A glycan gate controls opening of the SARS-CoV-2 spike protein

SARS-CoV-2 infection is controlled by the opening of the spike protein receptor binding domain (RBD), which transitions from a glycan-shielded (down) to an exposed (up) state in order to bind the human ACE2 receptor and infect cells. While snapshots of the up and down states have been obtained by cryoEM and cryoET, details of the RBD opening transition evade experimental characterization. Here, over 200 μs of weighted ensemble (WE) simulations of the fully glycosylated spike ectodomain allow us to characterize more than 300 continuous, kinetically unbiased RBD opening pathways. Together with biolayer interferometry experiments, we reveal a gating role for the N-glycan at position N343, which facilitates RBD opening. Residues D405, R408, and D427 also participate. The atomic-level characterization of the glycosylated spike activation mechanism provided herein achieves a new high-water mark for ensemble pathway simulations and offers a foundation for understanding the fundamental mechanisms of SARS-CoV-2 viral entry and infection.

Up and Down States During Slow Oscillations in Slow-Wave Sleep and Different Levels of Anesthesia

Slow oscillations are a pattern of synchronized network activity generated by the cerebral cortex. They consist of Up and Down states, which are periods of activity interspersed with periods of silence, respectively. However, even when this is a unique dynamic regime of transitions between Up and Down states, this pattern is not constant: there is a range of oscillatory frequencies (0.1–4 Hz), and the duration of Up vs. Down states during the cycles is variable. This opens many questions. Is there a constant relationship between the duration of Up and Down states? How much do they vary across conditions and oscillatory frequencies? Are there different sub regimes within the slow oscillations? To answer these questions, we aimed to explore a concrete aspect of slow oscillations, Up and Down state durations, across three conditions: deep anesthesia, light anesthesia, and slow-wave sleep (SWS), in the same chronically implanted rats. We found that light anesthesia and SWS have rather similar properties, occupying a small area of the Up and Down state duration space. Deeper levels of anesthesia occupy a larger region of this space, revealing that a large variety of Up and Down state durations can emerge within the slow oscillatory regime. In a network model, we investigated the network parameters that can explain the different points within our bifurcation diagram in which slow oscillations are expressed.

Modulation of cortical slow oscillatory rhythm by GABAB receptors: an experimental and computational study

Slow wave oscillations (SWO) dominate cortical activity during deep sleep, anesthesia and in some brain lesions. SWO consist of Up states or periods of activity interspersed with Down states or periods of silence. The rhythmicity expressed during SWO integrates neuronal and connectivity properties of the network and it is often altered in neurological pathological conditions. Different mechanisms have been proposed to drive the transitions between Up and Down states, in particular, adaptation mechanisms have been proposed to contribute to the Up-to-Down transition. Synaptic inhibition, and specially GABAB receptors, have also been proposed to have a role in the termination of Up states. The interplay between these two potential mechanisms, adaptation and inhibition, is not well understood and the role of slow inhibition is not yet clear regarding the full cycle of the slow oscillatory rhythm. Here we contribute to its understanding by combining experimental and computational techniques. GABAB receptors-blockade not only elongated Up states, but also affected the subsequent Down states, and thus the whole cycle of the oscillations. Furthermore, while adaptation tends to yield a rather regular behavior, GABAB receptors-blockade decreased the variability of the sequence of Up and Down states. Interestingly, variability changes could be accomplished in two different ways: either accompanied by a shortening or by a lengthening of the duration of the Down state. Even when the most common observation is the lengthening of the Down states, both changes are expressed experimentally and also in numerical simulations. Our simulations suggest that the sluggishness of GABAB receptors to follow the excitatory fluctuations of the cortical network can explain these different network dynamics modulated by GABAB receptors.

Up and Down States of Cortical Neurons in Focal Limbic Seizures

Recent work suggests an important role for cortical–subcortical networks in seizure-related loss of consciousness. Temporal lobe seizures disrupt subcortical arousal systems, which may lead to depressed cortical function and loss of consciousness. Extracellular recordings show ictal neocortical slow waves at about 1 Hz, but it is not known whether these simply represent seizure propagation or alternatively deep sleep-like activity, which should include cortical neuronal Up and Down states. In this study, using in vivo whole-cell recordings in a rat model of focal limbic seizures, we directly examine the electrophysiological properties of cortical neurons during seizures and deep anesthesia. We found that during seizures, the membrane potential of frontal cortical secondary motor cortex layer 5 neurons fluctuates between Up and Down states, with decreased input resistance and increased firing rate in Up states when compared to Down states. Importantly, Up and Down states in seizures are not significantly different from those in deep anesthesia, in terms of membrane potential, oscillation frequency, firing rate, and input resistance. By demonstrating these fundamental similarities in cortical electrophysiology between deep anesthesia and seizures, our results support the idea that a state of decreased cortical arousal may contribute to mechanisms of loss of consciousness during seizures.

Broadband slow-wave modulation in posterior and anterior cortex tracks distinct states of propofol-induced unconsciousness

A controversy 5 has developed in recent years over the role that frontal and posterior cortices play in mediating consciousness and unconsciousness. One hypothesis proposes that posterior sensory and association cortices are the principal mediators of consciousness, citing evidence that strong slow-wave activity over posterior cortex during sleep disrupts the contents of dreaming. A competing hypothesis proposes that frontal-posterior interactions are critical to ignite a conscious percept, since activation of frontal cortex appears necessary for perception and can reverse unconsciousness under anesthesia. In both cases, EEG slow-waves (< 1 Hz) are considered evidence that up- and down-states are disrupting cortical activity necessary for consciousness. Here, we used anesthesia to study the interaction between the slow-wave and higher frequency activity in humans. If slow-waves are derived from underlying up and down-states, then they should modulate activity across a broad range of frequencies. We found that this broadband slow-wave modulation does occur: broadband slow-wave modulation occurs over posterior cortex when subjects initially become unconscious, but later encompasses both frontal and posterior cortex when subjects are more deeply anesthetized and likely unarousable. Based on these results, we argue that unconsciousness under anesthesia comprises several shifts in brain state that disrupt the sensory contents of consciousness distinct from arousal and awareness of those contents.Significance StatementThe roles of frontal and posterior cortices in mediating consciousness and unconsciousness are controversial. Disruption of posterior cortex during sleep appears to suppress the contents of dreaming, yet activation of frontal cortex appears necessary for perception and can reverse unconsciousness under anesthesia. We studied the time course of regional cortical disruption, as mediated by slow-wave modulation of broadband activity, during anesthesia-induced unconsciousness in humans. We found that broadband slow-wave modulation covered posterior cortex when subjects initially became unconscious, but later encompassed both frontal and posterior cortex when subjects were deeply anesthetized and likely unarousable. This suggests that unconsciousness under anesthesia comprises several shifts in brain state that disrupt the contents of consciousness distinct from arousal and awareness of those contents.