Ultrafast photochemistry and electron-diffraction spectra in n → (3s) Rydberg excited cyclobutanone resolved at the multireference perturbative level.

We study the ultrafast time evolution of cyclobutanone excited to the singlet n → Rydberg state through non-adiabatic surface-hopping simulationsperformed at extended multi-state complete active space second-order perturbation (XMS-CASPT2) level of theory. These dynamics predict relaxation to the ground-state with a timescale of 822 ± 45 fs with minimal involvement of the triplets. The major relaxation path to the ground-state involves a three-state degeneracy region and leads to a variety of fragmented photoproducts. We simulate the resulting time-resolved electron-diffraction spectra, which track the relaxation of the excited state and the formation of various photoproducts in the ground state.

Electron and ion spectroscopy of azobenzene in the valence and core shells.

Azobenzene is a prototype and a building block of a class of molecules of extreme technological interest as molecular photo-switches. We present a joint experimental and theoretical study of its response to irradiation with light across the UV to x-ray spectrum. The study of valence and inner shell photo-ionization and excitation processes combined with measurement of valence photoelectron-photoion coincidence and mass spectra across the core thresholds provides a detailed insight into the site- and state-selected photo-induced processes. Photo-ionization and excitation measurements are interpreted via the multi-configurational restricted active space self-consistent field method corrected by second order perturbation theory. Using static modeling, we demonstrate that the carbon and nitrogen K edges of azobenzene are suitable candidates for exploring its photoinduced dynamics thanks to the transient signals appearing in background-free regions of the NEXAFS and XPS.

Photophysics of Deoxycytidine and 5-Methyldeoxycytidine in Solution: A Comprehensive Picture by Quantum Mechanical Calculations and Femtosecond Fluorescence Spectroscopy.

The study concerns the relaxation of electronic excited states of the DNA nucleoside deoxycytidine (dCyd) and its methylated analogue 5-methyldeoxycytidine (5mdCyd), known to be involved in the formation of UV-induced lesions of the genetic code. Due to the existence of four closely lying and potentially coupled excited states, the deactivation pathways in these systems are particularly complex and have not been assessed so far. Here, we provide a complete mechanistic picture of the excited state relaxation of dCyd/5mdCyd in three solvents-water, acetonitrile, and tetrahydrofuran-by combining femtosecond fluorescence experiments, addressing the effect of solvent proticity on the relaxation dynamics of dCyd and 5mdCyd for the first time, and two complementary quantum mechanical approaches (CASPT2/MM and PCM/TD-CAM-B3LYP). The lowest energy ππ* state is responsible for the sub-picosecond lifetime observed for dCyd in all the solvents. In addition, computed excited state absorption and transient IR spectra allow one, for the first time, to assign the tens of picoseconds time constant, reported previously, to a dark state (nOπ*) involving the carbonyl lone pair. A second low-lying dark state, involving the nitrogen lone pair (nNπ*), does significantly participate in the excited state dynamics. The 267 nm excitation of dCyd leads to a non-negligible population of the second bright ππ* state, which affects the dynamics, acting mainly as a "doorway" state for the nOπ* state. The solvent plays a key role governing the interplay between the different excited states; unexpectedly, water favors population of the dark states. In the case of 5mdCyd, an energy barrier present on the main nonradiative decay route explains the 6-fold lengthening of the excited state lifetime compared to that of dCyd, observed for all the examined solvents. Moreover, C5-methylation destabilizes both nOπ* and nNπ* dark states, thus preventing them from being populated.

Tracking the primary photoconversion events in rhodopsins by ultrafast optical spectroscopy.

Opsins are a broad class of photoactive proteins, found in all classes of living beings from bacteria to higher animals, which work either as light-driven ion pumps or as visual pigments. The photoactive function in opsins is triggered by the ultrafast isomerization of the retinal chromophore around a specific carbon double bond, leading to a highly distorted, spectrally red-shifted photoproduct. Understanding, by either experimental or computational methods, the time course of this photoisomerization process is of utmost importance, both for its biological significance and because opsin proteins are the blueprint for molecular photoswitches. This paper focuses on the ultrafast 11-cis to all-trans isomerization in visual rhodopsins, and has a twofold goal: (i) to review the most recent experimental and computational efforts aimed at exposing the very early phases of photoconversion; and (ii) discuss future advanced experiments and calculations that will allow an even deeper understanding of the process. We present high time resolution pump-probe data, enabling us to follow the wavepacket motion through the conical intersection connecting excited and ground states, as well as femtosecond stimulated Raman scattering data allowing us to track the subsequent structural evolution until the first stable all-trans photoproduct is reached. We conclude by introducing computational results for two-dimensional electronic spectroscopy, which has the potential to provide even greater detail on the evolution of the electronic structure of retinal during the photoisomerization process.

Outward rectifying potassium currents are the dominant voltage activated currents present in Deiters' cells.

Supporting cells in the cochlea are thought to maintain the homeostasis of the organ of Corti and contribute to the electrical and micromechanical environment of the hair cells. Of the different types of supporting cells, Deiters' cells form a structure that holds the outer hair cells (OHCs) at their base and apex. This structure may play an important role in modifying cochlear mechanics by influencing the force produced by sound induced motion of the OHCs which in turn may be modulated by ATP acting on ligand gated cation channels on the Deiters' cells. Also, a glia-like role of buffering external K+ concentration for the Deiters' cells has been suggested. We studied Deiters' cells' electrical properties and ion conductances using the whole cell variant of the patch clamp technique since they must play an important role in the function of these cells. It was found that isolated Deiters' cells possess a large voltage activated, outwardly rectifying K+ selective conductance. Voltage activated Ca2+ currents and non-selective currents were not detected and voltage activated inward currents were very small. The outward K+ currents were found to be dependent on voltage but not on Ca2+ for their activation. Nimodipine and 4-aminopyridine (4-AP) were shown to interact directly with the K+ channels in a voltage dependent manner. It is suggested that the K+ selective channels in Deiters' cells may be similar to the Kv1.5 type channel. However, based on the voltage dependence of the channels that was described by double Boltzmann equation and on the alteration of that dependence by 4-AP, it is possible that more than one type of K+ selective channel exists.

Pharmacological evidence that endogenous ATP modulates cochlear mechanics.

In the cochlea, outer hair cells (OHCs) and Deiters' cells most likely contribute to the generation of active cochlear mechanics. The presence of ATP receptors on these cells indicates that endogenous ATP may have a role in cochlear mechanics. To explore this possibility, the effects of ATP antagonists were studied both in vivo on distortion product otoacoustic emissions (DPOAEs) using cochlear perfusion and in vitro on isolated OHCs and Deiters' cells using the whole-cell configuration of the patch-clamp technique. Results show that extracellular application of 5-10 microM ATP to OHCs and Deiters' cells induced an inward current that was reduced by both suramin (100 microM) and cibacron (100 microM). Cibacron reduced the voltage gated currents in Deiters' cells and increased them in OHCs, while suramin had no effect. In addition, cibacron induced a hyperpolarizing shift of the half activation voltage of the whole cell currents in Deiters' cells. Suramin (0.1-1 mM) reversibly suppressed the 'slow decline' in the quadratic DPOAE that occurs during continuous stimulation with moderate level primaries. This effect of suramin may be evidence that endogenous ATP alters active cochlear mechanics.

Nitrosoglutathione suppresses cochlear potentials and DPOAEs but not outer hair cell currents or voltage-dependent capacitance.

Biochemical and pharmacological evidence support a role for nitric oxide (NO) and glutathione (GSH) in the cochlea. GSH combines with NO in tissue to form nitrosoglutathione (GSNO) that can act as a storage form for GSH and NO. Therefore, we tested GSNO on sound-evoked responses of the cochlea (cochlear microphonic, CM; summating potential, SP; compound action potential, CAP; cubic distortion product otoacoustic emission, DPOAE), on the endocochlear potential (EP), on isolated outer hair cell (OHC) currents and voltage-dependent capacitance, and on Deiters' cell currents. In vivo application of GSNO in increasing concentrations reversibly reduced low-intensity sound-evoked CAP, SP and DPOAEs starting at about 1 mM (CAP) and 3.3 mM (SP, DPOAE). However, even at 10 mM, GSNO had little effect on the EP. In vitro, salicylate (10 mM) but not GSNO (3 and 10 mM) suppressed the early capacitative transients of OHCs. GSNO (3 and 10 mM) had no effect on the whole cell currents of OHCs or Deiters' cells. Results show that GSNO suppresses cochlear function. This suppression may be due to an effect of GSNO on the cochlear amplifier. The actions of GSNO were different from those of other NO donors; therefore, the effects of GSNO may not be mediated by NO. The mechanisms underlying GSNO effects seem to be different from those of salicylate.

Outwardly rectifying currents in guinea pig outer hair cells.

Studies of K+ conductances in hair cells report that big-conductance Ca(2+)-dependent K+ (BK) channels carry parts of the outwardly rectifying currents. Lin et al. (1995) suggested that in guinea pig outer hair cells (OHCs) a portion of these currents is carried via a voltage-dependent and Ca(2+)-independent K+ channel. The present study tests the hypothesis that there are two separable current components of the outwardly rectifying currents by using patch clamp methods in OHCs to characterize the voltage dependence and sensitivity of the outwardly rectifying currents to channel blockers. Lowering of external Ca2+ caused no change in the currents while charybdotoxin (ChTx; 100 nM), a BK K+ channel blocker, and Cd2+ (200 microM), and L-type calcium channel blocker, abolished about 50% of the currents. Both ChTx and Cd2+ caused a depolarizing shift in the half-activation voltage paralleled by a decrease in the voltage sensitivity. 4-Aminopyridine (4-AP, 0.01 mM), an A-type and delayed rectifier type channel blocker, abolished about 50% of the currents and caused a hyperpolarizing shift in the half-activation voltage together with an increase in the voltage sensitivity. The outwardly rectifying currents were more sensitive to block by 4-AP at membrane voltages around 40 mV compared to voltages around -20 mV. The differences in the current characteristics may be due to two separate channel types, one of which is similar to the delayed rectifier type channels while the other may be similar to the BK Ca(2+)-dependent K+ channels. In addition, the largest outwardly rectifying currents were present in long OHCs with the smallest present in short OHCs.

Acetylcholine response in guinea pig outer hair cells. I. Properties of the response.

The properties of the ACh (acetylcholine) response in guinea pig outer hair cells (OHCs) are not well understood. It has been shown that the response to ACh involves the activation of a Ca2+ dependent K+ selective conductance (referred to as Ksub where sub stands for suberyldicholine). In the present study, we examined the voltage dependence, the time dependence, and the desensitization of the ACh response. In addition, we examined the K+ selectivity of K(sub). These properties are important for aiding in the determination of the type of K+ channels activated by ACh. Patch-clamp technique in the whole-cell mode was used to record from single OHCs isolated from adult pigmented guinea pigs. ACh (100 microM) was applied to the voltage-clamped OHCs and the ACh induced currents (IACh) were measured. A voltage dependence of the ACh response was found with the ACh induced currents decaying monoexponentially at potentials positive to -30 mV. The decay of the ACh induced currents was faster soon after establishing the whole-cell mode of recording when compared to the decay of the currents some time later. This effect, referred to as the time dependence, was different from the desensitization of the response upon prolonged application of ACh. The desensitization of the ACh induced currents was about 50% after 2 min of continuous application of 100 microM ACh. The examined characteristics of the ACh response in guinea pig OHCs indicate a voltage and time dependence of the response and strong K+ selectivity of the Ksub.

Acetylcholine response in guinea pig outer hair cells. II. Activation of a small conductance Ca(2+)-activated K+ channel.

The type of K+ channel involved in the acetylcholine (ACh) evoked response (Ksub; sub stands for suberyldicholine) in guinea pig outer hair cells (OHCs) is still uncertain. The present study tests the hypotheses that Ksub is one of the following: a big conductance Ca(2+)-dependent K+ channel (BK), a small conductance Ca(2+)-dependent K+ channel (SK), a KA type of K+ channel, or a Kn type of K+ channel. Patch-clamp technique in the whole-cell mode was used to record from single guinea pig OHCs. ACh (100 microM) was applied to voltage-clamped OHCs and the ACh-induced currents (IACh) were measured. Charybdotoxin (100 nM) had no effect on IACh, while apamin (1 microM) blocked more than 90% of IACh. Lowering the external Ca2+ concentration caused a hyperpolarizing shift of the IACh monitored as a function of the prepulse voltage. Increasing internal Mg2+ (Mgi2+) concentration caused a reduction in the outward IACh without affecting the inward IACh. The Ksub channel was found to be permeable to Cs+. In Cs+ solutions, IACh was 45% of the IACh in K+ solutions. The block of IACh by apamin, the dependence on extracellular Ca2+, the incomplete block of IACh by Cs+, and the ACh-induced Cs+ currents favor the hypothesis that Ksub belongs to the SK type of channels. An ionotropic/nicotinic nature of the ACh mechanism of action is favored. It is suggested that, in vivo, the amplitude of the ACh-induced hyperpolarization may depend on the Ca2+/Mg2+ ratio inside and outside the cell.

Noise exposure alters the response of outer hair cells to ATP.

The outer hair cells (OHCs) are one target of noise-induced effects. To date there are few studies which examine changes in the function of OHCs induced by noise exposure. There is increasing evidence that ATP may be a neuromodulator acting on OHCs. Therefore, we examined the possibility that the response to ATP may be altered by low-level noise exposure. ATP was tested on cation currents recorded from outer hair cells (OHCs) isolated from chronic noise-exposed guinea pigs and compared to currents recorded from normal control animals. The whole-cell variant of the patch-clamp technique was used. The incidence of response to 100 microM ATP was decreased in OHCs from noise-exposed animals as compared to controls when normal internal and external solutions were employed. When K+ was substituted by N-methyl-glucamine (NMG+) in the pipette solution, there were significant differences in the magnitudes of ATP-evoked currents between cells from noise-exposed and control animals. This was observed in both normal and 20 mM Ba2+ external solutions. In addition, the response to ATP exhibited a dependency on OHC length. In short OHCs (< 65 microns) from noise-exposed animals the magnitude of the response to ATP was significantly reduced. By contrast, the response in long OHCs (> 65 microns) from noise-exposed animals was increased. Results suggest that low-level noise exposure induces changes in OHCs which affect the response of the cell to ATP.

Nitroprusside suppresses cochlear potentials and outer hair cell responses.

Biochemical and pharmacological evidence supports a role for nitric oxide (NO) in the cochlea. In the present experiments, we tested sodium nitroprusside (SNP), an NO donor, applied by intracochlear perfusions on sound-evoked responses of the cochlea (CM, cochlear microphonic; SP, summating potential; EP, endocochlear potential; CAP, compound action potential) and in vitro on outer hair cell (OHC) voltage-induced length changes and current responses. In vivo application of SNP in increasing concentrations (10, 33, 100, 330 and 1000 microM) reduced all sound-evoked responses starting at about 300 microM. The responses continued to decline after a postdrug wash. At 1 mM SNP decreased EP slowly (approximately 80 min) whereas at 10 mM it reduced EP more rapidly (approximately 20 min). Ferricyanide (1 mM) and S-nitroso-N-acetylpenicillamine (SNAP; 1 mM) had no effect on sound-evoked cochlear potentials. Ferricyanide (1 mM and 10 mM) and ferrocyanide (10 mM) had no effect on EP. In vitro, SNP (10 mM) significantly reduced both OHC voltage-induced length changes and whole-cell outward currents. Results suggest that SNP, possibly acting by released NO, influences cochlear function through effects at the stria vascularis and at the OHCs.

ATP modulation of L-type calcium channel currents in guinea pig outer hair cells.

Ca2+ channel currents and their modulation by adenosine 5'-triphosphate (ATP) in acutely isolated guinea pig outer hair cells (OHCs) were investigated using the whole-cell patch-clamp technique. The current-voltage (I-V) relation of OHCs indicated that the Ca2+ channel opened near -30 mV, and the current reached a maximum at +10 and 0 mV in 20 mM Ca2+ and Ba2+ external solutions, respectively. BayK 8644 (BayK, 2 microM) caused a 3.5-fold increase in peak Ca2+ currents and shifted the I-V curves toward more negative potentials. These results suggest that the majority of Ca2+ channels in OHCs have L-type characteristics. The effects of ATP on Ca2+ channels of OHCs were heterogenous. ATP (100 microM) decreased Ca2+ channel currents by 31.7 +/- 5.6% at 0 mV and shifted Ca2+ tail activation curves toward more depolarized potentials in some cells (N = 6). By contrast, in others, ATP enhanced the currents by 43.5 +/- 12.5% at +10 mV (N = 6). In the presence of BayK, however, ATP-induced inhibition or enhancement of Ca2+ channel currents was attenuated. In addition, 100 microM ATP produced little effect on Ca2+ channel currents in another subpopulation of cells (N = 12). This heterogenous neuromodulation of Ca2+ channel currents by ATP may reflect a functional diversity among OHCs.

Acetylcholine activates a K+ conductance permeable to Cs+ in guinea pig outer hair cells.

Acetylcholine (ACh), the major neurotransmitter released by efferent nerve fibers in the cochlea, has been shown to activate a Ca(2+)-dependent K+ conductance in outer hair cells (OHCs). Previously we reported that this ACh operated conductance is permeable to Cs+. The purpose of the present study was to characterize further this Cs(+)-permeable channel and its dependency on Ca2+ using isolated OHCs and the patch clamp technique in the whole cell configuration. The changes in the ACh response were examined when Cs+, Ba2+, Cd2+, N-methyl-D-glucamine (NMG+) and tetraethylammonium (TEA+) were placed in the external or internal solutions. Cs+ substituted for K+ in carrying the ACh-evoked Ca(2+)-dependent K+ current. When NMG+/TEA+ was substituted for internal K+ ACh-evoked an inward and an outward current, and Cs+ substituted for external K+ blocked the outward but not the inward current evoked by ACh suggesting it was carried by K+. In the NMG+/TEA+ condition, when the cell was held at different Vh values for an extended period of time, the ACh-induced K+ current rectified. In Ba2+ (3 mM) with zero Ca2+ ACh failed to induce any detectable current and the ACh response slowly recovered from the Ba2+ block, suggesting a block at an intracellular site. Cd2+ (1 mM) readily and reversibly blocked ACh-induced currents even when carried by Cs+. This data suggests that ACh opens a channel selective for K+, conductive to Cs+ and dependent on Ca2+.

Laser damage tests of large flux-grown KTiOPO(4) crystals.

Large high-quality KTP crystals are grown from a high-temperature solution (the flux method). The laser damage threshold for these crystals is measured to be 3-3.5 GW/cm(2) for 1.064-microm light and 2.5-3 GW/cm(2) for 0.532-microm light. The damage thresholds and second-harmonic-generation conversion efficiency are comparable with and higher than those for hydrothermally grown KTP, respectively.