Céline Berbey

Bruno Allard

Electrically silent divalent cation entries in resting and active voltage-controlled muscle fibers

Keywords: calcium influx, Mn 2+ quenching of fura-2 fluorescence, voltage clamp, action potential, L-type channels, excitation-contraction coupling

Ca 2+ is known to enter skeletal muscle at rest and during activity. Except for the well characterized Ca 2+ entry through L-type channels, pathways involved in these Ca 2+ entries remain elusive in adult muscle. This study investigates Ca 2+ influx at rest and during activity using the method of Mn 2+ quenching of fura-2 fluorescence on voltage-controlled adult skeletal muscle cells. Resting rate of Mn 2+ influx depended on external [Mn 2+ ] and membrane potential. At -80 mV, replacement of Mg 2+ by Mn 2+ gave rise to an outward current associated with an increase in cell input resistance. Calibration of fura-2 response indicated that Mn 2+ influx was too small to be resolved as a macroscopic current. Partial depletion of the sarcoplasmic reticulum induced by a train of action potentials in the presence of cyclopiazonic acid led to a slight increase in resting Mn 2+ influx but no change in cell input resistance and membrane potential. Trains of action potentials considerably increased Mn 2+ entry through an electrically silent pathway independent of L-type channels which provided 24% of the global Mn 2+ influx at +30 mV under voltage-clamp conditions. Within this context, the nature and the physiological role of the Ca 2+ pathways involved during muscle excitation still remain open questions.

Introduction

In skeletal muscle, Ca 2+ ions triggering contraction are released from the sarcoplasmic reticulum (SR) in response to depolarization of the cell. In parallel to these massive variations of intracellular Ca 2+ , a Ca 2+ influx is also known to occur through the sarcolemma both in resting and working muscle. Early measurements using radiolabelled Ca 2+ have shown that Ca 2+ slowly enters skeletal muscle cells in resting conditions [START_REF] Clausen | The effect of hyperosmolarity and insulin on resting tension and calcium fluxes in rat soleus muscle[END_REF]. The physiological role of this Ca 2+ entry is poorly understood in normal muscle but several lines of evidence suggest that a chronic elevation of the resting influx of Ca 2+ might contribute to the initiation or progression of muscle cell degeneration in a number of muscle disorders [START_REF] Gailly | New aspects of calcium signaling in skeletal muscle cells: implications in Duchenne muscular dystrophy[END_REF]. Within this context of potential pathophysiological consequences of exacerbated Ca 2+ entry, the study of resting Ca 2+ influx and the search for the pathways through which it takes place are of fundamental interest. A widespread hypothesis suggests that Ca 2+ enters resting muscle cell through ion channels. Single channel recordings indeed revealed opening of poorly selective cation channels carrying inward current at negative membrane potentials in isolated mouse skeletal muscle fibers [START_REF] Haws | Developmental regulation of mechanosensitive calcium channels in skeletal muscle from normal and mdx mice[END_REF][START_REF] Hopf | A critical evaluation of resting intracellular free calcium regulation in dystrophic mdx muscle[END_REF][START_REF] De Backer | Long-term study of Ca 2+ homeostasis and of survival in collagenase-isolated muscle fibres from normal and mdx mice[END_REF][START_REF] Allard | Sarcolemmal ion channels in dystrophin-deficient skeletal muscle fibres[END_REF]. However, subsarcolemmal Ca 2+ load revealed by the measurement of Ca 2+activated K + channel opening was found to be independent of the activity of the spontaneously active Ca 2+ channels suggesting the existence of electrically silent sites of passive Ca 2+ influx [START_REF] Mallouk | Ca 2+ influx and opening of Ca 2+ -activated K + channels in muscle fibers from control and mdx mice[END_REF]. Additionally, certainly because of the absence of specific inhibitors of Ca 2+ channels active at rest and because of the drastic modifications of the electrophysiological muscle properties resulting from manipulation of external Ca 2+ , there is no experimental evidence available demonstrating the development of a Ca 2+ current at the whole cell level under resting conditions. Another way to monitor Ca 2+ influx at rest is to measure the changes in fluorescence of Ca 2+ dyes. Early works investigating plasmalemmal divalent cations influx in non-excitable cells have introduced the use of Mn 2+ as a surrogate of Ca 2+ to directly assess Ca 2+ entry (e.g. 8). Mn 2+ ions are assumed to penetrate cells through the same pathways as the ones used by Ca 2+ and, once within the cells, these ions are trapped in the cytosol and strongly quench the fluorescence emitted by Ca 2+ dyes. In adult skeletal muscle, this Mn 2+ quenching method has been used to confirm that the muscle sarcolemma has a substantial permeability to divalent cations under resting conditions but they were performed in the absence of voltage control and therefore did not establish whether Mn 2+ entry generated a measurable macroscopic membrane current whose properties could give evidence for the existence of ion channels open at rest, permeable to Mn 2+ and Ca 2+ (e.g. 9). The Mn 2+ quenching method has also been used to show that SR depletion induced in the presence of SR Ca 2+ -ATPase inhibitors gave rise to an increase in resting Ca 2+ influx in adult and fetal muscle fibers [START_REF] Kurebayashi | Depletion of Ca 2+ in the sarcoplasmic reticulum stimulates Ca 2+ entry into mouse skeletal muscle fibres[END_REF][START_REF] Collet | Calcium-dependent facilitation and graded deactivation of store-operated calcium entry in fetal skeletal muscle[END_REF]. However, using voltage clamp on adult muscle fibers, Allard et al. [START_REF] Allard | Sarcoplasmic reticulum Ca 2+ release and depletion fail to affect sarcolemmal ion channel activity in mouse skeletal muscle[END_REF] demonstrated that SR depletion was not associated with the generation of a resting inward current thus challenging the physiological relevance of a store-operated membrane conductance in adult skeletal muscle. In working adult muscle, depolarization elicits a well characterized Ca 2+ entry through L-type Ca 2+ channels early recorded as a voltage-activated current. But more recently, using the Mn 2+ quenching method in primary myotubes, Cherednichenko et al. [START_REF] Cherednichenko | Conformational activation of Ca 2+ entry by depolarization of skeletal myotubes[END_REF] demonstrated that membrane depolarization initiated a Ca 2+ entry independent of the voltage-gated L-type Ca 2+ current and of SR depletion. An increased Ca 2+ entry provoked by KCl depolarization was also recently described in adult muscle [START_REF] Cherednichenko | Enhanced excitation-coupled calcium entry in myotubes expressing malignant hyperthermia mutation R163C is attenuated by dantrolene[END_REF], but there was no attempt to determine if this Ca 2+ entry was independent of the voltage-activated L-type Ca 2+ current as found in myotubes.

There is also no available information about the behaviour of this Ca 2+ influx as a function of the membrane voltage which is yet recognized to tightly control Ca 2+ changes in skeletal muscle.

The present study is the first to combine the Mn 2+ quenching method and voltage control on adult mouse skeletal muscle cell to investigate sarcolemmal Ca 2+ influx under resting and active conditions. Monitoring of the background membrane current during Mn 2+ influx together with calibration of the fura-2 response to Mn 2+ led us to conclude that the Mn 2+ influx at rest and in response to partial depletion of the SR does not induce any resolvable macroscopic current. We also demonstrate that the rate of Mn 2+ entry is markedly increased by tetanic muscle activity principally through an electrically silent voltage-dependent Ca 2+ entry independent of the voltage activated L-type current.

Materials and methods

Preparation of muscle fibers

Experiments were performed on single skeletal fibers enzymatically isolated from interosseal muscles of adult male Swiss OF1 mice. All experiments were performed in accordance with the guidelines of the French Ministry of Agriculture (87/848) and of the European Community (86/609/EEC). Procedures for enzymatic isolation of single muscle fibers and partial insulation of the fibers with silicone grease were as described previously [START_REF] Jacquemond | Activation of Ca 2+ -activated K + channels by an increase in intracellular Ca 2+ induced by depolarization of mouse skeletal muscle fibres[END_REF]. In brief, the major part of a single fiber (between 0.5 and 1 mm length) was electrically insulated with silicone grease so that a fiber portion between 50 and 100 µm length was let free of grease. A micropipette was then inserted into the fiber through the silicone layer. Under these conditions, whole-cell voltage clamp could be achieved on the portion of the fiber extremity free of grease. The tip of the micropipette was then crushed into the dish bottom to allow intracellular dialysis of the fiber [START_REF] Weiss | Expression of the muscular dystrophy-associated caveolin-3 P104L mutant in adult mouse skeletal muscle specifically alters the Ca 2+ channel function of the dihydropyridine receptor[END_REF].

Electrophysiology

Fibers were voltage clamped with an RK-400 patch-clamp amplifier (Bio-Logic, Claix, France) used in the whole-cell configuration. Command voltage pulse generation and data acquisition were done using the pClamp10 software (Axon Instruments Inc., USA) driving an A/D converter (Digidata 1322A, Axon Instruments Inc., USA). Analog compensation was systematically used to decrease the effective series resistance. Voltage-clamp was performed with a microelectrode filled with the whole-cell intra-pipette solution (see Solutions). Cell capacitance was determined by integration of a current trace obtained with a 10-mV hyperpolarizing pulse from the holding potential and used to calculate the density of Mn 2+ currents (A/F). For voltage activated currents, leak currents were subtracted from all recordings using a 10-mV hyperpolarizing pulse preceding every test pulse from the holding potential supposing a linear evolution of leak current with depolarization. The voltage dependence of the mean Mn 2+ current density was fitted using the following equation:

I(V) = G max (V -V rev ) / ((1 + exp ((V 0.5 -V)/k)))
, where I(V) is the mean density of the current measured, V the test pulse, G max the maximum conductance, V rev the apparent reversal potential, V 0.5 the half-activation voltage and k a steepness factor. The voltage dependence of the normalized conductance was obtained by dividing I(V) by G max (V -V rev ).

Mn 2+ quenching experiments

Prior to experiments, cells were dialysed during 20 minutes with an internal pipette solution containing 100 µM fura-2 (see Preparation of muscle fibers) and imaged with an inverted microscope (Nikon Diaphot) equipped for epifluorescence using a 40x oil-immersion objective. Fura-2 fluorescence was measured with monochromatic excitation at a Ca 2+insensitive wavelength (360 nm) (T.I.L.L. Photonics, Martinsried, Germany). Images from a circular region of 20 µm diameter centred on the silicone-free extremity of the cell under study were captured with a Darkstar 800 device camera (Photonic Science, Robertsbridge, UK) at a frequency of 0.2 Hz or 0.1 Hz. This low sampling rate was chosen so that cells were exposed to the excitation beam very shortly (200 ms) during image capture to make photobleaching negligible. Image acquisition and processing were performed using the Axon Imaging Workbench (Axon Instruments Inc., USA). Fluorescence values were expressed as percentage of the initial fluorescence recorded before addition of Mn 2+ to the external medium, corrected for background fluorescence. Under resting conditions, the rate of fluorescence quenching was determined by fitting a linear regression to the fluorescence records. In response to a train of action potentials (AP) or to voltage pulses, given the low image acquisition frequency, the Mn 2+ influx rate was extrapolated from the absolute decrease in fluorescence between two fluorescence data points.

In vitro and in vivo calibration of Mn 2+ quenching

For in vitro calibration, the fura-2 fluorescence was measured on pulled glass tubes of 50 µm internal diameter filled with an aqueous solution containing 100 µM fura-2 and various [MnCl 2 ]. For in vivo calibration, cells dialyzed with an internal solution containing 100 µM fura-2 were exposed to a solution containing (in mM) 140 K-glutamate, 10 HEPES adjusted to pH 7.2 with K-OH and 20 µM ionomycin. Increasing concentrations of MnCl 2 were added in this external solution and the fura-2 fluorescence was measured after fluorescence intensity had reached a new steady state.

Estimation of Mn 2+ current intensity on the basis of Mn 2+ quenching rates

The membrane surface area was estimated by measuring the capacitance of the portion of the cell under study and assuming a specific capacitance of 4.5 µF.cm -2 (17). This surface value was used to estimate the cell volume into which Mn 2+ diluted once within the cell assuming an average cell diameter of 40 µm. The quenching rate (%.min -1 ) recorded in every cell was then converted into intracellular Mn 2+ concentration reached after one minute using the relationship obtained between the relative fluorescence intensity emitted by the fibers and the external concentration of Mn 2+ in the presence of ionomycin (see above and Fig. 4). This gave the quantity of divalent charges accumulated in the calculated cell volume after one minute which in turn, divided by 60 and by the value of cell capacitance, yielded the density of Mn 2+ current that should have flowed through the sarcolemma.

Solutions

For experiments in relation with Fig. 1 6 and 7A, 1.5 mM 4-aminopyridine was added in the external solution. The whole-cell intra-pipette solution contained (in mM) 120 K-glutamate, 5 Na 2 -ATP, 5 Na 2 -phosphocreatine, 5.5 MgCl 2 , 5 glucose, 5 HEPES adjusted to pH 7.2 with K-OH except for experiments in relation with Fig. 3 where the intra-pipette solution contained (in mM) 120 Cs-aspartate, 5 Na 2 -ATP, 5.5 MgCl 2 , 5 glucose, 0.1 EGTA, 5 HEPES adjusted to pH 7.2 with Cs-OH. Fura-2 was dissolved in water at 10 mM, cyclopiazonic acid (CPA), 9-anthracene carboxylic acid (9AC) and ionomycin were dissolved in dimethylsulfoxide at 50 mM, 100 mM and 1 mM respectively and diluted to the required concentration in the solutions. Cells were exposed to different solutions by placing them in the mouth of a perfusion tube from which flowed by gravity the rapidly exchanged solutions. Experiments were carried out at room temperature.

Statistics

Least-squares fits were performed using a Marquardt-Levenberg algorithm routine included in Microcal Origin (Microcal Software Inc., Northampton, MA). Data are given as means ± S.E.M and compared using unpaired, paired t-tests or ANOVA as indicated in the text. Differences were considered significant when P < 0.05.

Results

Divalent cation influx at rest

Figure 1 illustrates the quenching of fura-2 fluorescence in fibers voltage-clamped at -80 mV in response to the addition of various concentrations of Mn 2+ in Ca 2+ -free external solutions. Replacement of 3 mM Mg 2+ by 3 mM Mn 2+ in the external solution induced a marked reduction of the fura-2 fluorescence which remained at a stabilized lower value upon removal of Mn 2+ (Fig. 1A). As long as the fluorescence signal remained higher than 50 % of its initial value, it declined linearly with time so that the rate of quenching could be estimated by fitting the fluorescence decline with a linear regression. Fig. 1B shows that the rate of fluorescence decline was dependent on the external [Mn 2+ ]. On average, an elevation of the external concentration of Mn 2+ induced a significant gradual increase in the rate of quenching (Fig. 1C) (P = 0.0075, ANOVA). When 2 mM Ca 2+ was added to a 10 mM Mn 2+ containing Tyrode solution, the rate of quenching was approximately reduced by half (P = 0.02, unpaired t-test). A second set of experiments was performed in order to determine how the rate of quenching was affected by the membrane potential. To improve the control of the membrane potential, cells were bathed in an external solution containing 140 mM TEA-methanesulfonate. Figure 2A presents the drop of fluorescence induced by replacement of 3 mM external Mg 2+ by Mn 2+ in four different cells held at +40, 0, -40 or -80 mV. On average, Fig. 2B shows that the less negative the membrane potential, the lower the rate of quenching (P = 0.019, ANOVA). The effect of a change in membrane potential on the rate of Mn 2+ quenching could also be revealed on a same fiber. A hyperpolarization of 60 mV amplitude indeed induced an increase of the rate of quenching but which depended on the initial value of the holding potential (Fig. 2C). Figure 2D clearly illustrates that in average the more depolarized the initial holding potential, the lower the factor of increase of the quenching rate induced by the 60-mV negative voltage step.

Membrane current associated with resting cation influx

We next tried to determine if the influx of Mn 2+ ion responsible for the quenching of fura-2 fluorescence was associated with a measurable macroscopic inward current. Figure 3A shows that, at a holding potential of -80 mV, replacement of 3 mM Mg 2+ by Mn 2+ led to the development of an outward current associated with the quenching of fluorescence. In another cell held at +40 mV, the same manoeuvre gave rise to an inward current associated with a lower rate of quenching of fluorescence (Fig. 3B). The same respective behaviours were observed in 10 fibers tested at -80 mV and in 7 fibers tested at +40 mV. In order to determine whether the outward current which developed at -80 mV in response to replacement of Mg 2+ by Mn 2+ resulted from the opening or closing of ion channels, the input resistance was challenged by applying every 5 s voltage ramps bringing the holding potential from -40 to -100 mV. To minimize the contribution of Cl -and K + channels, cells were dialyzed with a Csaspartate containing solution and the Cl -blocker 9AC was added in the TEA-methanesulfonate containing external solution. Also, 10 mM Mg 2+ was replaced by 10 mM Mn 2+ to strengthen the inwardly directed Mn 2+ concentration gradient. Figures 3C andD show that replacement of Mg 2+ by Mn 2+ gave rise to a robust and reversible outward current associated with a decrease in the cell electrical conductance as evidenced by the decrease in the slope of the current induced by the voltage ramp. In 8 cells tested, the density of the inward background current at -80 mV was reduced from 3.13 ± 0.32 to 2.65 ± 0.26 A/F while the cell conductance was reduced from 50 ± 5 to 43 ± 4 S/F upon replacement of Mg 2+ by Mn 2+ which both represent a 14.6 ± 1.8 % decrease (P < 0.001, paired t-test). This result indicates that the main effect of external Mn 2+ is to block spontaneously active ion channels carrying inward currents at -80 mV rather to open channels carrying outward currents. It has to be mentioned that lower Mn 2+ concentrations (1 mM or 100 µM) which, as expected, induced a weaker blocking effect but concomitantly reduced the Mn 2+ concentration gradient, never elicited any discernable inward current at negative membrane potentials (not shown). The nature of the ion channels blocked by Mn 2+ was not further investigated. Another possible interpretation of these results is that the inward current generated by the Mn 2+ entry is to minute to be resolved under voltage clamp conditions and may be masked by a predominant blocking effect of Mn 2+ on resting conductances. To address this question, we measured the in vitro and in vivo potency of Mn 2+ to extinguish the fura-2 fluorescence (see Methods) so that the intracellular [Mn 2+ ] that should be reached and, consequently, the Mn 2+ current that should flow through the sarcolemma could be estimated. The Mn 2+ -induced quenching of fura-2 fluorescence obtained in glass tubes is shown in Fig. 4A (open symbols). Fitting the relationship between the mean relative change in fura-2 fluorescence and the [Mn 2+ ] with a Hill equation gave values of K 0.5 and N of 24 µM and 1. The Mn 2+ -induced quenching of fura-2 fluorescence was also measured in vivo by exposing fibers to various [Mn 2+ ] in the presence of the Ca 2+ ionophore ionomycin (Fig. 4B). Fitting the relationship between the mean relative change in fura-2 fluorescence and the [Mn 2+ ] with a Hill equation gave values of K 0.5 and N of 9.6 µM and 0.7 (Fig. 4A, closed symbols). The main difference compared to glass tubes was that fura-2 fluorescence within the cells never dropped below 20 % of the initial fluorescence even in the presence of millimolar Mn 2+ suggesting that part of the dye has been trapped in cell compartments out of reach of Mn 2+ . Using the in vivo relationship and the measured membrane surface area in each cell tested (see Methods), the quenching rates measured at different membrane potentials were converted into membrane current densities (Fig. 4C). At -80 mV the calculated average current density was 2.2 ± 0.6 mA/F and the amplitude of the membrane current in the 10 cells tested at -80 mV ranged from 0.8 to 9.8 pA, values which are far below the resolution capacity under voltage clamp conditions in the whole cell configuration.

Mn 2+ entry in active muscle

Another series of experiments was carried out in order to explore whether Mn 2+ entry could be modified by muscle activity. Trains of AP were evoked at tetanic frequency (50 Hz) under current clamp conditions in cells loaded with fura-2 and the rate of decrease of fluorescence induced by the Mn 2+ entry was simultaneously monitored (Fig. 5A). The fluorescence intensity was not changed in response to fiber stimulation in the absence of external Mn 2+ giving evidence that basal resting fluorescence was not altered by intracellular Ca 2+ changes in fibers excited at 360 nm. Then, during the continuous presence of Mn 2+ , two consecutive bursts of AP induced a large increase in the influx of Mn 2+ . The Mn 2+ influx was in average 29 ± 4 times higher (n = 18, P < 0.001, paired t-test) during a train of AP than under resting conditions. Previous studies performed on adult skeletal muscle cells have shown that Mn 2+ entry can be increased by depolarization [START_REF] Cherednichenko | Conformational activation of Ca 2+ entry by depolarization of skeletal myotubes[END_REF] and by depletion of the SR [START_REF] Kurebayashi | Depletion of Ca 2+ in the sarcoplasmic reticulum stimulates Ca 2+ entry into mouse skeletal muscle fibres[END_REF] which possibly occurs during activity. A first set of experiments aimed at exploring how store depletion could alter Mn 2+ quenching. As illustrated in Fig. 5A, trains of AP first induced an increase in the influx of Mn 2+ (Fig. 5B). After returning to resting conditions, addition of the potent inhibitor of the SR Ca 2+ -ATPase CPA to the external solution did not induce any major change in the rate of Mn 2+ influx in this cell although in average the drug evoked a slight but significant decrease of 23 ± 8 % in the quenching rate (n = 14, P = 0.014, paired t-test). In the continuous presence of CPA, a train of stimulations evoked an elevation of fura-2 fluorescence which likely resulted from the prolonged contracture of the cell that concentrated the dye. Upon repolarization, the resting influx of Mn 2+ was increased and in average it was 1.8 ± 0.4 times (n = 11, P = 0.045, paired t-test) that measured before stimulation. Also, measurement of the voltage drop resulting from hyperpolarizing pulses of 2 nA amplitude indicated that the input resistance of the cell was not significantly changed in the presence of CPA after (5.4 ± 0.5 MΩ) as compared to before stimulation (5.5 ± 0.5 MΩ) (n = 11, P = 0.11, paired t-test). The resting membrane potential was also not significantly changed (-83.1 ± 0.38 mV before and -82.6 ± 0.64 after stimulation, n = 11, P = 0.29, paired t-test). SR depletion was certainly only partial under our experimental conditions because fibers still contracted in the presence of CPA although they contracted weaker and failed to relax after stimulation. However, all our attempts to induce more severe depletion to the point of complete fiber exhaustion systematically led to an irreversible depolarization of the fiber associated with a large fall in the input resistance and a profound and irreversible increase in the influx of Mn 2+ that saturated the dye. Figure 5C illustrates such behaviour in a particular fiber which displayed irreversible damages as soon as stimulation was delivered in the presence of CPA. This result emphasizes that voltage control is essential to monitor cell integrity during quenching experiments.

Voltage dependence and Cd 2+ inhibition of Mn 2+ influx in active muscle

Another set of experiments was carried out to investigate how depolarization by itself initiates Mn 2+ influx. Mn 2+ influx together with membrane currents were measured in fibers stimulated by voltage pulses of increasing amplitudes in the presence of 3 mM Mn 2+ . In these experiments, the possible contribution of a store-operated Mn 2+ influx should be largely minimized since depolarization has been demonstrated to strongly inhibit store-operated Mn 2+ influx [START_REF] Kurebayashi | Depletion of Ca 2+ in the sarcoplasmic reticulum stimulates Ca 2+ entry into mouse skeletal muscle fibres[END_REF]. Figure 6 shows that 100 ms duration depolarizing pulses given to -50, -30 and -10 mV did not change the rate of Mn 2+ influx. A pulse to +10 mV induced an inward Mn 2+ current which reached -1.9 nA, associated with a 9.2 % decrease in fluorescence. A subsequent pulse to +30 mV gave rise to an inward Mn 2+ current which activated faster and reached -2.1 nA, accompanied by a 14.6 % drop in fluorescence. Finally, a last pulse to +50 mV induced an outward membrane current associated with a 12 % decrease in fluorescence. Inward currents evoked by membrane depolarization were completely blocked by the addition of 0.5 mM Cd 2+ in the external solution (Fig. 7A). Taken together, these data likely indicated that the recorded Mn 2+ current flowed through L-type Ca 2+ channels which are known to be permeable to Mn 2+ in skeletal muscle [START_REF] Almers | Slow calcium and potassium currents across frog muscle membrane: measurements with a vaseline-gap technique[END_REF]. Figure 7A presents the voltage-current relationship of the Mn 2+ current and Figure 7B superimposes the relationship between the magnitude of the fluorescence decrease induced by Mn 2+ entry and voltage and the voltage dependence of the relative conductance of the Mn 2+ current. It can be observed that the voltage-activated Mn 2+ current and the Mn 2+ influx were maximal around +30 mV. Fitting Boltzmann equations to these two relationships indicated that V 0.5 was 24 mV more negative for the Mn 2+ influx than for the voltage-activated current and that the voltage dependence of the Mn 2+ influx was steeper than the one of the normalized conductance (k was 4.7 as compared to 8.6) (Fig. 7B). Differences suggest that at least two processes displaying dissimilar voltage dependences may contribute to the influx of Mn 2+ : the L-type current and a parallel voltage-activated Mn 2+ entry. In order to determine to what extent each of these two processes contributed to the influx of divalent cations during physiological activation of muscle cells, the effects of a blockade of the L-type current by Cd 2+ were tested on the influx of Mn 2+ induced by trains of AP. As illustrated in Fig. 8C, the mean drop in fluorescence induced by the train of excitation in the presence of Cd 2+ (0.5 mM) was not significantly different from the one induced in its absence (n = 9, P = 0.39, paired t-test). This result can be interpreted in two ways. Either the L-type current is not activated during trains of AP, or the L-type current is activated, but the influx of Mn 2+ that this current generates does not contribute significantly to the quenching of fluorescence induced by depolarization. In order to discriminate between these two possibilities, we tested the effects of Cd 2+ on the Mn 2+ influx induced by depolarization. Figure 7D shows that the decrease in fluorescence in response to a depolarization to +30 mV that evoked an L-type current of maximal amplitude was greatly reduced in the presence of Cd 2+ . Figure 7B presents the mean changes in fluorescence induced by different depolarization pulses in the presence of Cd 2+ . On average, the Mn 2+ influx was significantly reduced by 72, 76 and 82 % for depolarization steps to +10, +30 and +50 mV in the presence of Cd 2+ but was not significantly affected for pulses to -10 mV (P = 0.46, unpaired t-test) (Fig. 7B). Figure 7B also indicated that a substantial Mn 2+ influx occurred through L-type channels at the reversal potential of the L-type current; however this reversal potential value was likely shifted toward negative voltages by contaminating outward currents so that there still was a driving force for Mn 2+ influx at +50 mV. Fitting a Boltzmann equation to the relationship between mean changes of fluorescence and membrane potential in the presence of Cd 2+ gave value for V 0.5 and k of -8 mV and 4 mV.

Discussion

Properties of divalent cation influx at rest and associated membrane current

In the first part of the present study, the combination of the Mn 2+ quenching method with a voltage clamp technique together with calibration of the fura-2 response to Mn 2+ allowed us to thoroughly characterize the influx of divalent cations at rest in isolated adult skeletal muscle fibers. At a holding potential of -80 mV and in the presence of 500 µM external Mn 2+ , the rate of Mn 2+ influx was found to be 4.5 %.min -1 in average, a value close to the values reported in other studies using the same external [Mn 2+ ] on adult mouse muscle fibers at rest [START_REF] De Backer | Long-term study of Ca 2+ homeostasis and of survival in collagenase-isolated muscle fibres from normal and mdx mice[END_REF][START_REF] Tutdibi | Increased calcium entry into dystrophin-deficient muscle fibres of MDX and ADR-MDX mice is reduced by ion channel blockers[END_REF][START_REF] Kurebayashi | Depletion of Ca 2+ in the sarcoplasmic reticulum stimulates Ca 2+ entry into mouse skeletal muscle fibres[END_REF][START_REF] Fraysse | Decrease in resting calcium and calcium entry associated with slow-to-fast transition in unloaded rat soleus muscle[END_REF]. The rate of quenching was found to depend on the Mn 2+ concentration and on the electrical gradient. The voltage dependence of the rate of quenching indicated that the influx of Mn 2+ was slightly favoured at negative membrane potentials, a result which explains that hyperpolarizing steps were less effective in increasing the quenching rate when they were applied from depolarized holding potentials. This apparent inward rectification may either result from intrinsic properties of the pathway involved or may be due to the fact that a Mn 2+ equilibrium potential can never be reached because Mn 2+ ions are supposed to be unable to move out of the cell. Finally, we observed that the quenching rate was reduced by more than half when Ca 2+ ions were present at physiological concentrations in the external solution, which indicates that Ca 2+ compete with Mn 2+ for permeating the influx pathway. The combined use of voltage clamp and Mn 2+ quenching techniques prompted us to try measuring the inward current which may be associated with Mn 2+ entry. Mn 2+ exerted a strong blocking effect on muscle background conductances which precludes revealing a putative inward current upon replacement of Mg 2+ by Mn 2+ . Nevertheless, our in vivo calibration experiments of the fura-2 response to Mn 2+ predicted that the inward current generated by the influx of Mn 2+ should have had amplitudes of a few picoamps in the presence of millimolar external [Mn 2+ ] which are clearly undetectable at the macroscopic level. At first sight, these results are hardly reconcilable with data obtained at the single channel level. Openings of spontaneously active Ca 2+ channels are indeed routinely recorded in cell-attached patches at negative voltages ( 6) and these channels were found to be permeant to Mn 2+ (5). However, no correlation was found between subsarcolemmal Ca 2+ load and activity of these channels suggesting that they are not involved in resting Ca 2+ influx [START_REF] Mallouk | Ca 2+ influx and opening of Ca 2+ -activated K + channels in muscle fibers from control and mdx mice[END_REF]. For obscure reasons, a parallel pathway which does not produce resolvable current could thus be more efficient in getting Ca 2+ in. Yet, the fact that Mn 2+ entry is driven by the concentration and electrical gradients strongly suggests that Mn 2+ and Ca 2+ enter muscle cells through ion channels spontaneously open at rest and necessarily of very low conductance. However, the involvement of electrically silent transport processes directly or indirectly influenced by the concentration and electrical gradients cannot be excluded and deserves further attention. Finally our study demonstrates that, because Mn 2+ accumulates in the cytosol, the Mn 2+ quenching method offers a higher resolution to monitor the entry of divalent cations at rest than the measurement of currents.

Store depletion-activated cation influx

We demonstrated here that a single burst of AP delivered in the presence of CPA significantly increased the resting rate of Mn 2+ influx. We observed that fibers still contracted in response to stimulation in the presence of CPA, indicating that store depletion was not complete under our experimental conditions. In our hands, attempts to provoke deeper depletion of the SR inevitably led to a huge influx of Mn 2+ and an irreversible depolarization of the fiber associated with a large decrease in the input resistance which gave evidence of irreversible damages of the sarcolemma, possibly produced by toxic elevation of the intracellular [Ca 2+ ]. As pointed out by Kurebayashi & Ogawa [START_REF] Kurebayashi | Depletion of Ca 2+ in the sarcoplasmic reticulum stimulates Ca 2+ entry into mouse skeletal muscle fibres[END_REF], severe Ca 2+ depletion to the point of complete exhaustion of contraction would certainly never occur in adult skeletal muscle under physiological conditions. Our experiments show that partial depletion does induce Ca 2+ entry as described by Launikonis & Rios (20) in skinned rat muscle fibers. Confirming our previous data [START_REF] Allard | Sarcoplasmic reticulum Ca 2+ release and depletion fail to affect sarcolemmal ion channel activity in mouse skeletal muscle[END_REF], these experiments also demonstrate that the store-operated Ca 2+ entry was not associated with the development of a measurable current as evidenced by the absence of a significant change in the cell input resistance and membrane potential. This series of experiments also indicates that experiments making use of SR Ca 2+ -ATPase inhibitors have to be interpreted with caution when they are performed in the absence of voltage control. Background current or membrane potential and input resistance measurement are very reliable indexes of cell integrity that may reveal massive damages of the sarcolemma not related to any store-operated pathway.

Cation influx activated by trains of AP

We found that trains of AP delivered at 50 Hz, which closely correspond to physiological conditions of stimulation, increased Mn 2+ influx to a much greater degree (29 fold in the presence of 3 mM external Mn 2+ ) as compared to store depletion. The control of the membrane potential allowed us to explore the Mn 2+ influx over a large range of membrane voltages. The following observations led us to conclude that the L-type voltage-activated Ca 2+ channels and a parallel distinct voltage-activated Mn 2+ pathway contributed independently to the Mn 2+ influx in response to depolarization: (i) the voltage dependence of the global Mn 2+ influx was steeper than the one of the L-type Mn 2+ current and shifted toward negative potentials; (ii) whereas Cd 2+ entirely inhibited the L-type Mn 2+ current, a Mn 2+ influx still occurred in response to depolarization with a voltage threshold and voltage dependence that were respectively lower and steeper than those of the L-type current. Our Cd 2+ experiments also allowed us to estimate the relative contribution of each of these divalent cations entries during muscle activation at different voltages. At -10 mV, the newly identified voltageactivated Mn 2+ influx pathway may contribute mainly to the global Mn 2+ influx while for higher depolarizations it may represent between 18 and 28 % of the total Mn 2+ influx. A striking observation was that no net inward current persisted when the L-type current was inhibited by Cd 2+ . Yet, if this influx represents 24 % of the global entry at +30 mV, we should have been able to record a net inward current after Cd 2+ blockade whose amplitude should also represent one quarter of the amplitude of the L-type current. A similar phenomenon identified as excitation-coupled Ca 2+ entry has been described in myotubes [START_REF] Cherednichenko | Conformational activation of Ca 2+ entry by depolarization of skeletal myotubes[END_REF] and, interestingly, current recordings on myotubes expressing a mutant isoform of type 1 ryanodine receptor also failed to reveal a current associated with this Mn 2+ entry although the magnitude of the Ca 2+ entry produced predicted that a current larger than the L-type current should have developed [START_REF] Hurne | Ryanodine receptor type 1 (RyR1) mutations C4958S and C4961S reveal excitation-coupled calcium entry (ECCE) is independent of sarcoplasmic reticulum store depletion[END_REF]. As postulated for myotubes, our results suggest that a voltageactivated Mn 2+ influx operates in an electrically silent manner in adult skeletal muscle. One possibility is that this Mn 2+ influx involves a non electrogenic exchanger, which up to now has never been described, but actually merits further consideration.

Physiological significance of activity-induced cation influx

A more striking result was that Cd 2+ did not affect the Mn 2+ influx induced by train of AP. Apart from the fact that this result confirms that a voltage-activated Mn 2+ influx insensitive to Cd 2+ does operate in adult skeletal muscle in parallel with the L-type current, these data also suggest that the L-type current does not contribute to the influx of divalent cations during physiological excitation. However, this last result must be taken with caution because the voltage dependence of the Mn 2+ current through L-type channels was shifted by 23 mV toward positive membrane potentials (V 0.5 = +22 mV) as compared to the voltage dependence of the Ca 2+ current in the presence of 2.5 mM external Ca 2+ (V 0.5 = -0.7 mV in 22). The membrane potential reached in response to trains of AP could thus be far above the peak value of the L-type Ca 2+ current but lower than the one of the Mn 2+ current so that the contribution of the L-type current may be minimized when Mn 2+ ions are flowing through the channels. Whatever the pathway involved, the physiological role of the Ca 2+ influx during excitation remains largely elusive in skeletal muscle. A current hypothesis suggests that the Ca 2+ entry induced by depolarization or by SR depletion may contribute to refill internal Ca 2+ stores especially under fatigue conditions. However, it was shown that the recovery of the releasable pool after fatigue occurred in the absence of extracellular Ca 2+ [START_REF] Kabbara | The role of calcium stores in fatigue of isolated single muscle fibres from the cane toad[END_REF] and growing evidences indicate that Ca 2+ is not extruded from the cells during fatigue but may remain as a non releasable pool possibly bound to phosphate within the SR [START_REF] Allen | Skeletal muscle fatigue: cellular mechanisms[END_REF]. Within this context, we believe that the nature and the physiological role of the Ca 2+ pathways involved during excitation still remain open questions in adult skeletal muscle which call for further attention. (B) In the continuous presence of a Tyrode solution containing 3 mM Mn 2+ , the fiber was stimulated by bursts of AP of 500 ms duration delivered at 50 Hz in the absence and in the presence of CPA. Fluorescence records were fitted with a linear regression in the presence of CPA with slopes of 4 %/min before and of 10 %/min after stimulation. (C) In this particular fiber, a single burst of AP given in the presence of CPA led to a brutal drop of fluorescence (the slope of the linear fit is 190 %/min) and an irreversible depolarization of the fiber. Note that membrane potential in lower traces was acquired at 200 Hz so that peak values of AP could not be resolved. show the membrane currents recorded in a same fiber in response to a 200-ms depolarization step given to +30mV from a holding potential of -80 mV in the absence and in the presence of 0.5 mM Cd 2+ (star) and the current-voltage relationship of the Mn 2+ current. The curve was fitted using the equation indicated in Methods with values for G max , V rev , V 0.5 and k of 127 S/F, +50 mV, +22 mV, and 7.9 mV (n = 9). (B) Relationships between the mean normalized conductance of the voltage-activated Mn 2+ current and membrane potential (closed symbols), between the mean Mn 2+ influx, expressed as percentage of decrease of fluorescence, and membrane potential in the absence (open circles) and in the presence of 0.5 mM Cd 2+ (open squares). The curves were fitted using a Boltzmann equation with values for V 0.5 and k of +22 mV and 8.6 mV for the normalized conductance, -1.9 mV and 4.7 mV for the Mn 2+ influx in the absence of Cd 2+ and -8 mV and 4 mV for the Mn 2+ influx in the presence of Cd 2+ . The number of cells tested at -10, +10, +30 and +50 mV is 8, 4, 4 and 2 in the absence of Cd 2+ and 7, 8, 8 and 1 in the presence of Cd 2+ . (C) In the left panel, a single train of AP (lower trace) was elicited under current clamp conditions by injection of positive supraliminar current pulses of 0.5 ms duration delivered at 50 Hz in the presence of 3 mM external Mn 2+ while fura-2 fluorescence was simultaneously recorded (open symbols). The same stimulation was applied in the same cell with 0.5 mM Cd 2+ added in the external medium (right panel). The two consecutive recordings were separated by a 2-min interval. (D) In the left panel, the cell was held at -80 mV and a 100-ms depolarizing pulse was given to +30mV as indicated by the step at the bottom in the presence of 3 mM external Mn 2+ . The same stimulation was applied in the same cell with 0.5 mM Cd 2+ added in the external medium (right panel). The two consecutive fluorescence recordings were separated by a 1-min interval. The fluorescence data points immediately before and after stimulations were linked by a straight line to make fluorescence drops more visible. The slopes of these lines were 30 and 29 %/min in the absence and in the presence of Cd 2+ in C, respectively, and 122 and 45 %/min in D, respectively. Mn 2+ Mn 2+ + Cd 2+

Figure 1 :

 1 Figure 1: Influence of the external Mn 2+ concentration on Mn 2+ influx at -80 mV. The bars indicate the periods during which the cell was exposed to an external Tyrode solution containing 3 mM Mn 2+ (A) and 1 mM or 10 mM Mn 2+ (B). The straight lines superimposed correspond to the linear regressions fitted to the fluorescence records with slopes of 27 %/min in A and 14 and 38 %/min in B. (C) The histogram shows the average values of quenching rate obtained in the presence of increasing external Mn 2+ concentrations. The numbers above each bar indicate the number of cells tested.

Figure 2 :

 2 Figure 2: Influence of membrane potential on Mn 2+ influx in the presence of 3 mM external Mn 2+ . (A) Effect of addition of Mn 2+ on fura-2 fluorescence at the indicated voltages in four different cells. The slopes of the linear fits are 8, 13, 20 and 29 %/min at +40, 0, -40 and -80 mV respectively. (B) Average values of quenching rate obtained at different membrane voltages. (C) Changes in the quenching rate induced by a 60mV-hyperpolarizing pulse given from a holding potential of +20 mV (upper trace) and -20 mV (lower trace). The slope of the linear fits increased from 9 to 13 %/min in the upper trace and from 15 to 29 %/min in the lower trace. (D) Effect of the holding potential on the change in the quenching rate induced by a 60mV-hyperpolarizing pulse.

Figure 3 :

 3 Figure 3: Membrane currents evoked by replacement of Mg 2+ by Mn 2+ . (A and B) Simultaneous recordings of membrane currents and fura-2 fluorescence at -80 mV and +40 mV. The slope of the linear fit is 24 and 12 %/min at -80 and +40 mV respectively. (C) Continuous recording of membrane current at -80 mV in response to replacement of 10 mM Mg 2+ by 10 mM Mn 2+ . The cell was challenged by voltage ramps given from -40 to -100 mV at a rate of 120mV/s every 5 s in the presence of 140 mM external TEA-methanesulfonate plus 9AC and 120 mM internal Cs-aspartate. (D) Current-voltage relationships obtained in the fiber illustrated in (C) in response to voltage ramps, the lower and upper traces corresponding to the left and right arrows in (C) respectively.

Figure 4 :

 4 Figure 4: Estimation of the sarcolemmal Mn 2+ current on the basis of the measurement of the changes in fura-2 fluorescence induced by Mn 2+ entry. (A) Relationships between the relative fluorescence of fura-2 and the concentration of Mn 2+ in glass tubes (open symbols, n = 9), and the concentration of Mn 2+ in the external solution bathing muscle fibers in the presence of ionomycin (closed symbols, n = 4). Curves were fitted using a Hill equation (see text for details). (B) Quenching of fura-2 fluorescence obtained in a fiber exposed to the indicated concentrations of Mn 2+ in the presence of ionomycin. (C) Relationship between the estimated resting Mn 2+ current and membrane voltage. The curve was drawn by eye.

Figure 5 :

 5 Figure5: Effects of trains of AP and CPA on Mn 2+ influx under current clamp conditions. (A) Simultaneous recordings of fura-2 fluorescence (open symbols) and membrane potential (lower trace) in a fiber stimulated by positive suprathreshold current pulses of 0.5 ms duration delivered at 50 Hz. The duration of the trains was 2 s in the left panel and 3 s in the right panel. Each burst of stimulation was preceded by a negative current pulse of 2 nA amplitude to measure the fiber input resistance (downward deflections of the voltage trace). The inset shows the first train of AP (star, left trace) and the first AP of this train (right trace) on an expanded scale. The recordings presented in the right panel were obtained in the same fiber 4 min after the ones presented in the left panel. The external Tyrode solution contained 2 mM CaCl 2 and 2 mM MgCl 2 in which 1 mM Mn 2+ was added. (B) In the continuous presence of a Tyrode solution containing 3 mM Mn 2+ , the fiber was stimulated by bursts of AP of 500 ms duration delivered at 50 Hz in the absence and in the presence of CPA. Fluorescence records were fitted with a linear regression in the presence of CPA with slopes of 4 %/min before and of 10 %/min after stimulation. (C) In this particular fiber, a single burst of AP given in the presence of CPA led to a brutal drop of fluorescence (the slope of the linear fit is 190 %/min) and an irreversible depolarization of the fiber. Note that membrane potential in lower traces was acquired at 200 Hz so that peak values of AP could not be resolved.

Figure 6 :

 6 Figure 6: Effect of depolarization steps of increasing amplitude on Mn 2+ influx under voltage clamp conditions. Fura-2 fluorescence (open symbols) and membrane currents (lower traces) were measured on the same muscle fiber in the presence of a 140 mM TEAmethanesulfonate Ca 2+ -free external medium containing 3 mM Mn 2+ . The cell was held at -80 mV and voltage steps of 100 ms duration bringing the membrane potential to the indicated values were applied between two fluorescence image captures at the indicated position of the steps. Note that the corresponding currents were positioned under the voltage steps on a different time scale.

Figure 7 :

 7 Figure 7: Voltage dependence of Mn 2+ influx and effect of Cd 2+ . (A) The upper tracesshow the membrane currents recorded in a same fiber in response to a 200-ms depolarization step given to +30mV from a holding potential of -80 mV in the absence and in the presence of 0.5 mM Cd 2+ (star) and the current-voltage relationship of the Mn 2+ current. The curve was fitted using the equation indicated in Methods with values for G max , V rev , V 0.5 and k of 127 S/F, +50 mV, +22 mV, and 7.9 mV (n = 9). (B) Relationships between the mean normalized conductance of the voltage-activated Mn 2+ current and membrane potential (closed symbols), between the mean Mn 2+ influx, expressed as percentage of decrease of fluorescence, and membrane potential in the absence (open circles) and in the presence of 0.5 mM Cd 2+ (open squares). The curves were fitted using a Boltzmann equation with values for V 0.5 and k of +22 mV and 8.6 mV for the normalized conductance, -1.9 mV and 4.7 mV for the Mn 2+ influx in the absence of Cd 2+ and -8 mV and 4 mV for the Mn 2+ influx in the presence of Cd 2+ . The number of cells tested at -10, +10, +30 and +50 mV is 8, 4, 4 and 2 in the absence of Cd 2+ and 7, 8, 8 and 1 in the presence of Cd 2+ . (C) In the left panel, a single train of AP (lower trace) was elicited under current clamp conditions by injection of positive supraliminar current pulses of 0.5 ms duration delivered at 50 Hz in the presence of 3 mM external Mn 2+ while fura-2 fluorescence was simultaneously recorded (open symbols). The same stimulation was applied in the same cell with 0.5 mM Cd 2+ added in the external medium (right panel). The two consecutive recordings were separated by a 2-min interval. (D) In the left panel, the cell was held at -80 mV and a 100-ms depolarizing pulse was given to +30mV as indicated by the step at the bottom in the presence of 3 mM external Mn 2+ . The same stimulation was applied in the same cell with 0.5 mM Cd 2+ added in the external medium (right panel). The two

  Figure 1

, 5B, C and 7C, the external saline corresponded to a standard Tyrode solution containing (in mM) 140 NaCl, 5 KCl and 10 HEPES adjusted to pH 7.2 with NaOH with no added Ca 2+ and various concentrations of MgCl 2 . MgCl 2 was replaced

  by MnCl 2 at equimolar concentrations to minimize surface charge effects. For experiments in relation with Fig. 5A, 1 mM MnCl 2 was added in a Tyrode solution containing 2 mM CaCl 2 and 2 mM MgCl 2 . For experiments in relation with Fig. 2, 3, 6, 7A, B and D, the external solution contained (in mM) 140 TEA-methanesulfonate, 10 HEPES adjusted to pH 7.2 with TEA-OH with no added Ca 2+ and various concentrations of MgCl 2 . MgCl 2 was replaced by MnCl 2 at equimolar concentrations. For experiments in relation with Fig.

Acknowledgements

This work was supported by the Université Lyon 1, the Centre National de la Recherche Scientifique, the Association Française contre les Myopathies and the Agence Nationale de la Recherche Maladies Rares. We are grateful to J-L. Barrat, M. Jospin and V. Jacquemond for helpful comments on the manuscript.


Maxime Pinaud

Georges Humbert

Sebastian Engelbrecht

Lionel Merlat

Bernd M Fischer

Aurelian Crunteanu
email: aurelian.crunteanu@xlim.fr

Terahertz Devices Using the Optical Activation of GeTe Phase Change Materials: Towards Fully Reconfigurable Functionalities

Keywords: phase-change materials, optical activation, reconfigurable terahertz metamaterials, polarizer

Multifunctional terahertz (THz) devices are crucial for the development of practical applications such as high-speed communications, spectroscopy and imaging, but their expansion is still requiring efficient agility functions operating in the THz domain.

Chalcogenide phase change materials (PCMs) with broadband responses, non-volatile and reversible transitions between a dielectric and metal-like phases, were successfully investigated as agile elements in photonics or electronics applications but their potential for controlling the THz waves is still under evaluation. We are demonstrating the optical control of specific states of the germanium telluride (GeTe) PCM and its integration as control element for enabling future optically-reconfigurable THz devices. The excellent contrast of material's THz properties in the two dissimilar states were used for optical-induced modulation of THz resonances of a metamaterial based on split-ring-resonator metallic structures integrating GeTe patterns. We experimentally confirm for the first time the feasibility of all dielectric GeTebased THz polarizers presenting broadband responses, high extinction ratios when the GeTe is in the metal-like phase and almost transparent when is amorphous. This highly functional approach based on optically-controlled multi-operational THz devices integrating PCMs, is extremely stimulating for generating future disruptive developments (field-programmable metasurfaces, dielectric coding metamaterials) with multifunctional capabilities for THz waves manipulation.

Introduction

Terahertz (THz) waves hold very promising applications such as high-speed wireless communication, security screening, chemical identification and non-destructive sensing. [START_REF] Tonouchi | Cutting-edge terahertz technology[END_REF] A prerequisite to the extensive development of these domains at THz frequencies is the development of reconfigurable devices (filters, spatial light modulators, lenses, frequency selective surfaces,…), which allow controlling the properties of electromagnetic waves such as their amplitude, phase, polarization and/ or spatiotemporal distribution of their wave fronts [START_REF] He | Metasurfaces for Terahertz Wavefront Modulation: a Review[END_REF] .

Reconfigurable devices are essential in reducing the cost, complexity and overall dimensions of THz systems. Reconfiguration strategies and the associated technologies already employed in the optical and microwaves domains are difficult or ineffective to transpose to THz frequencies [START_REF] Dragoman | Terahertz fields and applications[END_REF] . Current approaches for manipulating THz radiation are based on artificially engineered metasurfaces or metamaterials consisting of two-dimensional arrays of subwavelength metallic resonators [START_REF] He | Metasurfaces for Terahertz Wavefront Modulation: a Review[END_REF] . The topology or the electromagnetic properties of these individual building blocks can be modified by the integration in their design of different agility functions such as semiconductors, [START_REF] Kleine-Ostmann | Spatially resolved measurements of depletion properties of large gate two-dimensional electron gas semiconductor terahertz modulators[END_REF][START_REF] Chen | Active terahertz metamaterial devices[END_REF] liquid crystals, [START_REF] Savo | Liquid Crystal Metamaterial Absorber Spatial Light Modulator for THz Applications[END_REF][START_REF] Wang | Tunable optical negative-index metamaterials employing anisotropic liquid crystals[END_REF][START_REF] Zografopoulos | Tunable terahertz fishnet metamaterials based on thin nematic liquid crystal layers for fast switching[END_REF] MEMS devices, [START_REF] Zhengli | Tunable Terahertz Filter and Modulator Based on Electrostatic MEMS Reconfigurable SRR Array[END_REF] phase transition materials (VO2) [START_REF] Han | Broadband modulation of terahertz waves through electrically driven hybrid bowtie antenna-VO2 devices[END_REF][START_REF] Wang | Hybrid Resonators and Highly Tunable Terahertz Metamaterials Enabled by Vanadium Dioxide (VO2)[END_REF] or graphene [START_REF] Lee | Switching terahertz waves with gate-controlled active graphene metamaterials[END_REF][START_REF] Zhang | Tunable terahertz absorption in graphene-based metamaterial[END_REF] which has been proven as effective means to control the properties of THz waves.

In particular, vanadium dioxide (VO2) material has lately triggered a broad interest as active element in reconfigurable THz devices due to its ability to perform an insulator-to-metal phase transition at relatively low temperature (68°C) and under different applied stimuli (electrical or optical) [START_REF] Jeong | Dynamic Terahertz Plasmonics Enabled by Phase-Change Materials[END_REF][START_REF] Seo | Active Terahertz Nanoantennas Based on VO2 Phase Transition[END_REF] . Still, the low transition temperature of VO2, associated with the volatile phase transition (the material becomes insulator after removing the specific stimulus needed for the phase transition to the metallic state), although very practical for demonstration of specific functionalities, is inducing large non-linearity in material's properties and is limiting its operation at moderate temperatures.

In a similar way, most of the agile elements cited above have volatile-type responses requiring permanent optical or electrical bias, and complex integration technology with electrical/ optical control schemes over large areas. At the same time, they have to be integrated within the fixed topology of metadevices's for active manipulation of THz waves.

Chalcogenide phase change materials (PCMs) such as GeTe (germanium telluride) or Ge2Sb2Te5-GST (germanium antimony telluride) have been successfully integrated for realizing reconfigurable electrical or optical devices. The reconfiguration capability is based on the unique properties of PCMs undergoing a non-volatile phase transition on nanoseconds timescales from an amorphous (insulating) state to a crystalline (metallic), drastically changing their electrical and optical properties upon application of thermal, electrical or optical stimuli. [16-19] These PCM capabilities have been exploited over the years for realizing non-volatile memories by stacking the material between two electrodes and changing its state using pulsed current-Joule heating [START_REF] Ovshinsky | Reversible Electrical Switching Phenomena in Disordered Structures[END_REF][START_REF] Raoux | Phase change materials and phase change memory[END_REF] . More recently, GeTe and GST compositions have shown their ability to be used as high performance, bi-stable switching devices in the microwaves/ millimeter waves domains, holding high-promises for future multi-reconfigurable telecommunication systems [22,[START_REF] El-Hinnawy | A Four-Terminal, Inline, Chalcogenide Phase-Change RF Switch Using an Independent Resistive Heater for Thermal Actuation[END_REF] . Their implementation requires a planar electrodes indirect heating scheme using short voltage/ current pulses applied on a thin-film heater isolated through a dielectric film underneath the PCM films [22][START_REF] El-Hinnawy | A Four-Terminal, Inline, Chalcogenide Phase-Change RF Switch Using an Independent Resistive Heater for Thermal Actuation[END_REF][START_REF] Mennai | Bistable RF Switches Using Ge2Sb2Te5 Phase Change Material[END_REF] .

Compact optical metadevices based on PCMs, such as flat optics-like switchable/ tunable filters, multi-focus Fresnel zone plates, super-oscillatory lenses with subwavelength focus, and optical holograms have also been demonstrated [START_REF] Ohta | Overwritable phase-change optical disk recording[END_REF][START_REF] Dong | In PCRAMsim: System-level performance, energy, and area modeling for Phase-Change RAM[END_REF][START_REF] Karvounis | All-dielectric phasechange reconfigurable metasurface[END_REF][START_REF] Wang | Optically reconfigurable metasurfaces and photonic devices based on phase change materials[END_REF][START_REF] Singh | Characterization, Optimization, and Fabrication of Phase Change Material Germanium Telluride Based Miniaturized DC-67 GHz RF Switches[END_REF][START_REF] Gholipour | An all-optical, non-volatile, bidirectional, phase-change meta-switch[END_REF] . Furthermore, optical activation of PCMs between the two states has been widely used for rewritable optical disks storage memories [START_REF] Yamada | High-speed overwritable phase-change optical disk material[END_REF][START_REF] Chen | Compound materials for reversible, phase-change optical-data storage[END_REF] . Nevertheless, the large area capabilities of this technology are based on the association of mechanical movement of the storage medium and activation regions of PCMs limited by the laser beam size.

Most recently, it has been proven that PCMs can be used to realize all-dielectric, metal-free agile functions in the optical domain using optical control of GST-based metadevices. [START_REF] Karvounis | All-dielectric phasechange reconfigurable metasurface[END_REF][START_REF] Gholipour | Reconfigurable Ultraviolet and High-Energy Visible Dielectric Metamaterials[END_REF] Unlike electrical-based control schemes [START_REF] Ovshinsky | Reversible Electrical Switching Phenomena in Disordered Structures[END_REF][START_REF] Raoux | Phase change materials and phase change memory[END_REF][22][START_REF] El-Hinnawy | A Four-Terminal, Inline, Chalcogenide Phase-Change RF Switch Using an Independent Resistive Heater for Thermal Actuation[END_REF] , fast optical activation using femtosecond and nanosecond lasers of PCMs allows a more accurate control of the specific thermal diffusion effects associated with the crystallization and amorphization processes during the phase change mechanisms of the materials. [START_REF] Makino | Ultrafast optical manipulation of atomic arrangements in chalcogenide alloy memory materials[END_REF][START_REF] Liu | Crystallization of Ge2Sb2Te5 films by amplified femtosecond optical pulses[END_REF][START_REF] Lei | Refractive index modulation of Sb70Te30 phase-change thin films by multiple femtosecond laser pulses[END_REF] From the electrical point of view, in the amorphous state at low temperatures, GeTe is behaving like a disordered bandgap semiconductor with two main defect-induced band traps within the bandgap, located at ~0.26 eV and ~0.57 eV from the valence band edge 37 . It was previously shown that the electronic properties of amorphous GeTe are temperature-dependent, with nonlinear current-voltage behavior which can be modeled by space charge limited transport with uniform trap bands 37 . With increasing temperature and its transformation to the crystalline state, the material behave as a degenerate p-type semiconductor, with mobility and hole concentrations values up to 11 cm 2 /Vs and 8.8x 10 20 cm 3 , respectively, at 375K 37 . The THz conductivity of charge carriers in amorphous and crystalline phase change materials exhibits also a complex behavior, with a Lorentz-type resonance attributed to a phonon mode (grain boundaries tunneling) and losses due to free carriers (Drude relaxation conductivity term) 38 .

In the THz domain, the potential of PCMs as agile elements for controlling the properties of THz waves is still under evaluation. The few published studies report on their THz properties [38] , and on the possibility to integrate GeTe and GST stripes within THz metadevices which can adapt their response using thermal or electrical stimulus to change PCM's state only from amorphous to crystalline. [START_REF] Kodama | Tunable split-ring resonators using germanium telluride[END_REF] Recently, a THz metadevice based on 2x2 panels of metallic asymmetric split ring resonators (A-SRR) with dissimilar dimensions fabricated on a GST film has been reported to present multilevel states at THz frequencies using the electrical control (through a direct Joule heating scheme) of the crystallinity degree (and thus of the conductivity) of the GST material. [START_REF] Pitchappa | Chalcogenide Phase Change Material for Active Terahertz Photonics[END_REF] While the electrically induced nonvolatile and multi-state modulation of the THz transmission of the devices at multiple frequencies was validated for the amorphous-to-crystalline state of the GST material, the reverse operation of the system corresponding to the crystalline-to-amorphous state change of GST is yet to be proven. Electrical activation has obvious and numerous advantages for individual devices. However, the amorphization/ crystallization processes requires extremely localized areas of PCMs inserted between the command electrodes making it hardly compatible with design constraints of metadevices at THz frequencies.

In addition, the use of subwavelength metallic resonators as elementary building blocks of THz metadevices strongly limits the possibility to achieve large reconfiguration functionalities since the integration in their topology of active reconfigurable elements over wide areas (and associated electrical control) remain complex.

Although PCM-only metadevices made on GeTe-based split ring resonator arrays have been proposed in the past, they show very weak changes in their specific response at THz frequencies during GeTe phase transition from amorphous to the crystalline phase. [START_REF] Kodama | Tunable split-ring resonators using germanium telluride[END_REF] Nevertheless, THz devices fabricated with PCM active layers that are compatible with an optical activation between their dissimilar states, would lead to improved performances, simplicity of integration and may attain multiple reconfiguration functions [START_REF] Choi | Nanopattern enabled terahertz alloptical switching on vanadium dioxide thin film[END_REF] .

Here, we demonstrate for the first time to our knowledge an implementation scheme of PCMs and their associated optical control, foreshadowing the development of PCM-only THz devices with nonvolatile and reversible reconfiguration capabilities.

We validated the fabrication of GeTe phase change films with high contrast of their THz properties between amorphous and crystalline states allowing the first developments of all-PCM THz components. Furthermore, we experimentally show that these PCM films can be successively optically switched on large areas between their amorphous and crystalline phases using nanosecond laser pulses, while keeping an excellent contrast of their THz properties in the two dissimilar states. These performances were further used for demonstrating optical-induced THz response of a hybrid THz metadevice based of arrays of split ring resonator metallic structures integrating GeTe patterns. The significant amplitude contrast of the THz transmission between the two states of the GeTe film was finally exploited for developing all-PCM THz components by experimentally demonstrating optically activated GeTe-only THz polarizer devices with compelling performances to incident THz waves. This proof of principle based on the GeTe building elements and their associated activation method holds high-promises for future THz devices with fully reconfigurable transfer functions.

Thermal and optical activation of GeTe phase change material and its electrical performances at THz frequencies

We investigated the modification of electrical properties of GeTe films obtained on c-cut sapphire substrates, at DC and THz frequencies, for both direct plate heating or optical activation of phase changes (Figure 1a) (see also experimental section for details). As fabricated, the 500-nm thick GeTe film is amorphous with high DC resistivity of 800 Ω•cm. In the case of direct heating, the film's two-point resistance is gradually reducing with the temperature until ~175°C, when it is abruptly decreasing and stabilizes to the crystalline state resistance, as illustrated in Figure1b. The material retains its conductive state crystalline state with a resistivity value of 12x 10 -5 •Ω•cm, upon further annealing up to 300°C and subsequent cooling at ambient temperature, confirming its non-volatile electrical transition.

The GeTe phase change through thermal heating is inducing a large modification of the The thicknesses of investigated GeTe films are much smaller compared to the THz wavelengths and, taking also into account that the GeTe in the amorphous phase is almost transparent to the THz waves, it is very difficult to evaluate the complex permittivity of the 500 nm-thick GeTe films prepared in the two states, within acceptable errors. [START_REF] Withayachumnankul | Limitation in thin-film sensing with transmission-mode terahertz time-domain spectroscopy[END_REF] Still, the analysis of similar THz-TDS experimental data obtained for 1-µm thick GeTe films on sapphire substrates (supplementary material, Figure S1) allowed us to extract conductivity values of 1000 ±20% S/m for GeTe in the amorphous phase and (2.2 ±10%) x10 5 S/m for GeTe in the crystalline phase in the 0.2-1 THz frequency domain. These values are consistent with previous reported data on different phase change material compositions [38] . showing the spatial modulation of the THz transmission (the spectral profile of a section of the 2D image on the right-hand side panel).

An alternative and suitable method to achieve the amorphous -to-crystalline phase change in PCMs is their optical irradiation using nanosecond laser pulses (Figure 1a) [START_REF] Wang | Optically reconfigurable metasurfaces and photonic devices based on phase change materials[END_REF][START_REF] Yamada | High-speed overwritable phase-change optical disk material[END_REF][START_REF] Chen | Compound materials for reversible, phase-change optical-data storage[END_REF][START_REF] Makino | Ultrafast optical manipulation of atomic arrangements in chalcogenide alloy memory materials[END_REF] .

Compared to the plate heating method for which is extremely difficult to cycle in a reversible way the material between its dissimilar phases, the laser-induced phase change in PCMs has proven to be a method with fast switching capabilities, allowing the repetitive and sequential preparation of the material in a specific, desired state [START_REF] Wuttig | Phase-change materials for rewriteable data storage[END_REF] .

Our approach for optically switching the GeTe layers between the two states is employing the direct irradiation of the films using 35-ns long UV pulses from an excimer KrF laser operating at 248 nm (Figure 1a) [START_REF] Valdes | A Polarization Reconfigurable Patch Antenna in the Millimeter-Waves Domain Using Optical Control of Phase Change Materials[END_REF] . The normalized THz transmission of the optically-crystallized GeTe shows performances identical to those of the crystalline GeTe films prepared by the plate heating method (Figure 1e). The employment of the KrF excimer source for laser-induced phase change in GeTe has a major advantage: the convenient way of preparing a specific state of the material in a recurrent and reproducible manner over large areas (the imprint -beam dimensions on the irradiated surface-of the laser beam covering a surface as large as 1.5x 0.5 mm 2 ) by simple direct irradiation or using raster scanning. Alternatively, specific crystalline GeTe patterns can be prepared by laser irradiation of the layer through contact masks carrying a particular design. The specific THz transmission response of the GeTe film in the two dissimilar states was further confirmed by the THz 2D spatial mapping of the interface between the amorphous and crystalline phases after laser irradiation of the amorphous GeTe material (Figure 1f). Although THz imaging has much lower resolution than the optical microscopy, the THz image allow evaluating the spatial interface between the two GeTe regions with dissimilar properties, as perceived by the THz waves. The associated THz transmission at 1 THz (righthand side panel in Figure 1f, presenting the spectral profile of a section of the 2D image) shows that the optically transformed crystalline GeTe is highly blocking the THz waves propagation while the amorphous GeTe is transparent to the incident waves.

Thus, to crystallize an initially amorphous GeTe 500-nm thick layer, an UV laser pulse with a power density of 1.1 x 10 17 W/m 3 (peak power volume density) is required to heat the material above its crystallization temperature, TC (Figure 2a). For bringing back the crystalline GeTe film to its amorphous phase, a higher power laser pulse should be employed, with power densities around 8.4 x 10 17 W/m 3 , in order to rapidly heat the material above its melting temperature TM and quench it in a disordered (amorphous) phase (Figure 2b). This meltquenching step is essential for reversible change of PCMs between their two states and requires intense and fast thermal pulses (electrical or optical induced) which can be difficult to implement using usual thermal heating approaches involving heating plates or ovens [START_REF] Wuttig | Phase-change materials for rewriteable data storage[END_REF] .

The mechanism of the crystalline-to amorphous phase change is particularly complex, in which the temperature of the material above the melting temperature needs to drop down quickly, with cooling rates higher than 10 9 K/s, i.e. the fast quench should occur faster than the timescale for thermal diffusion from cell to neighboring cell, preventing reorganization into a crystalline structure [START_REF] Wuttig | Phase-change materials for rewriteable data storage[END_REF] . It is then possible to define a critical quench time (CQT) of PCM materials, defined as the longest thermal time constant (i.e. the slowest cooling) that still results in an amorphous PCM material after the layer has been melted. The material undergoing the cooling process will be recrystallized if the quench time is longer than CQT, its estimation is therefore very important for defining the design, power budget and thermal response constraints for a given device configuration. For a specific PCM composition, the CQT is both device geometry and stimulus dependent, but it is possible to evaluate it experimentally or by multi physics simulations. Although several reports found that optical-based melt-quenching can be performed at times shorter than 1 ns (with recrystallization times of about 13 ns on optimized multilayer structures), common values for CQT range from tens to hundreds of nanoseconds [START_REF] Yamada | High-speed overwritable phase-change optical disk material[END_REF][START_REF] Wuttig | Phase-change materials for rewriteable data storage[END_REF] .

In The evolution of the sheet resistance of the film following the sequential transformation from amorphous to crystalline states shows that crystallization and melt-quenched processes are reproducible and that the resistance values in each of the states are stable after more than 20 laser pulses. The initial, as-obtained, amorphous sheet resistance value of the film is higher than the values of the melt-quenched process which can be explained by the laser irradiation process performed in ambient atmosphere, without any capping or protective layer.

Indeed, additional capping layers placed on top of the PCM films will prevent the long-term degradation of the material during the melt-quench process. The atmosphere exposure may thus modify the composition of the film through oxidation, inter diffusion with adjacent materials or segregation or loss of tellurium from the alloy (for the materials lying on the (GeTe)/ (Sb2Te3) pseudo binary line), which affect its electrical performances. Potential capping layers like ZnS, ZnS:SiO2, silicon nitride-Si3N4 or alumina-Al2O3 films have proven to be highly effective for increasing the reliability of the optical switching of PCMs in optical data storage devices, providing an additional control of the thermal responses of the stacked layers within the devices [START_REF] Yamada | High-speed overwritable phase-change optical disk material[END_REF][START_REF] Njoroge | Influence of dielectric capping layers on the crystallization kinetics of Ag5In6Sb59Te30 films[END_REF] . Additionally, they may also act as anti-reflection coatings for a more efficient lasermatter interaction. Although for THz applications, some of the materials cited above may introduce additional adsorption losses, their influence can be minimized by the use of compositions that are quasi-transparent in the THz domain (e.g. Al2O3, SiO2).

Nevertheless, in our experiments, even without the use of a capping layer, the electrical contrast between the resistances values of the crystallized and melt-quenched GeTe phases is still higher than 10 5 , resulting in a high THz transmission ratio between the two phases (Figure 2d). Thus, at 1 THz, the mean normalized transmission of the optically crystallized GeTe layer is 1.5% and of 83% for the optically melt-quenched, amorphous phase.

The response times of the GeTe material upon laser irradiation were recorded by performing optical pump-electrical conductivity probe measurements. We used a simple electrical circuit in which the GeTe layer was connected using planar electrodes with a constant voltage source and a series resistance of fixed value. The conductivity variation of the GeTe layer subsequent to laser irradiation will largely modify the current circulating in the circuit and, consequently, will modify the voltage drop on the series resistance. The real-time recording of this voltage give useful insights on the dynamics of the material phase change. Thus, the measured response times following laser irradiation with 35-ns long laser pulses, for both amorphous-to-crystalline and crystalline-to amorphous phase changes, lie in the 45-50 ns range. Although these short switching times offer a preliminary indication on the temporal responses and electrical conductivity modification of the GeTe layer, further investigations are planned for evaluating the direct THz response timescales.

In the following sections, we will experimentally demonstrate that this optically-prepared high transmission ratio between the two dissimilar states of the GeTe film may be employed for recurrent non-volatile switching of THz waves. In addition, the presented technique shows high capabilities for the design of essential THz devices with high compatibility to reconfigurable functionalities for which the integration of active GeTe material is straightforward.

Integration of GeTe in hybrid reconfigurable metamaterial THz device

We exploit the optical activation and the high contrast of THz transmission between amorphous and crystalline phases of GeTe to demonstrate optical activation of a hybrid metamaterial acting as a notch filter in the THz frequency domain. The design of the device is based on a periodic collection of metallic square-shape split-ring resonators (SRR) for which the gap is closed by a GeTe pattern (Figure 3 a,b). Such metamaterial SRR-based topologies had proven their capacity to effectively manipulate the THz waves [START_REF] Crunteanu | Tunable THz metamaterials based on phase-changed materials (VO2) triggered by thermal and electrical stimuli[END_REF][START_REF] Al-Naib | Excitation of a high-Q subradiant resonance mode in mirrored single-gap asymmetric split ring resonator terahertz metamaterials[END_REF] , having sharp LC-type resonances for which the frequency and resonance amplitude can be conveniently adjusted by varying their dimensions, shape or and the dielectric environment in the gap of the structure [START_REF] Withayachumnankul | Metamaterials in the Terahertz Regime[END_REF][START_REF] Chen | Manipulation of terahertz radiation using metamaterials[END_REF] .

The simulated response of the proposed device based on a network of SRR unit cells (Figure 3b) spaced by 20 µm in both directions in the plane of the overall structure is shown on Figure (Figure 3d) and its performances were evaluated using the THz-TDS measurement technique in both configurations (GeTe patterns in the amorphous states and in the optically-crystallized one, respectively). For crystallizing the GeTe integrated in the SRR unit cells, the entire metamaterial structure was irradiated using a single laser pulse, as mentioned before.

The measured normalized THz transmissions of the fabricated metamaterial device are represented in Figure 3e for both amorphous and crystalline GeTe in the gap of the SRR structures and they are corresponding very well to the theoretical performances. Although some differences between the simulated and experimental results are still occurring, they can be explained by the imprecision in evaluating the exact electrical and optical properties of such very thin GeTe films and the slight dimension differences occurring in the microfabrication process between the simulated structure and the fabricated device. The performances of the fabricated device can be also evaluated in terms of the quality factor of each of the resonance peaks, defined as Q = f0/Δf, where f0 is the resonance frequency and Δf is its bandwidth at half minimum. When the GeTe is amorphous, Q= 9.36 (LC resonance at Amorphous GeTe Crystalline GeTe Measures 0.26 THz), while Q = 15.8 when the GeTe is transformed to its crystalline state (resonance occurring at 0.58 THz). These quality factors are similar to the ones corresponding to metalliconly SRR planar devices (replacing with air the amorphous GeTe and with pure gold patterns the crystalline GeTe in the corresponding designs). Although these figures are also designdependent, the introduction of GeTe material within the SRR unit structure does not alter their values, implying that the losses induced by the integration of GeTe in the amorphous state or the lower conductivity of crystalline GeTe (worst case scenario when compared to a pure metal conductivity) may be alleviated in such metamaterial hybrid implementations.

The same analysis was performed on identical devices fabricated on silica (SiO2) substrates (see supplementary Figure S7) and shows comparable performances, although the specific resonances of the device are appearing at different frequencies, given the different substrate permittivity.

We obtained quasi-identical responses for similar devices integrating GeTe patterns which were prepared to the crystalline state using direct thermal activation on a heating plate (as indicated on supplementary Figure S8) which is clearly indicating that the optical-induced phase change of GeTe within the metamaterial structure is a highly effective and much more appropriate method in modifying, on-demand, the THz response of such devices.

The strong variation of the transmission at 0.58 THz (higher than 35dB) of the GeTe implemented SRR device between the optically-induced two dissimilar phases is a strong representation of the capabilities of these type of materials and associated switching technique in the development of low consumption, highly integrated THz devices relevant for switching functions, filters etc. with a plethora of applications in telecommunication and imaging [START_REF] Cui | Coding metamaterials, digital metamaterials and programmable metamaterials[END_REF] .

All-dielectric reconfigurable THz polarizer based on GeTe phase change material

The strong contrast in THz amplitude signals transmitted through the GeTe films optically prepared in the two different states suggests that simple structures made only in GeTe (without additional metallic structures) could be feasible, offering interesting and promising opportunities for developing active THz reconfigurable devices. In particular, THz polarizers are essential devices, with critical roles in imaging and wireless communication applications [START_REF] Lu | A high extinction ratio THz polarizer fabricated by double-bilayer wire grid structure[END_REF][START_REF] Watanabe | Terahertz Polarization Imaging and Its Applications[END_REF][START_REF] Huang | High extinction ratio and low transmission loss thin-film terahertz polarizer with a tunable bilayer metal wire-grid structure[END_REF][START_REF] Wong | Active meta polarizer for terahertz frequencies[END_REF][START_REF] Yamada | Terahertz wire-grid polarizers with micrometer-pitch Al gratings[END_REF][START_REF] Deng | Extremely high extinction ratio terahertz broadband polarizer using bilayer subwavelength metal wire-grid structure[END_REF] . For demonstrating the potential capabilities of the GeTe material for these applications,

we develop what we believe being the first all-PCM THz polarizer, i.e. the first device using the metallic/ crystalline state of GeTe to produce the selection of the polarization state of a transmitted THz wave.

The design of the proposed polarization device is based on GeTe wire-grid parallel structures on a sapphire substrate and have reconfiguration capabilities (turning on or off its polarization behavior) brought by the optically-active GeTe material (Figure 4).

We designed three different polarizers with dissimilar wire widths (w) and periods (P) which were fabricated by patterning a 500-nm thick GeTe layer on a sapphire c-cut substrate (Figure 4b). The polarizer P1 was made with a period P = 10 µm and a wire width w = 3 µm (P1(10/3

on Figure 4b). The P2 device was realized with a smaller period P = 6 µm and the same wire width w = 3 µm while for the third design, the width of the wires was reduced to w = 2 µm and the period was fixed to P = 9 µm (denoted as P2(6/3) and P3 (9/2), respectively, on Figure 4b).

A fourth polarizer structure was realized using gold instead of GeTe to serve as a reference, with P = 6 µm and w = 3 µm(P4(6/3) on Figure 4c).

When the GeTe is amorphous, the wire-grid structures act as a transparent dielectric material, allowing TM and TE THz waves to be transmitted with identical results (see supplementary

Figure S5

). However, when the GeTe is brought to its crystalline, metal-like state using UV laser pulses, it induces a wire-grid polarizer effect, blocking a significant part of the TE-polarized waves [START_REF] Withayachumnankul | Metamaterials in the Terahertz Regime[END_REF] . This behavior is clearly visible on the simulated and measured performances of the GeTe-based devices shown on Figure 4c. Usually, the TE transmission of wire-grid polarizers is increasing with frequency and, while the TM transmission is roughly constant, this will results in a small extinction ratios at higher THz frequencies [START_REF] Yamada | Terahertz wire-grid polarizers with micrometer-pitch Al gratings[END_REF][START_REF] Deng | Extremely high extinction ratio terahertz broadband polarizer using bilayer subwavelength metal wire-grid structure[END_REF] (see supplementary material information Figure S9). Although the measurement results of the gold-based polarizer shown on Figure 4c are only roughly consistent with this behaviour, the difference can be explained by the poor signal to noise ratio of our THz-TDS measurement system, less adapted to the evaluation of high extinction polarizers. In addition, in the case of the GeTe-based polarizers, the rather constant evolutions with frequency of the TE transmissions can be also explained by the difference in conductivity between metallic gold and crystalline GeTe. Indeed, compared to gold, crystalline GeTe has metal-like properties with much lower conductivity (≈ 220 kS.m -1 ) than metallic gold (47000 kS.m -1 ).

When comparing the results for the P1 and P3 polarizers we may notice that a decrease of the fill factor (defined as the ratio of the width of a single GeTe line to the period of the wire grid) from 0.3 (P1) to 0.2 (P3) for a fixed period (P= 10 µm) is decreasing the attenuation of the TE mode from -13.04 dB to -10.9 dB while the TM transmission is slightly improving from -0.26 dB for the P1 device to -0.22 dB for the P3 polarizer. The best performances were obtained for the P2 device (having a fill factor of 0.5 and a smaller period value, of P= 6 µm), showing an experimentally-recorded contrast of 16.5 dB between TE and TM polarizations. This behavior is consistent with previous studies on THz polarizers showing that fill factors over 0.5 should be preferred for achieving high extinction ratios between the TE and TM modes in the THz domain [START_REF] Yamada | Terahertz wire-grid polarizers with micrometer-pitch Al gratings[END_REF][START_REF] Deng | Extremely high extinction ratio terahertz broadband polarizer using bilayer subwavelength metal wire-grid structure[END_REF] . The polarization performances of the GeTe-based devices induced by laser irradiation are similar with those of identical devices in which the GeTe was prepared to the crystalline phase by direct thermal heating (supplementary material Figure S10).

By comparing the performance of the GeTe-based P2 polarizer to the device having the same design but fabricated with gold instead of crystalline GeTe, we clearly notice the better performance of the latter (P4 on Figure 4c) which is expected, taking into account the lower conductivity of the crystalline GeTe compared to that of Au. Nevertheless, a gold-based structure is a fixed and passive structure whereas the GeTe structure can be tuned between its amorphous and crystalline states by optical stimuli while showing very good polarization performances of the THz waves. Thus, we have demonstrated the feasibility to develop all dielectric (metal free) THz polarizers using solely chalcogenide phase change materials which presents a broadband response, a high extinction ratio when in metal-like phase (up to 16.5 dB) and almost transparent in the amorphous phase, using an optically-induced non-volatile transition.

To our knowledge, this is the first experimental demonstration of such broadband devices based on GeTe PCM in the THz domain. As the overall performances of such THz devices can be further improved through design adjustments, the presented approach is highly functional, technological disruptive and extremely stimulating for fast THz waves control and manipulation using reconfigurable or switchable THz devices such as frequency-selective surfaces, polarizers, lenses and waveplates, spatial modulators, power limiters etc. [START_REF] Cui | Coding metamaterials, digital metamaterials and programmable metamaterials[END_REF]

Conclusion

In conclusion, we have demonstrated hybrid and all-dielectric THz devices based on GeTe phase change material integration, which can be optically switched between its two different amorphous and crystalline phases, in a reversible way and with zero-power holding capabilities by using nanosecond UV laser pulses. We experimentally realized optically-induced nonvolatile resonance switching by hybrid integration of GeTe with metallic elements within the unit cell of a metamaterial device. The good contrast of GeTe THz properties between the amorphous and the crystalline state, allowed us to design and prove for the first time the possibility to realize an all-dielectric reconfigurable THz devices based solely on GeTe which can achieve a strong modification of the polarization of incident THz waves (extinction ratios of 16.5 dB). The approach offers a great reconfiguration flexibility for multifunctional manipulation of THz waves, and can be easily adopted for implementing optical reconfiguration techniques capable to activate PCM structures within a particular THz device on large areas or on specific regions (through raster scanning or optical excitation of precise elements within the device) in order to program precise, on-demand functionalities of THz waves (arbitrary spatial profile of intensity, phase, and/or polarization distribution). This highly versatile approach that we are proposing, based on non-volatile optically controlled multi-operational THz devices integrating PCMs, will, most certainly, generate stimulating, disruptive developments in the THz domain like field-programmable metasurfaces, all-dielectric coding metamaterials with multifunctional capabilities for THz field manipulation.

EXPERIMENTAL AND METHODS

The THz hybrid metamaterial was fabricated in a cleanroom environment by standard photolithography and deposition procedures using a two-mask levels process. Firstly, a 500-nm thick GeTe layer have been obtained on a 500-µm thick c-cut sapphire substrate using DC magnetron sputtering from a GeTe (50-50 stoichiometry) target in an argon atmosphere. The GeTe elements were patterned using a lithographically-defined photo-resist mask and a wet etching process. The subsequently deposited metallic patterns (20/500-nm thick Ti/Au bilayers) defining the SRR structure were obtained by electron-beam evaporation of the respective metallic elements and an optical lithography step using the lift-off method. The GeTe-based THz polarizer was obtained by patterning a GeTe film using a one-mask lithographically step defining a photo-resist mask and a subsequent dry etching process while the metallic gold-based polarizer was fabricated in a similar matter from a Ti/Au (20-500 nm) film obtained by electronbeam evaporation.

For the optical reversible switching of the GeTe film between the two amorphous and crystalline states we employed the direct irradiation of the obtained GeTe films using pulses from an excimer KrF laser with a wavelength of 248 nm and a pulse duration of 35 ns. Laser pulses with energy densities ~90 mJ/cm 2 where employed for transforming the GeTe from the amorphous to the crystalline state while the reverse transition requires energy densities in the range 185-190 mJ/cm 2 . The conductivities of the GeTe films were measured using a four probes set-up.

The full-wave simulations of the THz devices were performed using the commercially available Ansys HFSS software package. For the GeTe materials parameters, we used the dc conductivities layer extracted in the two states by the four-probe method, while for the permittivity of the amorphous GeTe we used a value of 20. Similar permittivity values were reported previously for different PCM compositions [38] .

The THz characterization of the bare GeTe films and of the different fabricated THz devices was realized using a homemade THz time domain spectroscopy (THz-TDS) system.

Notes

The authors declare no competing financial interest and no conflict of interest.

Supporting Information

  material's THz properties. The transmitted temporal signals for both amorphous and crystalline 500-nm thick GeTe film recorded by terahertz time domain spectroscopy (THz-TDS) measurements are represented on Figure 1c. The corresponding Fast Fourier Transform (FFT)calculated frequency domain of THz transmission of the GeTe film in amorphous and crystalline states (spectra normalized by the substrate THz transmission) show normalized transmissions around 90% for the amorphous phase and of 1.7% for the crystalline state, with relatively constant values over the entire measured domain (Figure 1d). The metallic behavior of the crystalline phase of GeTe is manifested by a strong reflection of the incident THz radiation and therefore a drastically decrease of the amplitude of the transmitted THz pulse.

Figure 1 .crystalline

 1 Figure 1. (a) Amorphous-to-crystalline phase change of a 500-nm thick GeTe (initially amorphous) film obtained on a sapphire substrate (20x 20 mm 2 overall dimensions) using either

  our experiments, the finite elements multiphysics simulations indicate that the temperatures rise within the GeTe layers subsequent to the application of the two energy-dissimilar laser pulses (black curves in Figures2a and band temperature distribution over the entire laser pulse area on supplementary FigureS2) makes possible the reversible change of the GeTe film between the amorphous and crystalline phases. As the optical control of a specific phase of the GeTe material on a large area is critical for the realization of reconfigurable THz devices, we experimentally validated for the first time the reversible amorphous-to-crystalline phase changes of a GeTe layer obtained initially in the amorphous state on a sapphire substrate by recording its two-probe sheet resistance change subsequent to the application of alternating UV laser pulses with distinctive energies (Figure2c).

Figure 2 .

 2 Figure 2. a and b. Finite element simulations of temperature rise within a 500-nm thick GeTe

3c.

  When the GeTe pattern within the SRR gap is amorphous, it is almost transparent to the THz waves and the normal LC resonance of the SRR structure occurs at around 0.26 THz accompanied by higher order resonances at 0.73 and around 1.1 THz (red curve on Figure 3c). When the GeTe is crystalline, it electrically closes the gap of the SRRs and the LC resonance vanished, and different resonance appears at 0.58 THz, corresponding to the mutual coupling between each of the thus modified SRR structures. The electric field distributions of the SRR unit cell for both amorphous and crystalline states of the GeTe material, allowing identifying the origin of a specific resonance are displayed and extensively explained in the supplementary material associated with Figure S5 and Figure S6. The SRR metamaterial based on the hybrid integration of GeTe-metallic structures was further fabricated on a c-cut sapphire substrate

Figure 3 .

 3 Figure 3. (a) Schematic of the THz set-up for evaluating the performances of a metallic split-

Figure 4 .

 4 Figure 4. (a) Measurement scheme of the GeTe-only THz polarizer (b) Close-up optical images

Available: 1 .

 1 GeTe thin film conductivity extraction at THz frequencies 2. Multi physics simulation of thermal distribution within the GeTe film upon laser irradiation 3. Representation of the THz transmission curves of the GeTe material in logarithmic scale 4. Study of the SRR unit cell electromagnetic responses of the GeTe based metamaterial at different resonant frequencies 5. Comparison of thermal and optical activation performances of reconfigurable GeTe-based THz metamaterials on SiO2 substrates 6. Electromagnetic simulation of a gold-based polarizer structure 7. Performance analysis of thermal and optical activation of GeTe-based THz polarizers This material is available free of charge from the ACS Publications website and from the authors.

Acknowledgement

This work was partially supported by the France's Centre national de la recherche scientifique (CNRS), under the PEPS INSIS/DGA project AROMe. M.P. is kindly acknowledging the partial funding of his PhD grant from the French-German Research Institute of Saint-Louis (ISL).

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