A description is provided of the software algorithms developed for the CMS tracker both for reconstructing charged-particle trajectories in proton-proton interactions and for using the resulting tracks to estimate the positions of the LHC luminous region and individual primary-interaction vertices. Despite the very hostile environment at the LHC, the performance obtained with these algorithms is found to be excellent. For tt events under typical 2011 pileup conditions, the average trackreconstruction efficiency for promptly-produced charged particles with transverse momenta of p T > 0.9 GeV is 94% for pseudorapidities of |η| < 0.9 and 85% for 0.9 < |η| < 2.5. The inefficiency is caused mainly by hadrons that undergo nuclear interactions in the tracker material. For isolated muons, the corresponding efficiencies are essentially 100%. For isolated muons of p T = 100 GeV emitted at |η| < 1.4, the resolutions are approximately 2.8% in p T , and respectively, 10 µm and 30 µm in the transverse and longitudinal impact parameters. The position resolution achieved for reconstructed primary vertices that correspond to interesting pp collisions is 10-12 µm in each of the three spatial dimensions. The tracking and vertexing software is fast and flexible, and easily adaptable to other functions, such as fast tracking for the trigger, or dedicated tracking for electrons that takes into account bremsstrahlung.

Large scale transitions between active (up) and silent (down) states during quiet wakefulness or NREM sleep regulate fundamental cortical functions and are known to involve both excitatory and inhibitory cells. However, if and how inhibition regulates these activity transitions is unclear. Using fluorescence-targeted electrophysiological recording and cell-specific optogenetic manipulation in both anesthetized and non-anesthetized mice, we found that two major classes of interneurons, the parvalbumin and the somatostatin positive cells, tightly control both up-to-down and down-to-up state transitions. Inhibitory regulation of state transition was observed under both natural and optogenetically-evoked conditions. Moreover, perturbative optogenetic experiments revealed that the inhibitory control of state transition was interneuron-type specific. Finally, local manipulation of small ensembles of interneurons affected cortical populations millimetres away from the modulated region. Together, these results demonstrate that inhibition potently gates transitions between cortical activity states, and reveal the cellular mechanisms by which local inhibitory microcircuits regulate state transitions at the mesoscale.DOI: http://dx.doi.org/10.7554/eLife.26177.001

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Autonomic regulation of heart rate is largely mediated by the effect of cAMP on the pacemaker current I f , driven by hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. cAMP 2 enhances HCN open probability by binding to the CNBD (cyclic nucleotide binding domain). The Clinker transmits the cAMP-induced conformational change from the CNBD to the pore and is thus considered a passive element in the opening transition. Here we report the finding of an allosteric binding site in the C-linker of HCN4 that implies a regulatory function of this domain. By structural and functional analysis we show that cyclic dinucleotides, an emerging class of second messengers in mammals, bind to this C-linker pocket (CLP) and antagonize cAMP regulation of HCN4 channels. Accordingly, cyclic dinucleotides prevent cAMP regulation of I f in sinoatrial-node myocytes, reducing heart rate by 30%. The same effect is attained by Compound 11, a molecule selected by virtual docking to the CLP. Occupancy of the CLP hence constitutes an efficient mechanism to prevent -adrenergic stimulation on I f . Our results highlight the regulative role of the C-linker in HCN4 and identify an isoform-specific drug target within the HCN family. Furthermore, these data extend the signaling scope of cyclic dinucleotides in mammals, beyond their first reported role in innate immune system. IntroductionThe "funny" current (I f ) of cardiac pacemaker myocytes is an inward current activated by hyperpolarization of membrane voltage and controlled by intracellular cAMP 1 . Being activated and inhibited by -adrenergic and muscarinic M2 receptor stimulation, respectively, I f represents a basic physiological mechanism mediating autonomic regulation of heart rate and constitutes an ideal target for the pharmacological control of cardiac activity. The molecular determinants of I f are the Hyperpolarization-activated cAMP-gated (HCN) channels 2,3 . In these proteins, the transmembrane pore is connected at the N terminus to a voltage sensor domain and at the C-terminus to a cytosolic cyclic-nucleotide-binding domain (CNBD). The C-linker, anhelix folded domain of 90 amino acids, connects the CNBD to the pore. Structural studies showed that the cytosolic C-terminal fragment (C-linker + CNBD) assembles as a 4-fold symmetric tetramer in which the primary subunit interactions are provided by the linkers. The C-linkers form a ring in which the first two helices of one subunit (A' and B') form a helix-turn-helix motif that rests as an "elbow" on the "shoulder"formed by the second two helices, C' and D', of the neighboring subunit 4 . Enhancement of channel open probability by cAMP reflects the transition from the cAMP-unbound to the bound conformation of the 3 CNBD that induces a centrifugal rearrangement of the C-linkers with the shoulders twisting away from the elbows 5 . This movement in turn stabilizes the open conformation of the pore. Given the critical role of the Clinker in HCN channel modulation by ligands, it is interesting to note that this linker ...

Genetically encoded calcium indicators and optogenetic actuators can report and manipulate the activity of specific neuronal populations. However, applying imaging and optogenetics simultaneously has been difficult to establish in the mammalian brain, even though combining the techniques would provide a powerful approach to reveal the functional organization of neural circuits. Here, we developed a technique based on patterned two-photon illumination to allow fast scanless imaging of GCaMP6 signals in the intact mouse brain at the same time as single-photon optogenetic inhibition with Archaerhodopsin. Using combined imaging and electrophysiological recording, we demonstrate that single and short bursts of action potentials in pyramidal neurons can be detected in the scanless modality at acquisition frequencies up to 1 kHz. Moreover, we demonstrate that our system strongly reduces the artifacts in the fluorescence detection that are induced by single-photon optogenetic illumination. Finally, we validated our technique investigating the role of parvalbumin-positive (PV) interneurons in the control of spontaneous cortical dynamics. Monitoring the activity of cellular populations on a precise spatiotemporal scale while manipulating neuronal activity with optogenetics provides a powerful tool to causally elucidate the cellular mechanisms underlying circuit function in the intact mammalian brain.