Bidirectional optogenetic control of inhibitory neurons in freely-moving mice

Objective: Optogenetic manipulations of excitable cells enable activating or silencing specific types of neurons. By expressing two types of exogenous proteins, a single neuron can be depolarized using light of one wavelength and hyperpolarized with another. However, routing two distinct wavelengths into the same brain locality typically requires bulky optics that cannot be implanted on the head of a freely-moving animal. Methods: We developed a lens-free approach for constructing dual-color head-mounted, fiber-based optical units: any two wavelengths can be combined. Results: Here, each unit was comprised of one 450 nm and one 638 nm laser diode, yielding light power of 0.4 mW and 8 mW at the end of a 50 micrometer multimode fiber. To create a multi-color/multi-site optoelectronic device, a four-shank silicon probe mounted on a microdrive was equipped with two dual-color and two single-color units, for a total weight under 3 g. Devices were implanted in mice expressing the blue-light sensitive cation channel ChR2 and the red-light sensitive chloride pump Jaws in parvalbumin-immunoreactive (PV) inhibitory neurons. The combination of dual-color units with recording electrodes was free from electromagnetic interference, and device heating was under 7{\deg}C even after prolonged operation. Conclusion: Using these devices, the same cortical PV cell could be activated and silenced. This was achieved for multiple cells both in neocortex and hippocampus of freely-moving mice. Significance: This technology can be used for controlling spatially intermingled neurons that have distinct genetic profiles, and for controlling spike timing of cortical neurons during cognitive tasks.Comment: 11 pages, 9 figure

The Interaction of FABP with Kapα.

Gene-activating lipophilic compounds are carried into the nucleus when loaded on fatty-acid-binding proteins (FABP). Some of these proteins are recognized by the α-Karyopherin (Kapα) through its nuclear localization signal (NLS) consisting of three positive residues that are not in a continuous sequence. The Importin system can distinguish between FABP loaded with activating and non-activating compounds. In the present study, we introduced molecular dynamics as a tool for clarifying the mechanism by which FABP4, loaded with activating ligand (linoleate) is recognized by Kapα. In the first phase, we simulated the complex between KapαΔIBB (termed "Armadillo") that was crystallized with two NLS hepta-peptides. The trajectory revealed that the crystal-structure orientation of the peptides is rapidly lost and new interactions dominate. Though, the NLS sequence of FABP4 is cryptic, since the functional residues are not in direct sequence, implicating more than one possible conformation. Therefore, four possible docked conformations were generated, in which the NLS of FABP4 is interacting with either the major or the minor sites of Kapα, and the N → C vectors are parallel or anti-parallel. Out of these four basic starting positions, only the FABP4-minor site complex exhibited a large number of contact points. In this complex, the FABP interacts with the minor and the major sites, suppressing the self-inhibitory interaction of the Kapα, rendering it free to react with Kapβ. Finally, we propose that the transportable conformation generated an extended hydrophobic domain which expanded out of the boundary of the FABP4, allowing the loaded linoleate to partially migrate out of the FABP into a joint complex in which the Kapα contributes part of a combined binding pocket

The crystal structure of the complex between Armadillo and two SV40 large T antigen PKKKRKV peptides (1EJL.pdb).

<p>The N-terminal IBB domain of the Kapα, consisting of a 69 residues, was removed prior to crystallization. The two peptides (shown in VDW representation, are located at the major (left) and minor (right) sites. The C→N axis of the two peptides, as marked for the peptide at the major site, are antiparallel to that of the Armadillo.</p

The RMSD of the Armadillo bound to FABP and the NLS-peptide in different orientation and location (major and minor site).

<p>(A) FABP4 bound in the major site parallel to the Armadillo (2 simulations—left and right); (B) FABP4 bound in the major site anti-parallel to the Armadillo (2 simulations); (C) FABP bound in the minor site parallel to the Armadillo (2 simulations); (D) FABP bound in the minor site anti-parallel to the Armadillo (2 simulations); (E) NLS peptide bound in both sites (major and minor) to the Armadillo; (F)–free Armadillo.</p

The RMSF of the FABP4-Armadillo complex as calculated for the parallel orientation.

<p>The simulation of the free Armadillo is presented by the blue line; the Armadillo bound to the NLS peptide is in red; the Armadillo bound to the FABP is in green. (A) The FABP4 is bound to the major site. (B) The FABP4 is bound to the minor site. The locations of the Armadillo's WxxxN repeats are marked on the x axis.</p

Temporal distribution of clusters representing the structure of the peptide at the major (left) and the minor (right) sites.

<p>The upper frames denote the temporal distribution of the clusters where high values correspond with low probability of the cluster. The lower frames present the electrostatic (black) and Lennard-Jones (red) interaction energies between the peptide and the Armadillo. The interaction energies and the cluster number are presented on the same time scale. To reduce the noise, the energies were smoothed by a running-average over a window of 10 ps.</p

The most proximal pairs in the FABP4-Armadillo complex at the minor site in parallel orientation.

<p>The table lists all residues on the FABP4 and on the Armadillo for which the (geometric) average of the distance between them was less than 4 Å (marked in red) or less than 2.8 Å (blue). Residues on FABP4 marked with yellow are those that make a significant contribution to the stabilization energy of the complex. Residues of the Armadillo colored in orange are members of the NLS recognition sites (WxxxN). Residues marked in red are identified as Hot Spots [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132138#pone.0132138.ref041" target="_blank">41</a>]. The number of hydrogen bonds formed between two residues [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132138#pone.0132138.ref043" target="_blank">43</a>] is given in the table.</p

Rigidity index of Cα atoms for residues 10–60 of the FABP4 in different crystal structures.

<p>The blue line represents the Apo form of FABP4 (1ALB.pdb), the red line represents a complex with palmitate (1LIE.pdb) (both are non-transportable compounds) and the green line represents a complex with linoleate (2Q9S.pdb). The two helices (I and II) forming the lid are highlighted in light and dark blue, respectively.</p

The interaction energies between the residues on FABP4 with the Armadillo.

<p>The data were calculated for each of the five largest clusters representing the complex FABP4 (loaded with linoleate) located at the minor site in the parallel orientation.</p

Upper Frame: The structure of the Kapα Armadillo colored by its Arms.

<p>The WxxxN moieties of the major and minor sites, which are located on Arms 2, 3, 4 and 7, 8, respectively, are shown as black sticks. <u>The lower Frames</u> depict the electrostatic potential of the Armadillo, demonstrating the negative patches of the major and the minor sites, using the Pymol APBS tool [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132138#pone.0132138.ref038" target="_blank">38</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132138#pone.0132138.ref040" target="_blank">40</a>]. The two faces of the protein are presented by rotation 180⁰ of the protein, along its long axis.</p