Axons of cortical basket cells originating from dendrites develop higher local complexity than axons emerging from basket cell somata.

Neuronal differentiation is regulated by neuronal activity. Here, we analyzed dendritic and axonal growth of Basket cells (BCs) and non-Basket cells (non-BCs) using sparse transfection of channelrhodopsin-YFP and repetitive optogenetic stimulation in slice cultures of rat visual cortex. Neocortical interneurons often display axon-carrying dendrites (AcDs). We found that the AcDs of BCs and non-BCs were, on average, the most complex dendrites. Further, the AcD configuration had an influence on BC axonal development. Axons originating from an AcD formed denser arborizations with more terminal endings within the dendritic field of the parent cell. Intriguingly, this occurred already in unstimulated BCs, and complexity was not increased further by optogenetic stimulation. However, optogenetic stimulation exerted a growth-promoting effect on axons emerging from BC somata. The axons of non-BCs neither responded to the AcD configuration nor to the optogenetic stimulation. The results suggest that the formation of locally dense BC plexuses is regulated by spontaneous activity. Moreover, in the AcD configuration, the AcD and the axon it carries mutually support each other's growth.

Restoration of visual function by expression of a light-gated mammalian ion channel in retinal ganglion cells or ON-bipolar cells.

Most inherited forms of blindness are caused by mutations that lead to photoreceptor cell death but spare second- and third-order retinal neurons. Expression of the light-gated excitatory mammalian ion channel light-gated ionotropic glutamate receptor (LiGluR) in retinal ganglion cells (RGCs) of the retina degeneration (rd1) mouse model of blindness was previously shown to restore some visual functions when stimulated by UV light. Here, we report restored retinal function in visible light in rodent and canine models of blindness through the use of a second-generation photoswitch for LiGluR, maleimide-azobenzene-glutamate 0 with peak efficiency at 460 nm (MAG0(460)). In the blind rd1 mouse, multielectrode array recordings of retinal explants revealed robust and uniform light-evoked firing when LiGluR-MAG0(460) was targeted to RGCs and robust but diverse activity patterns in RGCs when LiGluR-MAG0(460) was targeted to ON-bipolar cells (ON-BCs). LiGluR-MAG0(460) in either RGCs or ON-BCs of the rd1 mouse reinstated innate light-avoidance behavior and enabled mice to distinguish between different temporal patterns of light in an associative learning task. In the rod-cone dystrophy dog model of blindness, LiGluR-MAG0(460) in RGCs restored robust light responses to retinal explants and intravitreal delivery of LiGluR and MAG0(460) was well tolerated in vivo. The results in both large and small animal models of photoreceptor degeneration provide a path to clinical translation.

Photoswitching of cell surface receptors using tethered ligands.

Optical probing and manipulation of cellular signaling has revolutionized biological studies ranging from isolated cells to intact tissues in the live animal. A promising avenue of optical manipulation is Chemical Optogenetics (or Optogenetic Pharmacology), an approach for engineering specific proteins to be rapidly and reversibly switched on and off with light. The approach employs synthetic photoswitched ligands, which can be reversibly photo-isomerized to toggle back and forth between two conformations in response to two wavelengths of light. We focus here on the photoswitched tethered ligand (PTL) approach in which the PTL is covalently attached in a site-directed manner to a signaling protein. For this a ligand anchoring site is introduced at a location which allows the ligand to dock only in one of the light-controlled conformations, thus enabling liganding to be rapidly switched. The ligand can be an agonist, antagonist or an active site (or pore) blocker. In principle, orthogonal chemistries of attachment would make PTL anchoring completely unique. However, extremely high specificity of remote control is also obtained by cysteine attachment because of the ligand specificity and precise geometric requirements for liganding. We describe here the design of light-gated ionotropic and metabotropic glutamate receptors, the selection of a site for cysteine placement, the method for PTL attachment, and a detailed protocol of photoswitching experiments in cultured cells. These descriptions can guide applications of Chemical Optogenetics to other receptors and serve as a starting point for use in more complex preparations.

Testing the diffusing boundary model for the helix-coil transition in peptides.

The dynamics of peptide α-helices have been studied extensively for many years, and the kinetic mechanism of the helix-coil dynamics has been discussed controversially. Recent experimental results have suggested that equilibrium helix-coil dynamics are governed by movement of the helix/coil boundary along the peptide chain, which leads to slower unfolding kinetics in the helix center compared with the helix ends and position-independent helix formation kinetics. We tested this diffusion of boundary model in helical peptides of different lengths by triplet-triplet energy transfer measurements and compared the data with simulations based on a kinetic linear Ising model. The results show that boundary diffusion in helical peptides can be described by a classical, Einstein-type, 1D diffusion process with a diffusion coefficient of 2.7⋅10(7) (amino acids)(2)/s or 6.1⋅10(-9) cm(2)/s. In helices with a length longer than about 40 aa, helix unfolding by coil nucleation in a helical region occurs frequently in addition to boundary diffusion. Boundary diffusion is slowed down by helix-stabilizing capping motifs at the helix ends in agreement with predictions from the kinetic linear Ising model. We further tested local and nonlocal effects of amino acid replacements on helix-coil dynamics. Single amino acid replacements locally affect folding and unfolding dynamics with a ϕf-value of 0.35, which shows that interactions leading to different helix propensities for different amino acids are already partially present in the transition state for helix formation. Nonlocal effects of amino acid replacements only influence helix unfolding (ϕf = 0) in agreement with a diffusing boundary mechanism.

Triplet-triplet energy transfer studies on conformational dynamics in peptides and a protein.

Peptides and proteins are highly dynamic systems, which can adopt more or less stable conformations. The dynamics of these molecules, particularly those on the nanosecond to tens of microsecond time scale, are difficult to assess with conventional techniques. This review summarizes experiments using TTET, a technique that reports on van der Waals contact formation between a triplet donor and acceptor group, and which is sensitive in this time range. TTET allows to directly measure the chain dynamics of unstructured model peptides, i.e. large-amplitude fluctuations on the nanosecond time scale. Furthermore, contact formation can be used as irreversible probing reaction to study the kinetics of conformational equilibria. This approach enabled us to measure local α-helix folding and unfolding in helical peptides, which gave new insight into the equilibrium dynamics of this fundamental secondary structure element. TTET has also been applied to study the dynamics both in the native and unfolded state of a protein, the villin headpiece subdomain. The contact formation kinetics between different positions revealed an unlocking and local unfolding reaction in the native state of this model protein, and gave information about the chain dynamics in the unfolded state ensemble.

Local conformational dynamics in alpha-helices measured by fast triplet transfer.

Coupling fast triplet-triplet energy transfer (TTET) between xanthone and naphthylalanine to the helix-coil equilibrium in alanine-based peptides allowed the observation of local equilibrium fluctuations in alpha-helices on the nanoseconds to microseconds time scale. The experiments revealed faster helix unfolding in the terminal regions compared with the central parts of the helix with time constants varying from 250 ns to 1.4 micros at 5 degrees C. Local helix formation occurs with a time constant of approximately 400 ns, independent of the position in the helix. Comparing the experimental data with simulations using a kinetic Ising model showed that the experimentally observed dynamics can be explained by a 1-dimensional boundary diffusion with position-independent elementary time constants of approximately 50 ns for the addition and of approximately 65 ns for the removal of an alpha-helical segment. The elementary time constant for helix growth agrees well with previously measured time constants for formation of short loops in unfolded polypeptide chains, suggesting that helix elongation is mainly limited by a conformational search.

Effect of thioxopeptide bonds on alpha-helix structure and stability.

Thioxoamide (thioamide) bonds are nearly isosteric substitutions for amides but have altered hydrogen-bonding and photophysical properties. They are thus well-suited backbone modifications for physicochemical studies on peptides and proteins. The effect of thioxoamides on protein structure and stability has not been subject to detailed experimental investigations up to date. We used alanine-based model peptides to test the influence of single thioxoamide bonds on alpha-helix structure and stability. The results from circular dichroism measurements show that thioxoamides are strongly helix-destabilizing. The effect of an oxo-to-thioxoamide backbone substitution is of similar magnitude as an alanine-to-glycine substitution resulting in a helix destabilization of about 7 kJ/mol. NMR characterization of a helical peptide with a thioxopeptide bond near the N-terminus indicates that the thioxopeptide moiety is tolerated in helical structures. The thioxoamide group is engaged in an i, i+4 hydrogen bond, arguing against the formation of a 3(10)-helical structure as suggested for the N-termini of alpha-helices in general and for thioxopeptides in particular.