Mechanisms underlying pacemaker activity in the sinus node remain controversial, with some ascribing a dominant role to timing events in the surface membrane (“membrane clock”) and others to uptake and release of calcium from the sarcoplasmic reticulum (SR) . Here we discuss recent evidence on mechanisms underlying pacemaker activity with a particular emphasis on the many roles of calcium. There are particular areas of controversy concerning the contribution of calcium spark-like events and the importance of I(f) to spontaneous diastolic depolarisation, though it will be suggested that neither of these is essential for pacemaking. Sodium-calcium exchange (NCX) is most often considered in the context of mediating membrane depolarisation after spark-like events. We present evidence for a broader role of this electrogenic exchanger which need not always depend upon these spark-like events. Short (milliseconds or seconds) and long (minutes) term influences of calcium are discussed including direct regulation of ion channels and NCX, and control of the activity of calcium-dependent enzymes . The balance between the many contributory factors to pacemaker activity may well alter with experimental and clinical conditions, and potentially redundant mechanisms are desirable to ensure the regular spontaneous heart rate that is essential for life. This review presents evidence that calcium is central to the normal control of pacemaking across a range of temporal scales and seeks to broaden the accepted description of the “calcium clock” to cover these important influences. The major determinants of pacemaker activity remain controversial, as illustrated by reviews from the Lakatta and DiFrancesco groups , since little was known on this aspect of pacemaker mechanisms at the time the review was written. In recent years much of the debate concerning the origin of pacemaker activity in the heart has been presented as a choice between two alternative mechanisms, a “membrane clock” in which I(f) activated by hyperpolarization plays the dominant role or a “Ca2+ clock” in which the timing of uptake and release of Ca2+ by the SR is the major determinant of the cardiac rhythm  can contribute to the timing of the membrane clock, that these events are potentially modulated by intracellular Ca2+ in a number of ways and that the relative importance of these pathways might vary under different physiological and clinical conditions. We consider data relating to the role of the Ca2+ clock under a range of conditions, and discuss whether such a clock needs to depend solely on spontaneous Ca2+ sparks or local calcium releases (LCRs) or whether other rhythmic Ca2+-dependent mechanisms should also be taken into account to form a complete picture. It appears that the Ca2+ clock could play a fundamentally important role for the timing mechanism of the cardiac pacemaker under particular conditions, but in many circumstances might play a cooperative interacting role with the membrane clock.This review seeks to discuss broader aspects of the influence of Ca2+ oscillators in smooth muscle and brain, and ideas concerning a Ca2+ clock are not unique to the heart  in SAN cells in comparison to atrial and ventricular myocytes and this allows very small ionic currents to exert a profound influence on membrane potential. In this regard, it is also relevant to consider that SAN myocytes exhibit small cell capacitance (of the order of 30–40 pF) in turn requiring only small currents to charge or discharge the membrane capacitance. The significance of the lack of IK1 in mammalian SAN pacemaker tissue was first demonstrated in an important paper from Irisawa . A “resting potential” of −38 mV was also described in rabbit SA node by Noma and Irisawa . This balance could lead to a “pseudo resting” potential of −30 to −40 mV. Chloride ions cannot be excluded and might also contribute [while a contribution of Cl− to “background current” was not detected by Hagiwara et al. . One possibility is that a major contributor to background conductance is not an ion channel but NCX in a leak mode as first suggested by Kang and Hilgemann  is carried by channels that allow passage of monovalent cations and the kinetics of this time and voltage-dependent pathway are sufficiently slow that it could play a role similar to that proposed for the “leak” or background conductance pathway. Under physiological conditions this pathway allows passage of both sodium and potassium ions, with a reversal potential for the current of approximately −20 mV. The single channel conductance of the I(st) pathway has been reported to be 13 pS, and to have an open probability that is regulated by protein kinase A (PKA). In rabbit SAN this pathway gives rise to an approximately 50 pA current at −50 mV in an isolated SAN myocyte  current activated by hyperpolarization will be discussed in more detail later, but it is worth mentioning that its kinetics are also slow at potentials close to −60 mV and it could feasibly contribute to conductance over many seconds as well as playing a role in “pacemaker depolarization” during a single cardiac cycle. In this context, Verkerk and Wilders have argued that de-activation of I(f) near the overshoot of the action potential, though fast, is not instantaneous, and significant I(f) can be present in their models at the start of diastolic depolarization, persisting from activation of this pathway during preceding action potentials  could influence heart rate through the generation of a background current that is modulated through beat to beat SR Ca2+ content changes   from the SR. CICR occurs as a global release event  arising from ryanodine receptor Ca2+ release channels. The global, substantial rise in subsarcolemmal Ca2+ accompanying CICR during the upstroke of the action potential will nevertheless be accompanied by a major component of Ca2+ extrusion through electrogenic NCX  occurs throughout the cycle including the most negative potential between beats. Thus, there could be a maintained depolarizing influence of NCX  adds two charges to the cell interior, while extrusion of each Ca2+ by NCX adds one charge , and it therefore follows that if the major component of Ca2+ extrusion is via NCX then the average depolarizing current through NCX in the steady state throughout the cardiac cycle must be approximately half that through the total Ca2+ entry mechanisms.The average current through the combined contribution of these three components of NCX is substantial since NCX is thought to be the major method of Ca2+ gradients close to these membranes. The comprehensive experimental data collected in this way led to the conclusion that the stoichiometry of NCX was 3.2 sodium ions to 1 Ca2+, rather than exactly 3:1. The explanation of a 3.2:1 stoichiometry was that there were additional “modes” of NCX. In particular, a mode with slower kinetics that allowed a single external Ca2+ together with a single external sodium ion to be exchanged for a single internal Ca2+, resulting in electrogenic sodium ion leak without net flux of Ca2+, was suggested as a possible contributor to “background current” in pacemaker cells.A fourth mechanism by which NCX could contribute to pacemaker activity is the leak pathway proposed by Kang and Hilgemann  that was2+on both sides of the membrane, but since there is no net movement of Ca2+ this mode is not dependent on the driving force for Ca2+ entry into the cell. It seems likely, however, that such a pathway could not operate if the cytosolic Ca2+ were too low to occupy the internal site on the NCX protein.The sodium “leak” mode of NCX proposed by Kang and Hilgemann requires the presence of Ca2+ balance of the cell (including Ca2+ content of the SR), and controlling Ca2+ concentrations in cytosolic microdomains within the cell that might influence the behavior/sensitivity of intracellular release channels such as ryanodine receptors . Relevant to the present discussion is the thoughtful review by Ottolia et al. . Although the interplay between NCX and degree of Ca2+ loading of the SR has not yet been studied in detail in pacemaker cells, it seems very likely that the mechanisms that operate in ventricular myocytes will have a close parallel in cardiac pacemaker tissue.The discussion above focuses on the importance of NCX as a direct contributor to membrane currents. However, in addition to its direct effects as a charge carrier, NCX may have additional functionally important roles in the context of pacemaker mechanisms. In particular these roles include influencing the overall Caa et al.  on the i2+-sensitive enzymes . Switch to low sodium does suppress the initiation of spontaneous action potentials, but this suppression can take many beats to establish in toad sinus venosus . Sanders et al. (2+ transients), and this cessation was maintained for the full several seconds of exposure. During this time cytosolic Ca2+ as measured by the fluorescent probe fluo-5F fell rather than rose  there may be outward currents through NCX that could complicate interpretation. However, immediate cessation of activity was also seen with smaller reductions of extracellular sodium to 50 mM and even 75 mM , which would be expected after prolonged exposure to abolish SR Ca2+ loading, slowed but did not abolish pacemaker activity, and switch to low sodium under these conditions again caused immediate cessation  during the period of exposure to low sodium. In the absence of CPA, when the SR was functional, Sanders et al consistently observed a reduction in cytosolic Ca2+ when beating was stopped by the switch to low sodium even though the major mechanism for Ca2+ extrusion was inhibited, and this was thought to be accompanied by SR Ca2+ uptake (see later).Another experimental point arising from the rapid switch experiments is that immediately after the switch back from low sodium to normal sodium the spontaneous rate was restored, but was transiently greater than that observed under control conditions  was first described by DiFrancesco and reviewed in DiFrancesco . SimilarTo answer the question whether or not I(f) is essential for pacemaking, it would be helpful either to block the current pharmacologically, or use the techniques of molecular biology to knock down expression of the channel.+ was observed to cause close to complete blockade of I(f) without effects on voltage-gated Ca2+ and potassium currents. This concentration reduced pacemaker rate by about 30% but did not stop the initiation of action potentials , and concluded that I(f) currents played an important role during the pacemaker depolarization, and that these currents were larger than would have been predicted by conventional voltage-clamp experiments applying only hyperpolarizing pulses, but that other inward currents besides I(f) contribute during this phase.There has been discussion in the past about whether the I(f) current is sufficiently large to make an important contribution to pacemaking, particularly in the context of conventional experiments in which the cell is held at a constant negative potential (commonly −40 mV) and hyperpolarizing voltage steps are applied. Although large currents are recorded when the cell is hyperpolarized to potentials that are not often experienced in normal physiology (such as −80 or −100 mV), the currents recorded at potentials less negative than −70 mV are small. Convincing evidence for a role of I(f) under conditions that approximate those of normal pacemaker activity is provided by “action potential clamp” experiments  in the mouse show similar effects in that as the severity of action increases to a maximal level the effect on spontaneous rate in the SAN appears to plateau at about a 50% reduction in rate.There may be other functions of I(f) current in the SAN than determining rate. HCN1 and HCN2 proteins are also present in SAN, and show different kinetics of activation and de-activation compared to HCN4  decrease in current amplitudes, while under the conditions of their experiments 10−7 M caused an increase. In experiments in which the cytosol was perfused with different concentrations of Ca2+, it appeared that a log(concentration)-response curve could be established showing increases in the amplitude of I(f) over the range of Ca2+ concentrations 10−10–10−6 M with the steepest part of the curve covering the range 10−8–10−7 M. Although the authors concluded that there was a direct effect of Ca2+ on I(f) since the effects of Ca2+ were not abolished by the calmodulin inhibitor calmidazolium, it seems likely that the concentration of this compound (10−6 M) was not sufficient to cause effective inhibition. This view is supported by the observations of Zaza et al. (2+ on I(f) in experiments on inside out patches. Rigg et al. . In addition, it is observed that under most conditions ryanodine slows but does not stop pacemaker activity and as expected this also suppresses LCRs . Note that when membrane potential is clamped at a fixed level close to the most negative potential following a period of normal spontaneous beating it is observed that Ca2+ transients can occur with a timing and frequency which is close to the frequency and timing that would have been adopted by spontaneous action potentials if firing had not been interrupted  could provide a driving mechanism for pacemaker activity, but may not be essential for pacemaker activity. The observation that spontaneous pacemaker activity in the form of action potentials is slowed but not stopped . The observed oscillations recorded during the development of the effect are best explained by a Ca2+ clock that is driving electrical activity presumably through NCX.Although under most conditions, ryanodine does not stop spontaneous activity, we have sometimes observed cessation when we have used Ca2+ clock  is correlated with the timing of the action potentials on the surface membrane recorded when spontaneous electrical activity is allowed to occur . The SAN region was studied using confocal microscopy and the Ca2+ probe fluo-4 at room temperature. The SAN from RyR2R4496C mice showed slowed pacemaker activity and reduced response to catecholamines as compared to myocytes from WT. There were also frequent pauses in the generation of spontaneous action potentials, while the overall frequency of Ca2+ sparks/LCRs was increased. Experiments were also carried in SAN preparations loaded with rhod-2 to measure Ca2+ and di-4-ANEPPS to measure membrane potential. Pauses interspersed with trains of spontaneous events were observed for both the Ca2+ and voltage signals. The pauses were associated with unusually high cytosolic Ca2+ concentrations, and it was suggested that the pauses may have resulted from inactivation of the L-type Ca2+ channels.An abnormality concerning ryanodine receptor Ca2+ handling is modified in mice lacking the SR protein calsequestrin (−/−Casq2). These mice showed bradycardia and beat to beat heart rate variability. SAN myocytes isolated from −/−Casq2 mice showed an unstable rate with frequent pauses between “trains” of action potentials and the abnormal behavior was associated with abnormal Ca2+ release from the SR. In particular the pauses were accompanied by elevated levels of diastolic Ca2+ concentration  inhibition of the SAN upstroke ICaL current from partially inactivated L-type Ca2+ channels, and/or (ii) decrease in the SR Ca2+ content below a threshold level required for the generation of spontaneous SR Ca2+ releases.”A recent paper from the Federov laboratory . This “store overload-induced Ca2+ release” (SOICR) is modified in ventricular myocytes when a point mutation is introduced in the proposed calcium sensing gate  on spontaneous action potentials in guinea pig SAN myocytes , but suppression of CaMKII activity by the peptide did cause a substantial reduction of the positive chronotropic response to isoprenaline. The responses that were reduced by inhibition of CaMKII included the amount of Ca2+ loaded in to the SR, the frequency of Ca2+ sparks/LCRs and the slope of the diastolic potential. However, interestingly the enhancement of L-type Ca2+ current amplitude by isoprenaline was maintained when CaMKII activity was suppressed by the inhibitor peptide. Another mouse model also showed little or no effect of CaMKII knockout on resting rate  is regulated by a Ca2+-calmodulin dependent mechanism that is not CaMKII was presented by Rigg et al. (2+ regulated adenylyl cyclase could provide such a mechanism. Application of the Ca2+ chelator BAPTA reduced I(f) as did calmodulin inhibitors including W7. Although W7 is known to have non-specific effects  although it was also pointed out that such a pathway might well have additional effects via PKA and phosphorylation of additional protein targets. Younes et al.  for the regulation of L-type Ca2+ currents has recently been demonstrated in atrial myocytes, and the observations may have relevance to SAN function  in stimulating adenylyl cyclases and regulating the magnitude of L-type Ca2+ currents. It is speculated that similar mechanisms may operate in the SA node.The importance of the Ca2+ dependent enzymes have the potential to influence pacemaker activity but have not yet been studied in detail. These include phophodiesterases, phosphatases and P21-activated kinase (PAK) enzymes that regulate phosphatases  in SAN myocytes since evidence supports actions of NO donors on pacemaker function  is regulated by a Ca2+-dependent process thought to be CaMKII in guinea-pig SA node. This current was found to be decreased when the cytosolic Ca2+ concentration was reduced from 10−7 to 10−10 M (buffered by EGTA in a ruptured patch conditions). When the cytosolic Ca2+ concentration was maintained at 10−7 M, addition of calmodulin via the patch pipette increased the current. Inhibitors of CaMKII (autocamtide-2 inhibitor peptide and the less selective KN-93) substantially reduced the current, while in the presence of KN-93 there was no effect of calmodulin addition. Taken together these observations provide convincing evidence that IKs is modulated by cytosolic Ca2+ and that CaMKII plays an important role in this modulation.Xie et al.  have recKr is thought to provide the major voltage-gated potassium current pathway . More evidence against Ca2+ regulation of IKr is provided by Wu and Anderson  a time when “global” spontaneous Ca2+ release would also have occurred. However, it is also possible that CICR can, and perhaps often does, occur without the accompaniment of spontaneous Ca2+ release. In this case Ca2+ released by CICR from the SR would be expected to drive a significant component of NCX that is independent of the Ca2+ clock.As was mentioned above, membrane and Ca2+ released from the SR by CICR that is independent of the Ca2+ clock could also drive Ca2+-dependent enzymes. These include CaMKII, AC1 and AC8 as described above. A role for SR-released Ca2+ (sensitive to ryanodine and thapsigargin) leading to increased L-type Ca2+ currents by a mechanism involving Ca2+-stimulated adenylyl cyclases was recently demonstrated in atrial myocytes  (by elevation of cAMP) and other ion channels such as I(st) and voltage-gated potassium channels (regulated by PKA).Ca2+ channels are a major component of the membrane oscillator providing explosive positive feedback for the upstroke of the action potential which in turn initiates repolarization as a consequence of activation of voltage-gated K+ channels. The positive feedback for activation of L-type Ca2+ channels underlies the fast upstroke and rapid spread of the action potential that synchronizes Ca2+ entry and CICR across the cell. The Ca2+ entry via L-type Ca2+ channels is a major determinant of the degree of Ca2+ loading of the SR. The action potential signal is essential for conduction of the pacemaker activity to neighboring myocytes. In addition, it appears that current through CaV1.3 channels can contribute to the late stage of diastolic depolarization .Voltage-gated L-type Ca2+ chelators in single cells, since in multicellular preparations it is difficult to be sure that uptake of Ca2+ chelator is complete. It is recognized that isolated myocytes might behave differently from those in the intact myocardium for a variety of reasons including different mechanical influences, lack of paracrine influences from neighboring cells and lack of electrical connections to adjacent myocytes.At the present time, it is probably only possible to investigate the effects of Ca2+ , spontaneous activity became unstable and ceased in the majority of cells several minutes after achieving the whole-cell configuration.” Another approach to apply the Ca2+ chelator is to expose the cells to the acetoxymethyl (AM) ester of the Ca2+ chelator applied in the extracellular solution. The membrane permeant ester enters the cell and the active chelator is liberated following the action of esterases in the cytosol. Application of EGTA-AM or BAPTA-AM in this way causes cessation of beating  that would be consistent with the chelating effects of BAPTA. Maltsev et al. . In support of this suggestion they note that in Figure 5 of Himeno et al. there is an upward deflection on the contraction trace following breakthrough that would be consistent with a small developing contraction associated with entry of Ca2+ from the extracellular solution via the leaky seal into the cytosol. Contribution of leakage current around a seal in sustaining pacemaker activity has been considered by other authors, and can make an important contribution under some conditions  acting on β-adrenoceptors and of sympathetic nerve stimulation are treated as equivalent, although some have argued that this is over-simplistic , the pacemaker period would only be reduced to 90% of the value before the increase in Ca2+ current (or a rate increase of 1/0.9 giving close to a 10% increase in rate). The increase in upstroke velocity would still be functionally useful in increasing the rate of conduction of the pacemaker message in the form of the action potential to surrounding tissue. However, in terms of the rate change it seems very likely that an important aspect of the β-adrenoceptor mediated increase in Ca2+ current is to increase entry of Ca2+ which in turn influences many of the Ca2+-stimulated processes that have been described above.A major effect of β-adrenoceptor stimulation by isoprenaline is to increase Ca2+ entry were to double, the cell would need, in the steady state, to remove twice as much Ca2+ and if the major mechanism for achieving this were NCX in its normal 3:1 mode then NCX would also double . Although this seems very likely to be the case, one additional point concerns another effect of β-adrenoceptor stimulation via cAMP and PKA. This is phosphorylation of phospholamban which will lead to increased Ca2+ uptake by SERCA into the SR. This might lead to a greater fraction of the Ca2+ to be taken up by the SR (or at least a greater rate of uptake into the SR depending on how quickly the Ca2+ is released), but in the steady state increased Ca2+ entry through the surface membrane must be accompanied by increased extrusion. In terms of the Ca2+ clock, the kinetics of Ca2+ uptake are expected to have a profound effect, and this might be one factor underlying the reduced effect of isoprenaline after ryanodine  could drive many Ca2+-dependent processes, such as Ca2+-stimulated enzymes and these are expected to contribute to the positive chronotropic effect. The Ca2+-stimulated enzymes include CaMKII as discussed by Wu and Anderson , I(st), IKs, IKr and possibly NCX, as well as ICaL.The additional CaAnderson , as wellKACh channels. The effects on I KACh channels are blocked by tertiapin Q. During parasympathetic nerve stimulation there is a rapid phase of slowing that is sensitive to tertiapin Q and therefore IKACh  was inhibited by 2 mM Cs+, and IKACh was inhibited by tertiapin Q, 1 μM ACh was still able to reduce pacemaker frequency by 72%. Under these conditions, there was a good correlation between the reduction in beating rate and the amplitude of the Ca2+ transient. In addition ryanodine and exposure to BAPTA-AM both facilitated ACh mediated slowing. They also showed that inhibition of the Ca2+ transient by ryanodine (3 μM) or BAPTA-AM (25 μM) exaggerated the ACh-mediated inhibition of cAMP content, consistent with the proposal that Ca2+ affects cAMP production in SAN cells. There may be additional effects of ACh on phosphodiesterases . The idea that the I(f) pathway is essential for pacemaker depolarization cannot be completely excluded because of the uncertainties concerning whether the pathway is totally suppressed in experiments using drug blockade or techniques of genetic modification, but it seems likely that I(f) is more an important modulator than an essential component of the timing mechanism. Ca2+ sparks/LCRs can sometimes contribute to the later stages of pacemaker depolarization preceding the action potential but are not essential for pacemaking. However, “global” properties of Ca2+ uptake and release by the SR across the whole myocyte  can give rise to a “Ca2+ clock” . The “Ca2+ clock” arising in this way frequently plays a modulatory rather than essential role, though there may be conditions in which it is absolutely necessary to re-initiate spontaneous activity if this were to stop. This would mean that Ca2+ released from the SR and driving electrogenic NCX may be essential to re-start spontaneous activity under particular conditions. Although a Ca2+ clock is not essential for pacemaker activity, it seems probable that under normal conditions there will be a cooperative interaction between membrane and Ca2+ clocks that has been referred to as a coupled clock mechanism. Under pathological conditions when the Ca2+ clock is not synchronized with the membrane clock the Ca2+ clock can lead to disturbed pacemaker rhythms. In normal physiology, Ca2+ is thought to drive a depolarizing influence of NCX throughout the cardiac cycle, including during the most negative potential of the pacemaker myocytes, at least under most conditions when the cytosolic Ca2+ concentration remains substantially higher than 100 nM. Ca2+ also plays important roles in maintaining and regulating pacemaker activity by activating a variety of Ca2+-dependent enzymes including CaMKII, AC1, and AC8. Ca2+ seems to exert a direct or indirect influence on most if not all of the proteins providing ionic pathways in the surface membrane.The above discussion leads to the view that pacemaker activity in the heart normally requires L-type CaThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Paramecium attempts to retreat from the dead-end of a long capillary that is too narrow for turning. After many trial-and-error episodes of short-term backward swimming (SBS), which is the conventional avoidance behavior exhibited in free swimming when an obstacle is faced, long-term backward swimming (LBS) that lasted five to ten times longer was developed. LBS may have a beneficial effect for complete withdrawal from the capillary space, although in our experiment it was impossible for the organism to do so due to the capillary length. In order to identify a physically possible mechanism for LBS, we propose model equations for the membrane potential of Hodgkin–Huxley type, which describe the control of ciliary movement. The physiological implications and physical mechanism of the development of LBS are discussed.We have observed how the ciliate  It is not trivial to study how unicellular organisms tackle a problem, as they often try different behaviors instead of previously unsuccessful behaviors to solve dilemmas. One of the most important issues to be addressed here is what types of behavior can be induced and how such behavioral options appear in terms of information processing in the cell. It is interesting to identify whether mechanisms of information processing are based on mechanical equations of motion, because in some sense the physical basis of an adaptation or learning process could be suggested  when an obstacle is faced during free swimming, we find emergent new avoidance behavior: long-term backward swimming (LBS) that lasted five to ten times longer than SBS. We next analyze the dynamical properties of the model equation for the membrane potential and consider a possible mechanism for the LBS. Finally, we discuss the physiological implications of episodes of SBS and LBS and propose a physical mechanism for the development of these two types of behavior.In this report, we describe how the ciliate Paramecium were grown at room temperature in a decocted liquid extracted from straw. This was exchanged with the assay medium  by using a narrow glass pipette 3 h before the test.Specimens of Paramecium in a dead-ended capillary of length 40–50 mm. The capillary had an internal diameter of 0.08 mm, which is about twice the width of the protozoan body, and was used in the horizontal orientation. At the beginning of a trial, an individual Paramecium was collected with a narrow glass pipet and placed in the capillary filled with the assay medium. The ends of the capillary were then closed with mineral oil in order to confine the Paramecium inside. Each specimen was used only once in all experiments.Figure The specimens were observed using a stereomicroscope (Olympus SZX16). Microscopic video images were taken using a CCD camera and recorded by a video camera. The video images were saved in AVI format on a personal computer before analysis with the free software NIH imageJ.Paramecium bumped against the capillary end, the specimen began to swim backward before reversing direction and swimming forward again. This behavior was repeated many times. Shortly after the first collision with the capillary end  with distances of 0.3–1 mm and periods of 2–10 s. The second type is long-term backward swimming (LBS) with distances of 3–4 mm and periods of 10–15 s.These results imply that Paramecium in a dead-ended capillary. Figure + concentration; at 2, 4, and 8 mM there were peaks at 3–9 s  might carry Ca2+ current after the action potential has gone, even though Ca2+ channels with fast and slow time constants are the same physically. Hinrichsen et al.  is the outward electric current, which corresponds to the mechanical stimulus applied by bumping against the end of the capillary in our experiment. ICa is the Ca2+ current, IK is the K+ current, and Ileak is the leak current. These three quantities can be expressed as:gCa and gK are the ionic conductance (the rate of passage of ions), the definition of which will be given later. gleak is the maximum ionic conductance of the leak ion channel. ECa and EK are the equilibrium potentials of Ca2+ and K+, respectively. These two quantities can be described by the Nernst equation (Equation 5) for the diffusion of electric charge. Eleak is the equilibrium potential of the leak ions, and is given approximately by Equation (6).Here R is a gas constant, T is the absolute temperature, z is the ionic valence, and F is the Faraday constant. The parameters [Xz+]o and [Xz+]i are the extracellular and intracellular concentrations of Xz+ ions, respectively. gLCa and gLK are the ionic conductance of the Ca2+ and K+ channels, which are insensitive to changes in the membrane potential.Here, 2+ and K+ currents are controlled by the conductance of the Ca2+ and K+ channels, respectively. The Ca2+ conductance (gCa) is described using the activation gate factor m and the deactivation gate factor h. The K+ conductance (gK) is described by the activation gate factor n. These factors are the variables that represent the gating of the voltage-sensitive ionic channels.The Ca2+ channel, because the duration time of backward swimming in Paramecium depends on the time for which Ca2+ current flows. We assume that the Ca2+ channels are in one of two modes, with a fast or slow time constant with respect to the deactivation process. We now introduce two more parameters, gCaS and P(t), to represent the conductance and occurrence rate of Ca2+ channels with the slow time constant, respectively. We include these parameters in the original equation for Ca2+ conductance in the Hodgkin–Huxley-type model for Paramecium reported by Naitoh and Sugino (P(t) will be given later. The total Ca2+ conductance is given as the sum of the products of the occurrence rate with the conductance of the Ca2+ channels with fast and slow time constants. The resulting equations for ionic conductance are as follows:gCaF is the Ca2+ conductance with the fast time constant. gCa and gK are the maximum Ca2+ and K+ conductance of the membrane when all the Ca2+ and K+ channels are activated, respectively. jF and jS  are the gate variables for the channels with fast and slow time constants. The equations for the gate variables are as follows:x is the opening rate of the gates. αx and βx are functions of the membrane potential, which are determined by fitting the rate constants using analytical expressions as follows:The occurrence of LBS requires long-term activation of the Cad Sugino . The defγ ≤ 1) is a constant. For each fixed V in Equation (11), the time constants ) and equilibrium values ) of the gate variables are given as follows:Here, γ (0 ≤ P (0 ≤ P ≤ 1) is the rate of the Ca2+ channel with the slow time constant in the deactivation process. The Ca2+ channels with the fast and slow time constants are the same physically, and it is assumed that the mode of the Ca2+ channel is changed by the stimulus-induced membrane potential response. The relevant equation is as follows:ti is the finishing time of the ith stimulation, ΔP is the maximum rate of the Ca2+ channel changing the mode, and τP is the decay time constant of P.et al and are as follows: Cm = 2 [μF/cm2], ECa = 125 [mV] , EK = −57 ~ 0 [mV] , gLCa = 0.1 [mS/cm2], gLK = 0.8 [mS/cm2], T = 298 [K], R = 2.0 , F = 23,000 , gCa = 0.6667 [mS/cm2], gK = 1.3333 [mS/cm2], gleak = 0.42 [mS/cm2], γ = 0.005–0.05, CP = 0.2, τP = 700.0, V(0) = −30 [mV], mF(V(0)) = 0.2911, mS(V(0)) = 0.01, hF(V(0)) = 0.162, hS(V(0)) = 0.1, n(V(0)) = 0.0163, P(0) = 0, Iapp = 100.The numerical parameters and initial values for our proposed model are based on the experimental conditions and the measurements of membrane potential reported by Naitoh 2+ current, the gate parameter mS, and the rate of the Ca2+ channel with the slow time constant when the outward electric current is injected periodically. Here it was assumed that [K+] = 4 mM (EK = −40 mV). When the outward electric current was injected before all the Ca2+ channels with the slow time constant had returned to the mode with the fast time constant (P > 0), the rate of the Ca2+ channel with the slow time constant increased gradually. This resulted in an increase in magnitude of the Ca2+ current after the action potential had passed.We simulated the membrane potential responses when the outward electric current is injected periodically, because LBS occurred by repeating the colliding with the end of capillary. Figure 2+ current flows (τb) on the number of trials (the number of collisions with the capillary end). The duration time is defined as the time for which the Ca2+ current is smaller than a threshold value. The duration time increased with the number of trials and eventually became saturated. The time for which Ca2+ current flows corresponds to the duration time of backward swimming in Paramecium, and the results of this simulation represent the behavior displayed during regions T2 and T3 in Figure Figure 2+ current flow on the equilibrium potential of the K+ ion. The threshold for this duration time was set to −0.1 (nA) based on the experimental data reported by Naitoh and Kaneko . As shown in Figure h, which influences the duration time of Ca2+ current flow, reached its maximum value when the membrane potential was −20 mV, which is the same value of the resting potential as EK and ECa were −40 and −125 mV, respectively. Therefore, when the threshold value for the duration time is sufficiently low, the Ca2+ current-flow duration time is maximum at EK = −40 mV as shown in Figure 2+ current-flow duration time decreases with increasing EK. The latter case was experimentally observed, as illustrated by the result in Figure The maximum value of the duration time of Ca2+ current, which is invoked by the Ca2+ channel with slow time constant, we analyzed the relationship between the change of membrane potential and the move of nullcline.To understand the mechanism of the long-term Ca2+ channels that have slow time constants with respect to the deactivation process. Figure Figures V nullcline  to (2), there was little change in the equilibrium values of the gate parameters  when the magnitude of the outward electric current was large enough. Therefore, the Ca2+ channels remained in the activated state.When the membrane potential became larger than 20 mV, V nullcline shifted from (2) to (3). As the time constant of mF was smaller than those of hF and n, the decrease of mF occurred before the decrease of n and the increase of hF. The K+ current was still carried after the Ca2+ current had stopped, and the membrane potential became hyperpolarized. The V nullcline then shifted from (3) to (4), and the membrane potential neared the resting state. Paramecium swims backward while Ca2+ current flows due to activation of the Ca2+ channels.The membrane potential decreased sharply after the outward electric current was gone, and then the V nullclines shifted successively from (5) to (9) in the presence of the Ca2+ channel with the slow time constant (P = 1). In Figure mS and hS in the deactivation process of the Ca2+ channel. As the time constants of mS and hS were larger than that of n, the Ca2+ current still flowed due to the slow convergence of m and h after the K+ current had stopped. Although the Ca2+ current flowed while the Ca2+ channels were activated, because the Ca2+ current was small [(7–8) in Figure 2+ channels with the slow time constant, our proposed model reproduces the Ca2+ current that invokes backward swimming in Paramecium after the action potential is gone.As shown in Figure Paramecium is well studied. Therefore, the behavior of Paramecium is a good example for the study of physical mechanisms of behavior in organisms in general. In this report, we have analyzed the mechanism of LBS using mechanical equations.The relationship between the membrane potential, which invokes ciliary motion, and the behavior of Paramecium bumps against the end of the capillary. As SBS is the short-term response with a period in the range 1–2 s, this is the conventional avoidance response when an obstacle is faced during free swimming. LBS is the long-term response with a period of 5–15 s, and thus represents a novel type of avoidance behavior. We have shown that LBS is invoked by the long-term activation of Ca2+ channels, because LBS occurred even when the potassium equilibrium potential was zero. Our proposed mathematical model reproduces the experimentally observed behavior.Our experiment shows that two types backward swimming are exhibited (SBS and LBS) when Paramecium. The Hodgkin–Huxley-type equation that describes the membrane potential for behavior in Paramecium does not obey the all-or-nothing law such as that in nerve cells because the equation has one stable state, which depends on the extracellular ionic concentration and the presence or absence of an outward stimulus current. Although one might consider that the membrane potential response can easily be determined, in practice a number of different responses can be expected due to the presence of three variables with different time scales in the non-steady state. We have shown that the long-term non-steady state imposed by the presence of Ca2+ channels with a slow time constant in the deactivation process invoke LBS, which is caused by an increase in the duration time of Ca2+ current flow.Our mathematical modeling allows us to discuss possible mechanisms for the development of LBS in 2+ channels in living organisms are unclear. However, channel proteins have rather complex structures and are involved in a number of different reactions, thus it is feasible that Ca2+ channels can have slow time constants. Although the experimental conditions differ from our experiment, Hennessey and Kung (2+ currents in Paramecium shows fast (10 ms range) and slow (10 s range) kinetics. It is expected that these properties will become more clearer through further electrophysiological measurements and the study of molecular dynamics.The properties of time constants related to Caand Kung  have rep2+ channel from the fast time-constant mode in the deactivation process to the slow time-constant mode. As this is a reversible physiological change, LBS in Paramecium is a type of adaptive behavior in a narrow space. We suggest that small changes in parameters such as the time constant of channel proteins enable the organism to adapt when confronted with problematic conditions.Our model proposes that the development of LBS is invoked by a change in the CaParamecia fold their bodies very tightly and somehow turn even in narrow spaces  “avoiding behavior” that was induced by the repetitive collisions to the top end, and (2) “reversal of swimming direction” that resulted in the success of escape .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


At the moment, one of the shortcomings of available technologies is their incapability of imaging multiple fast phenomena while controlling the biological determinants involved. In the near future, ultrafast deflectors can be used to rapidly scan laser beams across the sample, performing optical measurements of action potential and Ca2+ release from multiple sites within cardiac cells and tissues. The same scanning modality could also be used to control local Ca2+ release and membrane electrical activity by activation of caged compounds and light-gated ion channels. With this approach, local Ca2+ or voltage perturbations could be induced, simulating arrhythmogenic events, and their impact on physiological cell activity could be explored. The development of this optical methodology will provide fundamental insights in cardiac disease, boosting new therapeutic strategies, and, more generally, it will represent a new approach for the investigation of the physiology of excitable cells.Advances in the development of voltage sensitive dyes and Ca This process is known as excitation-contraction coupling (ECC)  Bers, . ECC abnC) Bers, . AlthougEvery heartbeat is triggered by a depolarizing event, the action potential (AP), which is spontaneously generated in the cardiac pacemaker and propagates through the conductive tissue and the working myocardium. In order to achieve a synchronized activation of all cardiomyocytes, the AP spreads from cell to cell throughout the lattice of interconnections called gap-junctions. Gap-junctions are specialized membrane structures, predominantly located at the longitudinal interdigitated ends of cardiomyocytes . Indeed, during each AP, depolarization-activated sarcolemmal Ca2+ channels open and allow a small amount of extracellular Ca2+ to enter the cell, thus generating an inward current (ICaL). Ca2+ entered via ICaL triggers the opening of Ca2+- activated SR Ca2+ channels , which are clustered to form Ca2+ release units and allow a massive release of Ca2+ from the SR. This cascade phenomenon, referred to as Calcium Induced Calcium Release (CICR), preferentially occurs in the TATS membrane, where most of Ca2+ channels are located and the spatial coupling with the SR cisternae is tightly established  , which is manly located in TATS membrane, leading to a slow diastolic depolarization  which may reach the threshold for a spontaneous premature AP  and they branch in the longitudinal direction  to form a complex system, named transverse axial tubular system (TATS). Indeed, the TATS of cardiac myocytes has been shown to be a network of interconnecting tubules in which the longitudinal elements  bridge 4–5 consecutive Z-lines and, similarly to the transverse components, form junctions with the SR  Marks, . DADs ar2+ waves start near the damaged region and propagate in a coordinated fashion into adjacent tissue  regions of the network Kleber, . The siz2+ fluxes and AP propagation. This investigation can be made possible thanks to the development of innovative optical methods capable of probing and stimulating cardiac samples in a multiple site modality EPPTEA. The random access multi-photon microscope . Moreover, the ability of ultra-fast scanning to stimulate multiple distant TATS regions within the cell will help understanding the amount of single spontaneous events that are required to generate a cell-spanning Ca2+ wave.The next generation of imaging systems can be also implemented with additional ultrafast scanning head for local Ca Rubart, . On the  Rubart, . In fact2+ waves propagation is caused by local Ca2+ release from the SR, followed by diffusion and subsequent activation of adjacent SR Calcium Release units, generating a cascade of events. Ca2+ waves are accompanied by DADs  that ranged from 0.1 to 15 mm/s, a speed that is too slow to be determined by active (1 m/s) or electrotonic membrane conduction (0.1 m/s)  or cyclopiazonic acid (to decrease Ca2+ buffering) can be administrated to enhance Vprop. Conversely, pharmacological blockers of cardiac RyR, such as JTV519 (Elliott et al., 2+ dynamics.Following a similar approach described above the same excitation wavelength can be used to simultaneously detect voltage and Ca2+ has been reported, disclosing in details their spatio-temporal relationship. The scenario is now getting even more exciting. Indeed, light can be employed even to control the two main determinants of cardiac function by using photosensitive ion channels and light-dependent Ca2+ caged compounds.Light microscopy together with functional fluorescent indicators is speeding up the comprehension of biological mechanisms in excitable cells. Cardiac research has been profoundly affected by the development of optical methodologies that allow non-invasive and fast imaging. Ultrafast scanning modalities have made possible multisite membrane potential recordings across single cardiomyocytes and multicellular preparations. More recently, multisite recording of membrane potential and CaThe opportunities that light may provide on cardiac research are not yet fully exploited. New light-based technologies can disclose cardiac function at different levels, from single molecule to the whole heart, as well as provide possible implications for future therapeutic applications.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background. To compare the early outcome of combined SMILE and collagen crosslinking (SMILE Xtra) with SMILE. Method. Prospective, comparative interventional study of 21 eyes receiving SMILE Xtra using a low energy protocol and 32 control eyes receiving SMILE only. The outcomes were compared at 1, 3, and 6 months postoperatively. Results. Both groups had myopia with spherical equivalent refraction (SEQ) > 4.00 D. The SMILE Xtra group had thinner preoperative central corneal thickness and residual stromal bed thickness (p < 0.021). At 6 months, no eyes lost more than 1 line in corrected distance visual acuity. The safety index was 0.96 ± 0.06 and 1.00 ± 0.00 in SMILE Xtra and control, respectively (p < 0.001). 89% and 94% of eyes were within ±0.50 D of target refraction, respectively, with the mean error in SEQ correction being −0.17 ± 0.26 D for SMILE Xtra and +0.03 ± 0.25 D for control (p = 0.021). The efficacy index was 0.88 ± 0.13 and 0.97 ± 0.06, respectively (p = 0.005). Conclusion. SMILE Xtra had good overall safety profile and predictability at 6 months. However, when compared with control, the safety index and efficacy index were statistically significantly lower in the early postoperative period. Small-incision lenticule extraction (SMILE) is a recently developed corneal refractive procedure that has been shown to be safe and effective in correcting myopia –5. The pIn LASIK, a rare but devastating complication is iatrogenic corneal ectasia, where there is progressive corneal steepening and thinning resulting in increasing myopia and astigmatism and decrease in visual acuity. Although post-LASIK ectasia can now be effectively treated with crosslinking , advanceA total of 21 consecutive eyes from 12 patients who have undergone SMILE Xtra between January and March 2015 at a private ophthalmic center in Hong Kong were recruited prospectively. A random sample of 32 eyes from 18 patients receiving SMILE alone (without CXL) during the same period were also recruited to serve as control. Inclusion criteria of the study included moderate to high myopia with baseline spherical equivalent refraction (SEQ) higher than 4 diopters, and all eyes had a plano target refraction. All patients had signed an informed consent before the refractive procedure. The study was approved by the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster (Reference number: UW 15-226) and adhered to the tenets of the Declaration of Helsinki.All patients underwent complete ophthalmic evaluation before the procedure and had no ocular comorbidities other than myopia and myopic astigmatism. All patients had preoperative CDVA of 20/20 or better. The ectasia risk factor score was also calculated based on the system proposed by Randleman, which took topographic pattern, age, CCT, RSB thickness, and SEQ into consideration . The addμm; cap diameter, 7.0–7.5 mm; lenticule diameter, 6.0–6.5 mm with a transition zone of 0.1 mm; cut energy, 1.4 μJ; spot and tracking distance, 2.0–3.0 μm. The back of the intrastromal lenticule was created from periphery to center of the cornea. The anterior lamellar cut was subsequently created from center to periphery of the cornea, which extended toward the surface to create a 2 mm incision located at 12 o'clock position (for left eye) or 10 o'clock position (for right eye), from which the stromal lenticule was extracted. A thin blunt spatula was used to separate the lenticule, which was then grasped with a pair of forceps and removed. The corneal interface was flushed with balanced salt solution. The same normogram was used for both groups. For cases with combined cross-linking, after lenticule removal, 0.22% riboflavin with saline  was instilled through the small incision into the interface and allowed for a soak time for 45 seconds. An additional 2 mm incision at the opposite site was created to allow for complete irrigation of the riboflavin solution. This was followed by ultraviolet A irradiation at 18 mW/cm2 for 45 seconds  using the CXL-365 variosystem .The same surgeon (GC) performed all the surgery under topical anesthesia. SMILE was performed using the 500-kHz VisuMax femtosecond laser  using an established technique . The folPostoperative medications included topical tobramycin 0.3% and dexamethasone 0.1% ophthalmic suspension four times a day for 1 week. Preservative-free artificial teardrops were used for 3 months postoperatively. All patients were examined preoperatively and at postoperative day 1, week 1, month 1, month 3, and month 6. Assessments included monocular uncorrected distance visual acuity (UDVA) and CDVA, manifest refraction, and slit-lamp and fundus examination. Corneal thickness was measured with a scanning-slit topography . Efficacy index was calculated as the ratio of postoperative UDVA over preoperative CDVA and safety index was determined as the ratio of postoperative CDVA over preoperative CDVA. Outcome measures included postoperative UDVA, CDVA, corneal thickness, efficacy index, safety index, and the predictability of the correction. Any complications arising from the procedure were reported.Statistical analysis was performed using SPSS version 21 .The patient demographics will be reported with descriptive statistics.U test for age, Fisher's exact test for gender, and linear mixed model analysis for other parameters to account for the effect of both eyes from the same patients. p < 0.05 was considered statistically significant.The parameters between SMILE Xtra and control group were compared with Mann-Whitney p = 0.966). The baseline parameters of both groups were shown in μm in SMILE Xtra and 311 ± 30 μm in control (p < 0.008). 14 SMILE Xtra eyes (66.7%) had an ectasia risk factor score of 4 or higher (high risk) and 4 eyes (19.0%) had a score of 3 (moderate risk). In the control group, 5 eyes (15.6%) were of high risk and 5 eyes (15.6%) had moderate risk.A total of 21 SMILE Xtra eyes (12 patients) and 32 SMILE eyes (18 patients) were included for analysis. All eyes aimed for a plano correction. The average age was 28.1 ± 6.1 in SMILE Xtra and 27.5 ± 5.2 in control . The mean spherical equivalence reduced from −7.08 D to −0.17 D at 6 months postoperatively in SMILE Xtra and from −6.56 D to +0.03 D in control. At 6 months, 94% and 97% of eyes in the SMILE group were maintained within ±0.50 D in the SEQ and astigmatism correction, respectively. In SMILE Xtra group, 89% and 94% of eyes were within ±0.50 D in SEQ and astigmatism correction, respectively . The meaμm, which was similar to control .The baseline CCT and RSB thickness were thinner in the SMILE Xtra group. However, at 6 months postoperatively, the CCT in SMILE Xtra group was 420 ± 39 Studies have demonstrated that SMILE is a safe procedure and has comparable outcome with femtosecond-LASIK , 4, 6–8.2. The rationale for adding simultaneous crosslinking for the patients in our SMILE Xtra group was similar to LASIK Xtra. Our SMILE Xtra eyes had a significantly thinner CCT and RSB thickness than the control, with an increased risk of ectasia. We applied the riboflavin directly to the stromal pocket after lenticule extraction, followed by UVA irradiation with a total energy of 0.8 J/cm2 (18 mW/cm2 for 45 seconds). This method of performing cross-linking was in fact very similar to the concept of intrastromal cross-linking in treatment of keratoconus, where a corneal stromal tunnel was created using femtosecond laser  [μm and total energy ranging from 3.6 to 6.3 J/cm2 have been shown to be safe and effective in stabilizing keratoconus [2 total energy, and good refractive outcomes were reported at two years [2 for 75 seconds  was used.In view of the weakening effect on the stroma after LASIK, the use of LASIK Xtra had gained popularity in the past few years and has reported good refractive and topographic stability comparable with LASIK alone –15. Howentation) , 24. Intatoconus , 24. Howatoconus , 26. Theatoconus , 28. Grawo years . Another2) was lower compared with those used in other LASIK Xtra or SMILE Xtra protocols (1.35–5.4 J/cm2). Previously, we have employed a stronger protocol with 60 seconds' soak time and 60 seconds' irradiation time. We found around 10% of cases still had clinical observable haze at 6 months (unpublished data). As a prophylactic treatment, a good balance between corneal restrengthening and visual outcome is preferred. Therefore, in this study, we employed a lower-energy protocol to minimize the postoperative haze. Unfortunately, due to limitation of resources, our study did not perform biomechanical studies to study the effect of the cross-linking. For the clinical effect, one would expect long-term studies (in terms of years) are needed to prove the benefit of SMILE, which has been shown to be a relatively stable procedure. A recently published 5-year SMILE outcome study showed stable refraction in SMILE with 0.48 D regression over 5 years [2) [The UVA energy dose in our SMILE Xtra series (0.8 J/cm 5 years . The rep 5 years . Therefoears [2) . This coOur SMILE Xtra results reported a high level of refractive predictability. At 6 months, 89% of our SMILE Xtra eyes were within ±0.50 D from target and 100% were within ±1.00 D, and this was also comparable with refractive predictability reported in the literature , 5. HoweIn our SMILE cases, the safety index at 3 and 6 months postoperatively was 1.00. This was comparable to the largest reported series by Ivarsen et al., where their safety index at 3 months was 1.05 . In our μm thickness at 140 μm depth) extraction resulted in significantly increased biomechanical properties compared with control non-cross-linked eyes [A limitation of the current work was unable to perform direct biomechanical measurements due to limited resources. Nevertheless, it had been shown that direct in vivo measurement of the corneal biomechanical property had low specificity and sensitivity . The sliked eyes . We did ked eyes  or in SMked eyes . Other lked eyes , we beli


While the amplitude of this Ca2+ signal is reduced following group I/II metabotropic receptor (mGluR) blockade, the duration is sustained. Based on previous studies it has been hypothesized that the molecular disassembly in astrocytic endfeet is caused by dystrophin cleavage mediated by Ca2+ dependent proteases. Using a newly developed genetically encoded Ca2+ sensor, the present study bolsters this hypothesis by demonstrating long-lasting, enhanced stimulation-evoked Ca2+ signals in astrocytic endfeet.Astrocytic endfeet are specialized cell compartments whose important homeostatic roles depend on their enrichment of water and ion channels anchored by the dystrophin associated protein complex (DAPC). This protein complex is known to disassemble in patients with mesial temporal lobe epilepsy and in the latent phase of experimental epilepsies. The mechanistic underpinning of this disassembly is an obvious target of future therapies, but remains unresolved. Here we show in a kainate model of temporal lobe epilepsy that astrocytic endfeet display an enhanced stimulation-evoked Ca Such a mechanism is plausible, as astrocytes activated by injury contain calpain  gene delivery of the genetically encoded Ca2+ indicator GCaMP5E  were used for all experiments. All procedures were approved by the animal use and care committee of the Institute of Basic Medical Sciences, University of Oslo, and the Centre for Comparative Medicine, Oslo University Hospital.SYNAPSIN-1 (SYN) promoter to generate the construct of pAAV-SYN-GCaMP5E. The human GFAP promoter /ml, TaqMan Assay, Applied Biosystems). For virus infection, adult mice were deeply anesthetized with a mixture of zolazepam (188 mg/kg body weight), tiletamine (188 mg/kg body weight), xylazine (4.5 mg/kg body weight) and fentanyl (26 µg/kg body weight) before viruses were stereotactically injected  was delivered.rAAVs serotype 1 and 2 were generated as described  kainate injection to elicit an initial status epilepticus (SE). Using this approach, more than 90% of injected animals developed recurrent behavioral seizures after a 5–8 day long latent period. For kainate injections, mice were anesthetized with a mixture of medetomidine  and ketamine  and kept on a heating blanket. A small craniotomy was performed in a stereotactic frame and kainate  was injected by a Hamilton pipette  at a depth of 1.7 mm at the following coordinates relative to Bregma: anteroposterior −2 mm, lateral +1.5 mm (right). Anesthesia was stopped with atipamezol  and SE was observed either clinically or by telemetric EEG recording and video monitoring. The animal model has been described in detail in a separate paper  and 4% paraformaldehyde  in PBS prior to decapitation. Brains were removed and fixed in ice-cold 4% PFA/PBS for 2 h, embedded in 2.5% Agarose (Invitrogen) in PBS and sliced on a Vibratome (Leica) into 70 µm sections. Immunostaining was performed with polyclonal rabbit anti-GFP , chicken anti-GFAP , rat anti-CD31  and FITC-coupled anti-rabbit, Cy3-coupled anti-chicken, Cy5-coupled anti-rat secondary antibodies . Confocal images were acquired on a Zeiss LSM5 PASCAL confocal laser scanning microscopy with 63x/1.4NA oil-immersion objective, equipped with an Argon laser  and a Helium Neon laser .Virus transduced mice were anesthetized with ~4% isoflurane and intracardially perfused with 1 × phosphate buffered saline , and brains were removed and cooled in artificial cerebrospinal fluid  containing (in mM): 124 NaCl, 2 KCl, 1.25 KH2PO4, 2 MgSO4, 1 CaCl2, 26 NaHCO3 and 12 glucose. Transverse slices (400 µm) were cut from the dorsal portion of each hippocampus with a Vibratome slicer (Leica) and placed in a humidified interface chamber at 30 ± 1°C and perfused with ACSF containing 2 mM CaCl2. In some experiments the group I/II mGluR antagonist α-methyl-4-carboxyphenylglycine  or the mGluR5 selective antagonist 2-methyl-6-phenylethynyl pyridine hydrochloride  were added to the ACSF. Two glass electrodes filled with ACSF and positioned 200–300 µm away from each other in CA1 stratum radiatum served as stimulation and recording electrodes, respectively. Orthodromic synaptic stimulations at 20 Hz for 10 s were delivered and excitatory postsynaptic potentials (fEPSPs) were monitored. Neuronal stimulation-evoked  astrocytic GCaMP5 fluorescence signals were recorded by a two-photon laser scanning microscope , as described previously  side. Images were recorded with a model “XLPLN 25 × WMP” 1.05NA, water-immersion objective  at 900–910 nm laser pulses. The laser was a model “Chameleon Vision II” . The recording was done either with 1 Hz or 4 Hz frame rate, the images were 512 × 512 px or 256 × 256 px, respectively.Experiments were performed on hippocampal slices prepared 3–4 weeks after injection of rAAV-Time-series of fluorescence images were first imported into Fiji ImageJ (Fiji), and regions of interest (ROIs) were manually selected based on morphology. Astrocytic cell bodies were identified by their projecting branches and endfeet by their characteristic circular pattern around transversely cut vessels and elongated, linear appearance along obliquely cut vessels. ROIs over processes were chosen at least 5 µm away from the perimeter of the soma. The relative change in fluorescence (ΔF/F) in each ROI, the individual traces and the histograms were all calculated and plotted by MATLAB  with custom written scripts. Standard deviation (SD) images were generated from time-lapse image recordings by Fiji ImageJ.t-test was used for comparison before and after wash-in with MCPG and MPEP. P < 0.05 was considered statistically significant.Statistical analyses were performed using Prism . One-way ANOVA with Tukey multiple comparisons test was used for comparison of GCaMP5E fluorescence changes in astrocytic somata, processes and endfeet following stimulation of Schaffer collaterals/commissural fibers. Paired Injection of the rAAV-GFAP-GCaMP5E construct into the hippocampus yielded robust and selective GCaMP5E expression in hippocampal astrocytes, as revealed by immunolabeling with antibodies against green fluorescent protein (GFP) and glial fibrillary acidic protein .Stimulation  of Schaffer collateral/commissural fibers (Scc) in acute hippocampal slices from the control side of rAAV-s Figure , as repos Figure . Compare2+ signal amplitudes were still significantly elevated in astrocytic somata and endfeet, but not in processes . At day 7 the Ca2+ signal amplitudes in all astrocytic compartments had returned to control values. At day 1 post kainate injection the duration of the stimulation evoked Ca2+ signal was significantly increased in the astrocytic endfeet compared to the non-injected side , while no changes were observed in other astrocytic processes or in the cell bodies. The latency from start of Scc stimulation to Ca2+ fluorescence increase was similar in all astrocytic subcompartments and not affected by kainate injection. However, kainate injection increased the Ca2+ signal rise rate in all compartments . The duration and latency of the Ca2+ signals were not affected by MCPG, whilst rise rate was significantly reduced only in processes , processes  and endfeet . MPEP reduced the amplitudes of the Ca2+ transients by 30–40%, i.e., to the level at the non-injected side (Figure 2+ transients to the same extent, suggesting that mGluR5 alone is mediating the enhanced Ca2+ signal amplitude in the latent phase. Similarly to MCPG, MPEP did not affect the duration and latency of the Ca2+ signals, and had inconsistent effects on transient rise rate in the three compartments (Figure As mGluR5 receptors have been shown to mediate enhanced astrocytic Cae Figure . The nons Figure .Neither MPEP nor MCPG significantly affected the fEPSP amplitudes Figures .2+ signaling analogous to that found in neurons. With the advent of genetically encoded Ca2+ sensors this possibility can be experimentally explored. A key question is whether Ca2+ signaling in the astrocytic endfeet could play a role in epileptogenesis, by initiating a sequence of events that lead to disassembly of the DAPC in the endfoot plasma membrane. This complex, known to be critical for K+ and water homeostasis in brain, is lost in patients with mesial temporal lobe epilepsy (Eid et al., Astrocytes are highly polarized cells, structurally as well as functionally, opening for the possibility of a compartmentation of Ca2+ signal in the endfeet that outlasts the Ca2+ signal in the astrocytic cell bodies. This underlines the idea that endfeet are distinct subcompartments of astroglia (Nagelhus and Ottersen, Here we show that intracortical kainate application leads to a stimulation evoked Ca2+ signal in endfeet is attenuated by blockade of group I/II mGluRs and thus dependent on Ca2+ mobilization from intracellular stores. However, mGluR blockade does not cancel out the difference between endfeet and cell bodies when it comes to the duration of the Ca2+ signal. This suggests that the increased signal duration primarily reflects reduced clearance of Ca2+. An uncoupling of astrocytes could contribute to reduced clearance (Bedner and Steinhäuser, The CaA disassembly of the DAPC in astrocytic endfeet and the loss of astrocyte polarization that this entails now emerge as a signature event in mesial temporal lobe epilepsy and epilepsy models (Nagelhus and Ottersen, 2+ signals necessary for activation of calpain or any other Ca2+ dependent protease with affinity for dystrophin or dystrophin associated molecules. The present study fills this void and shows that endfeet display Ca2+ signals that even outlast those in the astrocytic cell bodies. The cascade of events underlying the molecular disassembly in endfeet is an obvious target for future therapies.It has been proposed that the molecular disassembly in astrocytic endfeet is caused by calpain activation (Nagelhus and Ottersen, KS designed experiments, acquired, analyzed and interpreted data, and wrote the paper; KH conceived the study and supervised experiments, acquired, analyzed and interpreted data, and wrote the paper; WT designed experiments, acquired, analyzed and interpreted data, and wrote the paper; VJ designed and supervised experiments, interpreted data, and wrote the paper; RE analyzed and interpreted data, and wrote the paper; PB designed the animal model, interpreted data and commented upon the manuscript; CS designed the animal model, interpreted data and commented upon the manuscript; ET conceived the study, interpreted data and commented upon the manuscript; OPO conceived the study, interpreted data and wrote the manuscript; EAN conceived the study and supervised experiments, interpreted data and wrote the manuscript. All authors revised the work critically and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Calcium sensing receptors (CaSR) interact with 14-3-3 binding proteins at a carboxyl terminal arginine-rich motif. Mutations identified in patients with familial hypocalciuric hypercalcemia, autosomal dominant hypocalcemia, pancreatitis or idiopathic epilepsy support the functional importance of this motif. We combined total internal reflection fluorescence microscopy and biochemical approaches to determine the mechanism of 14-3-3 protein regulation of CaSR signaling. Loss of 14-3-3 binding caused increased basal CaSR signaling and plasma membrane levels, and a significantly larger signaling-evoked increase in plasma membrane receptors. Block of core glycosylation with tunicamycin demonstrated that changes in plasma membrane CaSR levels were due to differences in exocytic rate. Western blotting to quantify time-dependent changes in maturation of expressed wt CaSR and a 14-3-3 protein binding-defective mutant demonstrated that signaling increases synthesis to maintain constant levels of the immaturely and maturely glycosylated forms. CaSR thus operates by a feed-forward mechanism, whereby signaling not only induces anterograde trafficking of nascent receptors but also increases biosynthesis to maintain steady state levels of net cellular CaSR. Overall, these studies suggest that 14-3-3 binding at the carboxyl terminus provides an important buffering mechanism to increase the intracellular pool of CaSR available for signaling-evoked trafficking, but attenuates trafficking to control the dynamic range of responses to extracellular calcium. CaSR is crucial to establishing and maintaining organismal Ca2+ homeostasis, regulating parathyroid hormone synthesis and secretion, intestinal Ca2+ uptake, renal Ca2+ resorption, and aspects of bone Ca2+ uptake and release  over the range from 0.5 to 5 mM to determine the effects on total CaSR expression by western blotting; a representative experiment is in 2+ results in an increase in net cellular CaSR. Cells expressing wt CaSR and the S899 site mutants have substantially higher levels of cellular CaSR at all extracellular [Ca2+] when compared to the 5A mutant (2), there were no significant differences between wt CaSR and CaSR-5A expression levels . However, prolonged exposure to 0.5 mM Ca2+ bath had a significant differential effect on wt CaSR and CaSR-5A protein levels. Net wt CaSR after 18 hrs of 0.5 mM Ca2+ was comparable to that at 2 hrs , while CaSR-5A was significantly reduced after 18 hrs in 0.5 mM Ca2+ . Further, cells expressing the 5A mutant require significantly higher extracellular Ca2+ to induce an increase in net CaSR expression  intracellular Ca2+ responses , then fixed and permeabilized. The SEP moiety was stained with anti-GFP antibody, and confocal imaging was used to assess both intrinsic SEP fluorescence and anti-GFP immunoreactivity. BS-wt had significant SEP fluorescence after 60 min exposure to either 0.5 or 10 mM Ca2+, and staining with the anti-GFP antibody was equally robust, showing strong correlation in the merged images  . Prolong2+ . To recod images . In 0.5 rescence , with a ed image . These r2+, then TIRFM imaging was begun, and cells were exposed to 10 mM Ca2+. At three intervals during sustained exposure to elevated extracellular Ca2+ (marked by bars), imaging was briefly halted to label cells with BgTx-A594. TIRFM imaging of both SEP and BgTx-A594 fluorescence was then resumed. Data are presented as the average ± S.D. of 4 cells, and representative of 3 independent experiments. Plasma membrane BS-wt SEP fluorescence (green) was constant during the 55 min experiment, and each application of BgTx-A594 elicited a peak followed by mono-exponential decay of fluorescence indicative of endocytosis , plateau (%), and time constant (tau)) ) . The req2+ causes net changes in cellular CaSR, and whether this is altered by mutations at the 14-3-3 protein binding site. Cells expressing FLAG-wt or FLAG-5A were treated with DMSO/0.5 mM Ca2+, tunicamycin in 0.5 or 5 mM Ca2+ for 2, 4, 8 or 18 hrs, then lysed and subjected to western blotting. We next determined whether extracellular Ca2+ in the presence of tunicamycin further accelerates loss of the 140 kDa form and increases production of the 120 kDa form for wt CaSR. Comparison of the dynamics of the 120 and 140 kDa forms for wt and the 5A mutant demonstrates the importance of 14-3-3 binding in controlling CaSR synthesis and trafficking. Stimulation of wt CaSR with 0.5 mM Ca2+ leads to a significantly greater loss of the 140 kDa and synthesis of the 120 kDa forms at 8 hrs , although this difference is lost by 18 hrs. The 5A mutant, on the other hand, shows a significant lag in induction of the 120 kDa form, although addition of 5 mM Ca2+ doubles the rate of synthesis . The rate of loss of the 140 kDa form of the 5A mutant is not sensitive to extracellular Ca2+, Exposure to 5 mM Ca2+. An inability to bind 14-3-3 proteins leads to higher basal levels of plasma membrane CaSR despite a reduction in net cellular CaSR. Signaling is commensurate with plasma membrane levels of receptors, i.e., we observed left-shifted sensitivity to extracellular Ca2+ for activation of steady changes in intracellular Ca2+, as well as increased Ca2+-stimulated ERK1/2 phosphorylation. Loss of 14-3-3 binding did not, however, result in complete loss of agonist-mediated increases in plasma membrane CaSR. The ADIS response, as monitored by periodic BgTx-A594 labelling, is significantly higher for the CaSR-5A mutant than wt CaSR at elevated extracellular Ca2+. Converse results, observed for the CaSR-S899A mutant, argue that tight binding of 14-3-3 proteins leads to enhanced intracellular retention of receptors, and thus reduced signaling. Independent confirmation of these differences is clearly demonstrated by the reduced CaSR-S899A and elevated CaSR-5A localizations at the plasma membrane in 0.5 mM Ca2, as measured by ELISA. Cellular Ca2+ homeostasis is tightly controlled, and its dysregulation can contribute to pathologies ranging from diabetes to Parkinson’s disease [2+ within physiological limits. Mutations in the 14-3-3 protein binding region of CaSR, including R896H and R898Q, have been identified in patients with pancreatitis [This report characterizes the role of 14-3-3 proteins in regulating CaSR signaling in elevated extracellular Ca disease –36. For reatitis  and idioreatitis , and a lreatitis ,23. We hreatitis , support2+ to conclude that both wt CaSR and CaSR-5A are subject to additional regulatory interactions which determine maximal trafficking rates [2+. While the original conclusion remains valid, i.e., there are additional as yet undefined determinants of the ADIS response, the present results establish the importance of 14-3-3 proteins in decreasing the lability of the intracellular, ADIS-mobilizable pool of CaSR. The complete lack of ADIS response of the truncation mutant, CaSRΔ868 [One of the most striking results from the present study is the evidence for enhanced lability of the intracellular, mobilizable pool of CaSR caused by loss of 14-3-3 protein binding. We used the relative plasma membrane SEP and BgTx-A594 fluorescence intensities to characterize this lability and its effect on the magnitude of ADIS responses for BS-wt and 14-3-3 protein binding mutants. When normalized plasma membrane SEP and BgTx-A594 fluorescence intensities are comparable, then the dwell time in intracellular, pre-plasma membrane compartments must be sufficient for full maturation of the SEP chromophore. Estimates of chromophore maturation rates for GFP variants range from hours for wild-type GFP to less than an hour for enhanced folding mutants –32, argung rates . In thisCaSRΔ868 , and the2+. Tunicamycin, which rapidly blocks endoplasmic reticulum exit of newly synthesized CaSR [2+. Results suggest that CaSR expression levels are modulated by ambient extracellular Ca2+ in a feed-forward mechanism, i.e., increased extracellular Ca2+ activates CaSR signaling, which increases net cellular CaSR levels, stabilized by 14-3-3 protein binding. An inability to bind 14-3-3 proteins does not, paradoxically, attenuate signaling, but enables rapid targeting of newly synthesized receptors to the plasma membrane, leading to increased coupling between biosynthesis and signaling.A second major result of these studies is the requirement for ongoing biosynthesis to maintain CaSR signaling during prolonged exposure to elevated extracellular Cazed CaSR ,28, indu2+ also increases net cellular levels of CaSR, ensuring that the intracellular, ADIS-mobilizable pool is sufficient to maintain signaling. It should be noted that the present study interrogates only translational and post-translational mechanism(s) contributing to establishment and maintenance of cellular levels of CaSR, since transcription is driven by the vector CMV promoter. Prolonged stimulation in vivo may be additionally regulated at the transcriptional level. We here demonstrate that mechanism(s) contributing to regulation of CaSR protein are poised to maintain a constant level of maturely glycosylated CaSR, at the expense of the immaturely glycosylated, endoplasmic reticulum-localized form. In this context, the net level of the 160 kDa form is not equivalent to plasma membrane CaSR levels, but rather is the sum of CaSR localized to Golgi, plasma membrane and post-plasma membrane sites. Nevertheless, the most striking result of these studies is that cellular biosynthesis and trafficking of CaSR is poised to maintain plasma membrane levels of CaSR despite alterations in cell signaling and/or cell stress, induced in these studies by tunicamycin [2+, to facilitate depletion of pre-plasma membrane pools of CaSR. Finally, prolonged exposure to elevated extracellular Ca2+ leads to net cellular increases in CaSR, mediated by an increased rate of CaSR biosynthesis [2+ superimposed on a constant baseline of Ca2+ [Finally, biochemical approaches demonstrate that prolonged stimulation with elevated extracellular Caicamycin ,38. Thesynthesis  counterbynthesis ,40. Prolynthesis –48. In cIn summary, these studies use novel approaches to demonstrate that 14-3-3 protein binding at the endoplasmic reticulum stabilizes and decreases the lability of the intracellular, ADIS-mobilizable pool of CaSR. CaSR binding to 14-3-3 proteins supports ongoing CaSR signaling while maintaining tight control of the dynamic range of signaling responses.


LRPPRC gene, which has previously been associated with the neurodegenerative French Canadian Leigh Syndrome, results in a decrease in the production of mitochondria-encoded subunits of complex IV, thereby causing a reduction in complex IV activity. Previously we have shown that reducing complex IV activity triggers a compensatory and conserved mitochondrial hyperfusion response. We now demonstrate that LRPPRC knock-down in mammalian cells leads to an imbalance between mitochondria-encoded and nuclear-encoded subunits of complex IV and that this imbalance triggers the mitochondrial unfolded protein response (UPRmt). The inactivation of the LRPPRC-like gene mma-1 in C. elegans also induces UPRmt, which demonstrates that this response is conserved. Furthermore, we provide evidence that mitochondrial hyperfusion and UPRmt are coordinated but mediated by genetically distinct pathways. We propose that in the context of LRPPRC mma-1 knock-down, mitochondrial hyperfusion helps to transiently maintain mitochondrial ATP production while UPRmt participates in the restoration of mitochondrial proteostasis. Mitochondrial proteostasis is not only critical in pathophysiology but also during aging, as proteotoxic stress has been shown to increase with age. Therefore, we speculate that the coordination of these two mitochondrial stress responses plays a more global role in mitochondrial proteostasis.The inactivation of the  Mitochondria are essential eukaryotic organelles that participate in processes such as cellular energy production, cell signaling and apoptosis -3. The pmt). This response leads to the increased production and import into mitochondria of chaperones (such as HSP60 and HSP70) and proteases (such as ClpP), which help misfolded proteins to fold properly or cause their degradation, respectively  (see for review ). The acr review ). Finalloduction -12.LRPPRC gene encodes a leucine-rich pentatricopeptide repeat containing protein that is imported into mitochondria and that is mutated in patients with French Canadian Leigh Syndrome, a neurodegenerative disorder associated with complex IV deficiency ) that expresses mitochondrial matrix-targeted GFP in body wall muscles, which allows us to monitor steady-state mitochondrial morphology in these cells [mma-1(RNAi) causes mitochondrial hyperfusion  causes mitochondrial hyperfusion in animals homozygous for the atfs-1 loss-of-function mutation tm4525 . Finally, mma-1(RNAi) also causes mitochondrial hyperfusion in animals homozygous for the haf-1 loss-of-function mutation ok705 . Compared to the response in the wild-type and atfs-1(tm4525) background, the response in the haf-1(ok705) background is slightly reduced; however, the difference is not statistically significant (p = 0.22 by Student t-test). Based on these results we conclude that mma-1(RNAi)-induced mitochondrial hyperfusion is independent of ATFS-1 and HAF-1.In order to test whether the mitochondrial hyperfusion response induced by the inactivation of se cells . In an omma-1(RNAi) causes synthetic embryonic lethality and synthetic midlarval arrest in the fzo-1(tm1133) loss-of-function mutant background, indicating that mitochondrial hyperfusion is essential for viability in response to mma-1(RNAi) [mt is also essential for viability in response to mma-1(RNAi), we inactivated mma-1 in animals carrying a loss-of-function mutation in the atfs-1 gene (atfs-1(tm4525)) [mma-1(RNAi) does not cause any obvious embryonic lethality or midlarval arrest in atfs-1(tm4525) mutant animals (afts-1(RNAi)+mma-1(RNAi)) and did not observe any obvious synthetic lethality or arrest (mma-1(RNAi), mitochondrial hyperfusion is essential for viability while ATFS-1-dependent UPRmt is not.We previously showed that e fzo-1tm33 loss-otm4525)) . We foun animals . We alsor arrest . This inmt, mitochondrial hyperfusion or mitophagy) can be activated in order to protect mitochondrial function [LRPPRC or mma-1 function leads to an evolutionary conserved co-activation of UPRmt and mitochondrial hyperfusion. Depletion of LRPPRC or MMA-1 protein results in a decrease in the production of mitochondria-encoded subunits of complex IV and thereby causes a reduction of complex IV activity [mt occurs when this balance and, hence mitochondrial proteostasis, is disrupted . This finding suggests that the accumulation of unassembled complex IV subunits is most likely the signal that triggers UPRmt. Furthermore, our data indicate that UPRmt is transiently activated until mitochondrial proteostasis is restored . We, therefore, propose that the transient activation of UPRmt participates in the restoration of mitochondrial proteostasis by either proteolytic degradation of unassembled subunits by ClpP or assisted folding of nascent subunits by HSP70. In addition, the response may also involve the down-regulation of the genes encoding nuclear-encoded subunits of complex IV.Depending on the type and the severity of the stress to which mitochondria are exposed, different mito-chondrial stress responses  [mt is not essential for viability upon inactivation of mma-1. Furthermore, we show that ATFS-1-dependent UPRmt is also not essential for viability in response to spg-7(RNAi). One possible explanation for this finding is the existence of an ATFS-1-independent UPRmt pathway. Indeed, Haynes and co-workers have shown that while spg-7(RNAi) induces the up-regulation of 685 genes, 294 of these genes are still up-regulated in atfs-1(tm4525) mutant animals [mma-1 inactivation, the ATFS-1-independent UPRmt in combination with the mitochondrial hyperfusion response may be sufficient for the completion of embryonic and larval development.We previously showed that the mitochondrial hyperfusion response is essential for viability in response to -1(RNAi) . In cont animals . Hence, mt on aging remains controversial. While Houtkooper et al reported an extension of lifespan in response to the activation of UPRmt [et al observed no clear correlation between UPRmt and lifespan [C. elegans and in rat that mitochondrial fragmentation increases with age and that this fragmentation is accompanied with a loss of mitochondrial volume [mt and the mitochondrial hyperfusion response has not only the potential to increase our knowledge of how cells respond to mitochondrial stress under physiological and pathophysiogical conditions (i.e. French Canadian Leigh Syndrome) but also during aging.The effect of the activation of UPRof UPRmt , Bennettlifespan . We and l volume , 27. Intl volume . While tl volume . Consequl volume . In concC. elegans strains were cultured as previously described [haf-1(ok705) (OMRF Knockout Group); (LG V) atfs-1(tm4525) . A summary of the transgenic lines is provided in escribed . Bristolescribed  except: 600nm/ml of concentrated mma-1(RNAi) or spg-7(RNAi) bacterial clones from the Ahringer library [mock(RNAi) bacteria (HT115 bacteria transformed with the empty RNAi feeding vector pPD129.36) or atfs-1(RNAi) bacteria. 24 hours later, L4 larvae were inoculated on the RNAi plates and incubated at 20°C for 4 days. Mitochondrial morphology in L4 larvae of the F1 generation was analyzed by fluorescent microscopy as described [C. elegans development, three wild-type (+/+), three atfs-1(tm4525) and 15 fzo-1(tm1133) L4 animals  genotype were used since the broodsize of these animals is smaller) were inoculated on mock(RNAi), mma-1(RNAi) or spg-7(RNAi) plates and incubated at 20°C for 18 hours. For the double RNAi experiment, three wild-type L4 larvae were inoculated on mock(RNAi), atfs-1(RNAi), mma-1(RNAi) (diluted 1:5 with mock(RNAi) or atfs-1(RNAi)) or spg-7(RNAi) (diluted 1:5 with mock(RNAi) or atfs-1(RNAi)) plates and incubated at 20°C for 18 hours. The adults were then transferred onto freshly seeded RNAi plates and incubated for an additional three hours at 20°C. After removing the adults, the embryos laid during these three hours were counted. 24 hours later, the number of unhatched embryos was counted to determine the embryonic lethality. The number of animals that reached adulthood by day 5 and the number of animals that underwent a midlarval arrest were also counted.RNAi by feeding was performed as previously described  with the library  or dilutescribed . For pro2. For RNA interference, cells were transfected reversely with control stealth siRNA medium GC or stealth siRNA oligos targeting LRPPRC using Lipofectamine RNAiMax in OPTI-MEM (Invitrogen). 24 hours after transfection, fresh culture medium (25 mM glucose) was added. For the analysis of mitochondrial morphology, cells were kept in normal culture medium until harvested. For the analysis of UPRmt, the normal culture medium was replaced with medium containing only 5 mM glucose 24 hours before analysis.SH-SY5Y cells were cultivated in DMEM:F12 supplemented with 15% FBS (Sigma), 1% non essential amino acids and 1% penicillin/streptomycin (Invitrogen); HEK293T cells were cultivated in DMEM supplemented with 10% FBS (Sigma) and 1% penicillin/streptomycin (Invitrogen); both cell lines were passaged twice a week and kept at 37°C with 5% CO472QEAKTAEEPKKEQN  by Thermo Fisher Scientific, as previously described [mock(RNAi). Only samples for which mma-1 knock-down was at least 20% were used for the statistically analysis. Statistical analysis was performed by using one sample t-test. Normality of the data was assessed by Shapiro-Wilk Normality test.Rabbit polyclonal anti–HSP-6 antibodies were generated using the antigenic peptide Cescribed  and usedescribed  (1:2000)SH-SY5Y and HEK293T cells were harvested with pre-warmed 1x Laemmli-sample buffer and boiled for 10 minutes at 95°C and then subjected to SDS/PAGE and Western. The following antibodies were used: mouse monoclonal anti-β-Actin , mouse monoclonal anti-LRP130 , goat polyclonal anti-HSP60 , rabbit polyclonal anti mortalin/mitochondrial HSP70 , rabbit polyclonal anti-ClpP , goat polyclonal anti-COXI  and goat polyclonal anti-COXIV  .Mitochondrial morphology was analyzed as previously described with the following modifications . Cells g