations bind filaments at two discrete and specific sites positioned between adjacent subunits along the long-pitch helix of the filament (19, 21). These cation binding sites are referred to as “polymerization” and “stiffness” sites based on their roles in filament assembly and mechanics, respectively. These discrete sites bind both monovalent and divalent cations with a range of affinities (low millimolar for divalent and tens of millimolar for monovalent cations) (19, 21) but are predominantly occupied by Mg²⁺ and K⁺ under physiological conditions. Here we demonstrate that cation release from the stiffness site plays a central role in filament severing by vertebrate cofilin, both in vitro and in cells.     

Differential effect of aneuploidy on the X chromosome and genes with sex-biased expression in Drosophila

Global analysis of gene expression via RNA sequencing was conducted for trisomics for the left arm of chromosome 2 (2L) and compared with the normal genotype. The predominant response of genes on 2L was dosage compensation in that similar expression occurred in the trisomic compared with the diploid control. However, the male and female trisomic/normal expression ratio distributions for 2L genes differed in that females also showed a strong peak of genes with increased expression and males showed a peak of reduced expression relative to the opposite sex. For genes in other autosomal regions, the predominant response to trisomy was reduced expression to the inverse of the altered chromosomal dosage (2/3), but a minor peak of increased expression in females and further reduced expression in males were also found, illustrating a sexual dimorphism for the response to aneuploidy. Moreover, genes with sex-biased expression as revealed by comparing amounts in normal males and females showed responses of greater magnitude to trisomy 2L, suggesting that the genes involved in dosage-sensitive aneuploid effects also influence sex-biased expression. Each autosomal chromosome arm responded to 2L trisomy similarly, but the ratio distributions for X-linked genes were distinct in both sexes, illustrating an X chromosome-specific response to aneuploidy.Changes in chromosomal dosage have long been known to affect the phenotype or viability of an organism (1–4). Altering the dosage of individual chromosomes typically has a greater impact than varying the whole genome (5–7). This general rule led to the concept of “genomic balance” in that dosage changes of part of the genome produce a nonoptimal relationship of gene products. The interpretation afforded these observations was that genes on the aneuploid chromosome produce a dosage effect for the amount of gene product present in the cell (8).However, when gene expression studies were conducted on aneuploids, it became known that transacting modulations of gene product amounts were also more prevalent with aneuploidy than with whole-genome changes (9–14). Assays of enzyme activities, protein, and RNA levels revealed that any one chromosomal segment could modulate in trans the expression of genes throughout the genome (9–15). These modulations could be positively or negatively correlated with the changed chromosomal segment dosage, but inverse correlations were the most common (10–13). For genes on the varied segment, not only were dosage effects observed, but dosage compensation was also observed, which results from a cancelation of gene dosage effects by inverse effects operating simultaneously on the varied genes (9, 10, 14–18). This circumstance results in “autosomal” dosage compensation (14, 16–18). Studies of trisomic X chromosomes examining selected endogenous genes or global RNA sequencing (RNA-seq) studies illustrate that the inverse effect can also account for sex chromosome dosage compensation in Drosophila (15, 19–21). In concert, autosomal genes are largely inversely affected by trisomy of the X chromosome (15, 19, 21).The dosage effects of aneuploidy can be reduced to the action of single genes whose functions tend to be involved in heterogeneous aspects of gene regulation but which have in common membership in macromolecular complexes (8, 22–24). This fact led to the hypothesis that genomic imbalance effects result from the altered stoichiometry of subunits that affects the function of the whole and that occurs from partial but not whole-genome dosage change (8, 22–25). Genomic balance also affects the evolutionary trajectory of duplicate genes differently based on whether the mode of duplication is partial or whole-genome (22, 23).Here we used RNA-seq to examine global patterns of gene expression in male and female larvae trisomic for the left arm of chromosome 2 (2L). The results demonstrate the strong prevalence of aneuploidy dosage compensation and of transacting inverse effects. Furthermore, because both trisomic males and females could be examined, a sexual dimorphism of the aneuploid response was discovered. Also, the response of the X chromosome to trisomy 2L was found to be distinct from that of the autosomes, illustrating an X chromosome-specific effect. Genes with sex-biased expression, as determined by comparing normal males and females, responded more strongly to trisomy 2L. Collectively, the results illustrate the prevalence of the inverse dosage effect in trisomic Drosophila and suggest that the X chromosome has evolved a distinct response to genomic imbalance as would be expected under the hypothesis that X chromosome dosage compensation uses the inverse dosage effect as part of its mechanism (15).     

Structural basis for the sheddase function of human meprin β metalloproteinase at the plasma membrane

Ectodomain shedding at the cell surface is a major mechanism to regulate the extracellular and circulatory concentration or the activities of signaling proteins at the plasma membrane. Human meprin β is a 145-kDa disulfide-linked homodimeric multidomain type-I membrane metallopeptidase that sheds membrane-bound cytokines and growth factors, thereby contributing to inflammatory diseases, angiogenesis, and tumor progression. In addition, it cleaves amyloid precursor protein (APP) at the β-secretase site, giving rise to amyloidogenic peptides. We have solved the X-ray crystal structure of a major fragment of the meprin β ectoprotein, the first of a multidomain oligomeric transmembrane sheddase, and of its zymogen. The meprin β dimer displays a compact shape, whose catalytic domain undergoes major rearrangement upon activation, and reveals an exosite and a sugar-rich channel, both of which possibly engage in substrate binding. A plausible structure-derived working mechanism suggests that substrates such as APP are shed close to the plasma membrane surface following an “N-like” chain trace.Physiological processes in the extracellular milieu and the circulation require finely tuned concentrations of signal molecules such as cytokines, growth factors, receptors, adhesion molecules, and peptidases. Many of these proteins are synthesized as type-I membrane protein variants or precursors consisting of a glycosylated N-terminal ectoprotein, a transmembrane helix, and a C-terminal cytosolic tail. Their localization at the cell surface restricts their field of action to autocrine or juxtacrine processes. However, to act at a distance in paracrine, synaptic, or endocrine events, they have to be released from the plasma membrane into the extracellular space as soluble factors through “protein ectodomain shedding” (1, 2). This entails limited proteolysis and is a major posttranslational regulation mechanism that affects 2–4% of the proteins on the cell surface, occurs at or near the plasma membrane (3), and apparently follows a common release mechanism (2). It may also proteolytically inactivate proteins to terminate their function on the cell surface (4). Peptidases engaged in such processing are “sheddases” and the most studied transmembrane sheddases are members of the adamalysin/ADAMs (4, 5) and matrix-metalloproteinase (MMP) (6) families within the metzincin clan of metallopeptidases (MPs) (7–9). These include ADAM-8, -9, -10, -12, -15, -17, -19, -28, and -33 (1, 4) and membrane-type 1 (MT1)-MMP, MT3-MMP, and MT5-MMP (2, 6, 10). Other confirmed transmembrane sheddases are the aspartic proteinases BACE-1 and -2 (ref. 11 and references therein) and the malarian parasite serine proteinases, PfSUB2, PfROM1, and PfROM4 (12). Distinct sheddases may participate in intercalating processes, with disparate physiological consequences: ADAM-9, -10 (α-secretases), and -17 contribute to the nonamyloidogenic pathway of human amyloid precursor protein (APP) processing, whereas BACE-1 (β-secretase) participates in the amyloidogenic pathway. Whereas the former generates innocuous peptides, the latter gives rise to the toxic β-amyloid peptides believed to be responsible for Alzheimer’s disease (11). In several instances, shedding at the membrane surface is followed by a “regulated intramembrane proteolysis” step within the membrane (1). This is the case for the processing of Notch ligand Delta1 and of APP, both carried out by γ-secretase after action of an α/β-secretase (11), and for signal-peptide peptidase, which removes remnants of the secretory protein translocation from the endoplasmic membrane (13).Recently, human meprin β (Mβ) was found to specifically process APP in vivo, which may contribute to Alzheimer’s disease (14, 15). It was also reported to activate cell-anchored α-secretase ADAM-10 and to be widely expressed in brain, intestine, kidney, and skin (14, 16–18). Disruption of Mβ in mice affects embryonic viability, birth weight, and renal gene expression profiles (19). The enzyme was further identified as a sheddase or proteolytic regulator at the plasma membrane of interleukin-1β (20), interleukin-18 (21), tumor growth factor α (22), procollagen III (23), epithelial sodium channel (24), E-cadherin (25), tenascin-C (26), and vascular endothelial growth factor A (27). Further substrates include fibroblast growth factor 19 and connective tissue growth factor. Altered expression and activity of the enzyme are associated with pathological conditions such as inflammatory bowel disease (28), tumor progression (29), nephritis (30), and fibrosis (23).Mβ is a 679-residue secreted multidomain type-I membrane MP that belongs to the astacin family within the metzincins (7, 9, 16, 31, 32). The enzyme is glycosylated and assembles into either disulfide-linked homodimers or heterodimers with the closely related meprin α-subunit (33). Mβ homodimers are essentially membrane bound but may also be shed from the surface by ADAM-10 and -17 (34, 35). To assess function, working mechanism, and activation of Mβ, we analyzed the structure of the major ectoprotein of mature Mβ (MβΔC) and of its zymogen, promeprin β (pMβΔC). With regard to transmembrane sheddases, to date only the structures of the isolated monomeric catalytic domains of ADAM-17 [Protein Data Bank (PDB) access code 1BKC], ADAM-33 (PDB 1R55), MT1-MMP (PDB 1BUV), MT3-MMP (PDB 1RM8), BACE-1 (PDB 1FKN), and BACE-2 (PDB 2EWY) have been described. Accordingly, this is a unique structural report of a multidomain oligomeric transmembrane sheddase. This report has allowed us a better understanding of the structural basis for latency and activation of this MP and to derive a plausible working mechanism for shedding of glycosylated type-I membrane substrates such as APP at the extracellular membrane surface.     

Frequency-invariant temporal ordering of interneuronal discharges during hippocampal oscillations in awake mice

Endogenous brain rhythms occurring at various frequencies and associated with distinct behavioral states provide multiscale temporal windows that enable cells to time their spiking activity with high precision, which is thought to be important for the coding of information in neuronal circuits. However, although the selective timing of GABAergic inputs to specific spatial domains of principal cells are known to play key roles in network oscillations, the in vivo firing patterns of distinct hippocampal interneurons in awake animals are not known. Here we used a combination of juxtacellular labeling techniques with recordings from anesthesia-free, head-fixed mice running or resting on a spherical treadmill to study the oscillation-dependent discharges by two major interneuronal subtypes, the perisomatically projecting parvalbumin-positive basket cells (PVBCs) and distal dendritically projecting oriens lacunosum moleculare (OLM) cells. Recordings of the spiking activity of post hoc-identified CA1 interneurons during theta (5–10 Hz), gamma (25–90Hz), epsilon (“high-gamma”; 90–130 Hz), and ripple (130–200 Hz) oscillations revealed both cell type- and behavioral state-dependent entrainments of PVBC and OLM cell discharges in awake mice. Our results in awake mice differed in several respects from previous data on interneuronal discharge patterns in anesthetized animals. In addition, our results demonstrate a form of frequency-invariant, cell type-specific temporal ordering of inhibitory inputs in which PVBC-derived perisomatic inhibition is followed by OLM cell-generated distal dendritic inhibition during each of the network oscillation bands studied, spanning more than an order of magnitude in frequencies.Brain state-specific network oscillations in the theta, gamma, epsilon, and ripple frequencies reflect the temporally structured, coordinated activation of principal cell populations in the hippocampus and its connected structures (1–5). These distinct oscillations with their characteristic frequency bands occur during different behaviors. Theta oscillations, which often appear with nested gamma and epsilon oscillations, are prominent during locomotion and rapid eye movement (REM) sleep, whereas ripple waves are present mostly during consummatory states, quiet wakefulness, and slow-wave sleep (6–11). The various oscillatory patterns likely serve distinct computational roles in the circuit. For example, the differential phase coupling of epsilon and gamma oscillations to theta oscillations in the CA1 has been suggested to route information selectively from the entorhinal cortex and CA3 (ref. 12, but also see refs. 13 and 14). In general, network oscillations occurring at different timescales are associated with the encoding, consolidation, and retrieval of information, and experimental perturbation of the phase-locked firing during distinct oscillations results in functional deficits (15, 16).It has been recognized that different GABAergic cell types innervating specific postsynaptic domains release GABA at particular times during behaviorally relevant network oscillations (17), underscoring the fundamental unity of neuronal space and time as reflected in the recently coined term “chronocircuit” (18). However, a better understanding of the rules governing the organization and functions of hippocampal chronocircuits has been hindered by the lack of data on the in vivo spike timing of anatomically and neurochemically identified interneurons during hippocampal network oscillations from awake, anesthesia-free animals. The reason for the difficulty in obtaining unambiguous data from identified interneurons in awake animals is technical in nature. Multiple single-unit recordings from freely moving animals cannot identify the recorded units definitively as belonging to particular interneuronal subtypes, because the axo-dendritic structure cannot be visualized, and the expression of defining cellular markers cannot be determined. In addition, even when multiunit recordings are combined with Cre-line–based optogenetics (19), cell identification has been limited to broad categories such as parvalbumin- or somatostatin-expressing cells (Discussion).Here we report the brain state-specific discharge patterns of two major dendritically or perisomatically projecting interneuronal subtypes in the CA1 region. The results were obtained using juxtacellular recording and labeling techniques (20) in anesthesia-free, head-fixed mice that were running or resting on a spherical treadmill (21, 22), followed by post hoc identification of the recorded cells. We find that parvalbumin-positive basket cells (PVBCs) and most oriens lacunosum moleculare (OLM) cells are significantly modulated by theta, gamma, epsilon, and ripple oscillations, with the degree of modulation dependent on both the cell type and the oscillatory frequency. In addition, the data indicate a consistent temporal ordering of PVBC and OLM discharges during individual oscillatory cycles regardless of the oscillatory frequency, with the firing of PVBCs preceding the discharges of OLM cells from the slowest (theta) to the fastest (ripple) oscillations. These findings provide insights into the mechanisms of generation of theta, gamma, epsilon, and ripple oscillations and indicate that delayed inhibitory input onto the distal dendrites of pyramidal cells may play a critical role for hippocampus-dependent computations across a broad range of oscillations and related brain states.     

Coherent delta-band oscillations between cortical areas correlate with decision making

Coherent oscillations in the theta-to-gamma frequency range have been proposed as a mechanism that coordinates neural activity in large-scale cortical networks in sensory, motor, and cognitive tasks. Whether this mechanism also involves coherent oscillations at delta frequencies (1–4 Hz) is not known. Rather, delta oscillations have been associated with slow-wave sleep. Here, we show coherent oscillations in the delta frequency band between parietal and frontal cortices during the decision-making component of a somatosensory discrimination task. Importantly, the magnitude of this delta-band coherence is modulated by the different decision alternatives. Furthermore, during control conditions not requiring decision making, delta-band coherences are typically much reduced. Our work indicates an important role for synchronous activity in the delta frequency band when large-scale, distant cortical networks coordinate their neural activity during decision making.Studies of the neural correlates of decision making in behaving monkeys have mainly been based on the analysis of firing rate patterns of neurons in individual cortical circuits, recorded one by one in succession, while trained monkeys perform sensory, motor, and cognitive tasks (1–3). These studies showed that the neuronal activities distributed across parietal and frontal lobe cortices correlate with processes that lead to decision making (4–7). However, how these spatially distant, cortical circuits coordinate their activities into a unified functional network during decision making remains poorly understood.It has been proposed that coherent oscillations of neuronal activities constitute a putative dynamical mechanism for mediating the interaction between different subsets of brain areas (8–11). Simultaneous recording from multiple intracortical areas in monkeys showed that the coherent higher frequency (beta and gamma bands) oscillations are linked to a broad variety of cognitive functions (12–18). Cortical oscillations at lower frequencies (theta and alpha bands) have also been discussed in terms of long-range integrative processes (19, 20). In fact, recent evidence showed theta-band coupling between visual area V4 and prefrontal cortex during short-term memory (21) and among the rat prefrontal cortex, ventral tegmental area, and hippocampus during working memory (22). It remains, however, probing whether coherent delta-band oscillations play a functional role in the interaction between cortical circuits. Delta-band oscillations are typically associated with slow-wave sleep (SWS; ref. 23), but an important question is whether delta-band oscillations during SWS and waking states represent the same underlying phenomenon (24). Recent findings associated delta-band oscillations in individual cortical areas with attention (25). In monkey primary visual cortex (26) and human motor cortex (27), delta-band oscillations entrain to the rhythm of external sensory events in an attention-dependent manner.Here, we examined whether coherent oscillations coordinate the activity of five simultaneously recorded cortical areas in the monkey performing a somatosensory discrimination task (7). We specifically focused on whether coherent delta-band oscillations play a significant functional role in linking cortical circuits during decision making.     

Drosophila seminal protein ovulin mediates ovulation through female octopamine neuronal signaling

Across animal taxa, seminal proteins are important regulators of female reproductive physiology and behavior. However, little is understood about the physiological or molecular mechanisms by which seminal proteins effect these changes. To investigate this topic, we studied the increase in Drosophila melanogaster ovulation behavior induced by mating. Ovulation requires octopamine (OA) signaling from the central nervous system to coordinate an egg’s release from the ovary and its passage into the oviduct. The seminal protein ovulin increases ovulation rates after mating. We tested whether ovulin acts through OA to increase ovulation behavior. Increasing OA neuronal excitability compensated for a lack of ovulin received during mating. Moreover, we identified a mating-dependent relaxation of oviduct musculature, for which ovulin is a necessary and sufficient male contribution. We report further that oviduct muscle relaxation can be induced by activating OA neurons, requires normal metabolic production of OA, and reflects ovulin’s increasing of OA neuronal signaling. Finally, we showed that as a result of ovulin exposure, there is subsequent growth of OA synaptic sites at the oviduct, demonstrating that seminal proteins can contribute to synaptic plasticity. Together, these results demonstrate that ovulin increases ovulation through OA neuronal signaling and, by extension, that seminal proteins can alter reproductive physiology by modulating known female pathways regulating reproduction.Throughout internally fertilizing animals, seminal proteins play important roles in regulating female fertility by altering female physiology and, in some cases, behavior after mating (reviewed in refs. 1–3). Despite this, little is understood about the physiological mechanisms by which seminal proteins induce postmating changes and how their actions are linked with known networks regulating female reproductive physiology.In Drosophila melanogaster, the suite of seminal proteins has been identified, as have many seminal protein-dependent postmating responses, including changes in egg production and laying, remating behavior, locomotion, feeding, and in ovulation rate (reviewed in refs. 2 and 3). For example, the Drosophila seminal protein ovulin elevates ovulation rate to maximal levels during the 24 h following mating (4, 5), and the seminal protein sex peptide (SP) suppresses female mating receptivity and increases egg-laying behavior for several days after mating (6–10). However, although a receptor for SP has been identified (11), along with elements of the neural circuit in which it is required (12–14), SP’s mechanism of action has not yet been linked to regulatory networks known to control postmating behaviors. Thus, a crucial question remains: how do male-derived seminal proteins interact with regulatory networks in females to trigger postmating responses?We addressed this question by examining the stimulation of Drosophila ovulation by the seminal protein ovulin. In insects, ovulation, defined here as the release of an egg from the ovary to the uterus, is among the best understood reproductive processes in terms of its physiology and neurogenetics (15–27). In D. melanogaster, ovulation requires input from neurons in the abdominal ganglia that release the catecholaminergic neuromodulators octopamine (OA) and tyramine (17, 18, 28). Drosophila ovulation also requires an OA receptor, OA receptor in mushroom bodies (OAMB) (19, 20). Moreover, it has been proposed that OA may integrate extrinsic factors to regulate ovulation rates (17). Noradrenaline, the vertebrate structural and functional equivalent to OA (29, 30), is important for mammalian ovulation, and its dysregulation has been associated with ovulation disorders (31–38). In this paper we investigate the role of neurons that release OA and tyramine in ovulin’s action. For simplicity, we refer to these neurons as “OA neurons” to reflect the well-established role of OA in ovulation behavior (16–20, 22).We investigated how action of the seminal protein ovulin relates to the conserved canonical neuromodulatory pathway that regulates ovulation physiology (39–41). We found that ovulin increases ovulation and egg laying through OA neuronal signaling. We also found that ovulin relaxes oviduct muscle tonus, a postmating process that is also mediated by OA neuronal signaling. Finally, subsequent to these effects we detected an ovulin-dependent increase in synaptic sites between OA motor neurons and oviduct muscle, suggesting that ovulin’s stimulation of OA neurons could have increased their synaptic activity. These results suggest that ovulin affects ovulation by manipulating the gain of a neuromodulatory pathway regulating ovulation physiology.