In vitro, cerebellar Purkinje cells can intrinsically fire action potentials in a repeating trimodal or bimodal pattern. The trimodal pattern consists of tonic spiking, bursting, and quiescence. The bimodal pattern consists of tonic spiking and quiescence. It is unclear how these firing patterns are generated and what determines which firing pattern is selected. We have constructed a realistic biophysical Purkinje cell model that can replicate these patterns. In this model, Na+/K+ pump activity sets the Purkinje cell's operating mode. From rat cerebellar slices we present Purkinje whole cell recordings in the presence of ouabain, which irreversibly blocks the Na+/K+ pump. The model can replicate these recordings. We propose that Na+/K+ pump activity controls the intrinsic firing mode of cerbellar Purkinje cells. The cerebellum coordinates the execution and adaptation of motor behaviours +/K+ pump controls intrinsic Purkinje cell activity. The Na+/K+ pump uses the energy of one ATP molecule to exchange three intracellular Na+ ions for two extracellular K+ ions In vitro, in the absence of synaptic input, Purkinje neurons can spontaneously fire action potentials in a repeating trimodal pattern that consists of tonic spiking, bursting and quiescence +/K+ pump controls the transition from tonic to burst firing by regulating the extracellular K+ concentration ([K+]o), which rules a Kv1.2 K+ channel “gate” to bursting. And the Na+/K+ pump's electrogenicity generates the quiescent mode, upon regulation by the intracellular Na+ concentration ([Na+]i). During quiescence the ionic concentrations reset and the trimodal cycle starts again from the tonic phase. The Na+/K+ pump, as an enzyme, adheres to Michaelis-Menten kinetics + (KK) sets the duration of tonic firing and its affinity for internal Na+ (KNa) sets the total duration of firing (tonic+bursting). If these affinities are modulated to make the total firing duration shorter than the tonic, then the model cell fires in an experimentally observed bimodal pattern repeat of tonic spiking and quiescence Na is set equal to (or lower than) the basal [Na+]i, then the model cell becomes locked in quiescence  Act 1986 and associated guidelines. We had approval to conduct these experiments from the Biological Ethics Sub-Committee, of the Research Ethics Committee, at the University of Warwick. This committee scrutinizes in accordance with the appropriate guidance for such committees from the Home Office, the RSCPA and the Laboratory Animal Science Association (LASA). This committee ensures that staff and students are trained and experienced, and that the potential benefits of the research outweigh the effects on the animals concerned. The committee is committed to the promotion of the 3Rs . At the University of Warwick, highly qualified and experienced veterinary staff are actively involved in (1) the ethical scrutiny of research (2) the welfare, care and husbandry of animals and (3) provide advice and support to all staff and students involved in research using animals.m] , moderately TEA sensitive (IK_mid) and TEA insensitive (IK_slow) voltage-gated K+ currents, a BK voltage-and-Ca2+-gated K+ current (IBK), a resurgent Na+ current (INa-R), a P-type Ca2+ current (ICaP), a hyperpolarization activated cation current (IH), a leak current (IL) and an intracellular Ca2+ dynamics abstraction - all sourced from Khaliq et al. 2+-gated K+ current (ISK) CaT), Class-E (ICaE) and P-type (ICaP) voltage-gated Ca2+ currents; a leak current (IL); A-type (IKA), D-type (IKD), M-type (IKM), and delayed rectifier (IDR) voltage-gated K+ currents; BK (IBK) and K2 (IK2) type voltage-and-Ca2+-gated K+ currents and an intracellular Ca2+ dynamics abstraction - all sourced from Miyasho et al. H) + current (IKv1) The model incorporates twenty-one gated ion channels with descriptions established from the literature. The soma has highly TEA sensitive  transports 3 Na+ out (ispump_Na) for every 2 K+ in (ispump_K) + concentration ([Na+]i). The Na+ affinity constant (KNa) is 40 mM is) is 1 mM V represents the membrane potential in mV as a dimensionless quantity.+/K+ pump  has the same 3Na+:2K+ stoichiometry but no voltage dependency and a hyperbolic relation to extracellular K+ concentration ([K+]o) + affinity constant (KK) is 2.245 mM + concentration ([Na+]i) is initiated at 10 mM t according to the relationship:spump_Na and Ispump_Na are Na+/K+ pump Na+ currents at the soma, set by Na-R is the voltage-gated Resurgent Na+ current, Isex_Na is the Na+/Ca2+ exchanger Na+ current  and Na+ current pumped out of the soma by the Na+/K+ pump  is a factor that we have introduced and is not found in the original Canavier description + stimulates the Na+/K+ pump and this lag τ accounts for the duration of sodium's diffusion from channels to pumps. It aligns with the concept of a “fuzzy space” under the pump where the Na+ concentration differs from other parts of the cell + diffusion abstractly, with this τ parameter, because a more explicit account would be ill constrained by the literature and too computationally expensive; intracellular diffusion processes have a much shorter spatial scale than electrical signalling and so their modeling requires a higher nseg value  to the dendritic compartments is initiated at 2 mM and then changes in time t according to the relationship:F is the Faraday constant, wid is the thickness of an extracellular region around the compartment that K+ accumulates in (70*10−3 µm), Q is a K+ accumulation factor (0.143) and K_netI , A-type (IKA), M-type (IKM), delayer rectifier (IDR), BK (IBK), K2 (IK2) and Kv1 (IKv1) K+ currents. The Na+/K+ pump K+ current in the dendrites: idpump_K is set by dpump_K is set by Extracellular Kd IK_net  is the d+ accumulation is set physiologically by the glial buffer system +]o is not permitted to exceed 3.03 mM +]o>3.03) [[K+]o = 3.03].A “ceiling” for extracellular K+ accumulation equation employed by Durstewitz et al. wid setting is the same (70*10−3 µm) but Durstewitz et al. Q value of 2 as opposed to our employed 0.143. We adjusted Q as a free parameter in our model tuning because this arbitrary factor is not constrained by the experimental literature. Durstewitz et al. +/K+ pump mechanism in their model and hence no IK_in parameter, only having an IK_out parameter. Their formulation has an additional term on the right hand side (RHS), setting a decay to the extracellular K+ accumulation, where [K+]eq is the equilibrium/resting value of [K+]o and τK is the time constant with which it approaches this resting value. This term is an abstractive capture of cellular processes acting against extracellular K+ accumulation, primarily the action of the Na+/K+ pump (IK_in). In our work, we model the Na+/K+ pump explicitly and so this term is redundant and dropped from our description of extracellular K+ dynamics.Formula +/K+ pump mechanisms. One has already been described , inwardly passing 3 singly positive Na+ ions (3*[+1]) for the extrusion of every doubly positive Ca2+ ion (1*[+2]) +/K+ pump current  in its transport of 3 Na+ out (3*[+1]) for every 2 K+ in (2*[+1]).dex and gdpump are Na+/Ca2+ exchanger and Na+/K+ pump membrane current densities (respectively) in the dendrites and their equality at 0.0021 mA/cm2 ensures an electrical counterbalance So, the model dendrites have a Na+/Ca2+ exchanger current and an electrically counterbalancing Na+/K+ pump current:+/Ca2+ exchanger mechanism (sex) and a counterbalancing, simple Na+/K+ pump mechanism (spump). Thus, the soma compartment, like those of the dendrites, has both a detailed Na+/K+ pump description  which we can represent as  where superscript [s] denotes a density at the soma and superscript [d] denotes a density in the dendrites. dpump represents the density of a detailed Na+/K+ pump formalism  and the dendrites have a detailed and simplified Na+/K+ pump formalism (with densities [ddpump] and [gdpump] respectively).Convention permits inward (depolarizing) currents to be denoted negative and outward (repolarising) currents to be denoted positive cription  and a sicription  in paral mA/cm2]  which peon refer .(17)The ormalism  as opposcription . So, the+/K+ pump equations are each valid and founded in previously published Na+/K+ pump descriptions. They capture different aspects of Na+/K+ pumping. The first captures [Na+]i and voltage dependency  by functions of time, t (in seconds): Ouabain simulation is arbitrarily initiated at t = 3(s)−2 s−1; c1s = 0.002 mA cm−2 s−1; c1d = 0.01 mA cm−2 s−1; M = 0.1 mA cm−2 s−1; c2s = 0.011 mA cm−2 s−1; c2d = 0.055 mA cm−2 s−1Ouabain irreversibly blocks the Naxpump,. gxpump, x = s,d) from occurring.Catch coding, which is code of the form: if (x<0) [x = 0], is used to prevent negative values of  GABAergic stellate inputs make inhibitory synaptic contacts upon the model dendrites; two inputs to every smooth dendrite compartment and one input to every spiny dendrite compartment +/K+ pump and Na+/Ca2+ exchanger densities, the synaptic weight, Q, τ and the parameters of Na+/K+ pump density decline used in the simulation of ouabain application . These were all ill constrained by the literature and tuned manually 2+ channel block switched the model from trimodal to bimodal firing, as has been observed experimentally (refer All parameters were established by prior literature (as referenced) except the twenty-one current densities, the Naly refer  section.a) R) of an equivalent cylinder is given by:ir are the radii of the collapsed branches for that equivalent cylinder. The length (l) of an equivalent cylinder is taken as an average of the lengths of the collapsed branches (li) for that cylinder, weighted by their respective radii (ri):Cd), which rescales the current/pump/exchanger density values (gi) and membrane capacitance (Cm) in the dendrites such that:Cd is the ratio of the total surface area of the dendritic segments to their equivalent cylinders. The model's somatic compartment is not introduced into the collapsing algorithm and so in the reduced model it has the same dimensions as in the full model, and the Cd correction factor is not applied to any of its parameters.To generate a simpler version of the described Purkinje cell model, the dendritic arbour was collapsed into fewer compartments with a reduction algorithm that conserves axial resistance . The reduced model's 40 dendritic compartments were allocated smooth and spiny  in an arbitrary mirroring of the full model . Note that in our approach, smooth and spiny dendrites are not distinguished from one another by the actual modeling of dendritic spines but by an abstraction, with a larger specific membrane capacitance (Cm) for spiny dendrites  and the dimensions of the 40 dendritic compartments are shown in We used this “Rcitance Cm for spiQ parameter is a setting in the model's description of extracellular K+ dynamics and in the full model it is 0.143. This parameter isn't set or constrained by any feature of the reduction algorithm used and is not constrained by any experimental literature. It is a free parameter. We manually tuned this parameter to confer a best fit between reduced and full model output, ultimately assigning it a value of 0.033. This is reasonably distinct from the value of 0.543 obtained by a Cd manipulation  but this is of no consequence, as there is no directive in the literature for applying Cd to such a parameter.The We strove for the smallest number of compartments, but through testing we found that 41  was the smallest number that could behave equivalently to the full 1089 compartments. This is because the collapsing algorithm does not conserve dendritic length fully, and Purkinje functioning requires a degree of uncoupling (distance) between somatic and dendritic events. 41 compartments is the smallest number that confers the sufficient distance (uncoupling) with a dendritic length of ∼502 µm. Without this sufficiency of length, for instance if the reduced model is the full model transposed by the algorithm to just 3 compartments (dendritic length of ∼129 µm), the tonic mode is absent in the trimodal firing pattern i.e. the model cannot replicate the Purkinje cell's trimodal firing pattern. By contrast, the reduced model of 41 compartments can replicate this activity motif.n it is as though there are [n] mathematically identical compartments, including the entire sub-tree, connected to that compartment. This is a method of adding load to a dendritic tree without adding load to computation time. However, increasing rallbranch numbers could not decrease the compartment number requirement and thus it is not a load requirement that is limiting.We confirmed that the problem with the 3 compartment model was a lack of dendritic distance, rather than a lack of dendritic load, because increasing dendritic load could not enable a compartment number lower than 41. The “rallbranch” variable is unique to the NEURON simulator a conservation” algorithm). We then overcome the dendritic distance issue by linking these 3 dendritic compartments to the somatic compartment through a coupling compartment that had a length of our setting, which we ensured was sufficient. This coupling compartment had the same passive and conductance properties as the dendritic compartments and, indeed, can just be thought of as component to the reduced model's dendritic tree. The coupling compartment and the next dendritic compartment along were set as smooth dendrites  and the dimensions of the 4 dendritic compartments are shown in With another round of simplification, we produced a 5 compartment model that could reproduce the behavior of the full 1089 compartment model. Firstly, we collapsed the dendritic tree of the full 1089 compartment model to just 3 compartments . This is ∼51* faster than the full model and ∼3* faster than the 41 compartment model.In the 2+, low Ca2+ aCSF, composed of (mM): 127 NaCl, 1.9 KCl, 7 MgCl2, 0.5 CaCl2, 1.2 KH2PO4, 26 NaHCO3, 10 D-glucose (pH 7.4 when bubbled with 95% O2 and 5% CO2). Slices were stored in normal aCSF  at room temperature for 1–6 hours before recording.Parasagittal slices of cerebellum (250 µm) were prepared from male Wistar rats, at postnatal days 28–35 (P28–35), with methods based on −1) with a CSF, which was bubbled with 95% O2 and 5% CO2. Whole-cell patch-clamp recordings were made from visualized Purkinje cells using an EPC 8 amplifier  controlled via a Digidata 1322a interface  using Clampex . Patch-pipettes  were fire-polished, and had resistances of 1.5–4 MΩ when filled with an intracellular solution containing (mM): 135 K gluconate, 7 NaCl, 10 HEPES, 0.5 EGTA, 2 Na2-ATP 0.3 Na2-GTP and 10 mM Na phosphocreatine (adjusted to pH 7.2 with KOH and osmolarity adjusted to 300 mOSM with sucrose). Aliquots of intracellular solution were stored frozen at −20°C and thawed on the day of recording.Individual slices were viewed on a Zeiss FS Axioskop microscope with a 40× water immersion objective and Nomarski differential interference optics, at a total magnification of 640×. Slices were maintained at 30–32°C and continuously perfused (1–5 ml minDrugs were dissolved at 1–100 mM in deionised water: bicuculline methiodide (Sigma), TTX  and ouabain (Sigma). Aliquots of these stock solutions were stored frozen at −20°C, thawed and diluted in perfusion medium on the day of recording. All drugs were bath applied.t), bursting (b) and silence (s)  C. SigniThere is some uncertainty as to whether the trimodal firing pattern is intrinsically generated or a function of neuromodulatory input + spikes (2+ spikes (t) is somatic spiking in the absence of dendritic spiking. Whilst the model's burst mode (b) is produced by dendritic spikes propagating to the soma to drive somatic bursting. Somatic bursts have a stereotypic waveform and each somatic burst is co-incidental with a dendritic spike  permits a higher frequency of dendritic firing .Kv1.2 is low-voltage gated. In addition to Kv1.2, the model has other KThe control of the tonic to burst transition by Kv1.2 channels enables the model to replicate an experimental investigation in which Kv1.2 channel block dramatically shortened the tonic phase within the trimodal pattern F [9].+/K+ pump hyperpolarizes the membrane potential with a stoichiometry of three internal Na+ ions exchanged for every two external K+ ions (methods). The Na+ concentration in a “fuzzy space” underneath the pump ([Na+]i) rises during the tonic and burst firing modes . Eventually, during the bursting mode, the pump generates such a hyperpolarizing current that firing stops and the model cell is driven to quiescence. In this case, without any spike associated Na+ entry, [Na+]i would be expected to stop rising. However, [Na+]i continues to increase as the Na+ influx is lagged by a parameter τ (refer Methods), which phenomenologically encodes the long duration of Na+ diffusion from the Na+ channel to Na+/K+ pump (refer Discussion for more detail and justification). When τ expires, Na+ influx stops whilst the pump maintains Na+ efflux. This decreases [Na+]i, which reduces hyperpolarizing pump activity and eventually permits the model's spontaneous firing to resume. Firing resumes in the tonic spiking mode, rather than in the bursting mode which preceded the quiescence, as during the quiescence the Na+/K+ pump resets the extracellular K+ concentration and the Kv1.2 channels block to bursting. Thus, the trimodal pattern is reset for another cycle.The Nang modes , as a fuThe trimodal repeat length is the duration of a single repeat of the trimodal pattern. It is described as a constant for an individual Purkinje cell but can reportedly vary from 20 seconds to 20 minutes between different cells +/K+ pump's affinity for internal Na+ (KNa model parameter) sets the “firing length”, which is the combined duration of the tonic and bursting modes. The lower KNa, the smaller the quantity of intracellular Na+ that is needed to stimulate the Na+/K+ pump to a level of pumping that can hyperpolarize the cell to silence, which equates to a shorter period of Na+ entry i.e. a shorter “firing length” .The Na length” B. Repor+/K+ pump's affinity for external K+ (KK model parameter) sets the duration of tonic firing. The higher KK, the shorter the tonic mode  set the duration of the quiescent mode. The smaller τ, the shorter the quiescent phase  (H) is essential to the model's dendritic spontaneity. Without IH in the dendrites, the dendrites cannot fire Ca2+ spikes spontaneously. They can only fire in the presence of somatic drive (data not shown). The importance of IH in the Purkinje cell's electrical response to TTX has been shown experimentally The model has a soma capable of spontaneously firing Na by TTX;  (TTX bloty to 0) . These dty to 0) B propagty to 0) A. Hyper2+ spike activity alternating with periods of quiescence +/K+ pump . In this TTX simulation, although there is no voltage-gated Na+ entry through the resurgent Na+ current, stimulatory Na+ to the Na+/K+ pump can still enter through the Na+/Ca2+ exchanger.In the presence of TTX, somatic activity is bimodal with periods of CaSome Purkinje cells express the trimodal firing pattern without pharmacological manipulation while others express it only upon the condition that GABAergic synaptic inputs are blocked 2+ spike generation. Thus, they prevent the tonic to bursting transition and switch the model from firing in a trimodal pattern  D. This 2+ channel complement  and setting KNa to 10 mM, which is a realistic value Without synaptic input, not all Purkinje cells fire spontaneously - some are quiescent +/K+ pump irreversibly +/K+ pump densities by arbitrary functions of time (Methods), which switched the model's trimodal firing pattern into a continuous burst mode that converged upon depolarization block. This same transition was observed experimentally in whole cell patch clamp recordings from Purkinje cells  following the application of 2.5 µM ouabain (with 10 µM bicuculline present to block GABAergic inputs) (n = 4). After noting that the model and experimental response to ouabain matched, validating the model, we then tuned the model's pump density decline functions to generate a best fit between data . After a time, this continuous bursting attained a shallow gradient of depolarization that eventually converged upon somatic depolarization block . During the progression to somatic depolarization block, the number of Na+ spikes per burst decreased (2+ spikes (from the dendritic arborisation) were observed .In the Purkinje cell response to ouabain , the duration of the trimodal pattern's silent periods became shorter until firing was continuous A Fig. A. The don block A  resistant Na+ entry in the Purkinje cell + entry is through the Na+/Ca2+ exchanger, which has been shown present in Purkinje cells +/Ca2+ exchanger is net depolarizing (−1), inwardly passing 3 Na+ ions for the extrusion of every Ca2+ ion +/K+ pump is net hyperpolarizing (+1) with a stoichiometry of [3 Na+ out: 2 K+ in] +/Ca2+ exchanger and an electrically counterbalancing Na+/K+ pump mechanism (Methods). With Na+/K+ pump loss the counterbalance to the depolarizing force of the Na+/Ca2+ exchanger is lost and depolarization ensues. The sizable Na+/Ca2+ exchanger density at the soma, when not counterbalanced by Na+/K+ pump, puts the soma into depolarization block. However, the lesser Na+/Ca2+ exchange density in the dendrites, when not counterbalanced by Na+/K+ pump, cannot put the dendrites into depolarization block.In this matching experimental and model response to Nain vitro  using slower functions of decline for the model's Na+/K+ densities. To elaborate on point (2) - the effect of ouabain, which blocks a greater and greater fraction of Na+/K+ pumps over time, is replicated in the model by decreasing the model's Na+/K+ densities over time. The model parameters that set this rate of decline  could be changed to pace the decline over ∼1000 seconds rather than ∼10 seconds – they are free parameters, unconstrained by any prior literature. However, the model takes 15 days to simulate ∼1000 seconds  and it would take many trial and error runs  to tune these free parameter values to give a best fit between model and reality. We believe that the model captures the processes that occur in real Purkinje cells under ouabain block, but that these processes run faster and take less time in the model i.e. the model abstracts on the time course. Biological modelling is typically abstractive, often because of computational constraints, and this can be permissible if it is clear where abstraction has been placed.Unfortunately, the ouabain response of the real and model Purkinje cell was not in complete alignment. The model's deflections in the membrane potential, furrowing the depolarization block, are smaller than those observed experimentally. Also, the model's timescale of response to ouabain , presumably because the pump block progression was slower. And with higher ouabain concentrations (10–20 µM), the progression to depolarization block was rapid and the transition through the aforementioned sequence was not observed .In experiments with a lower ouabain concentration (1.5 µM) the same sequence of events occurred but took longer  and which fire in a repeating bimodal pattern of tonic spiking and quiescence. Ouabain (2.5 µM) initially switches their firing from bimodal to trimodal, before a switch to continual bursting and an eventual depolarization block (n = 3). This indicates that the pump is the quiescence generating mechanism for the bimodal pattern of firing as well as for the trimodal pattern. The model is able to accurately reproduce this experimentally observed behaviour (Ouabain block of the Naon block  (n = 3).ehaviour 9.2+ spike activity and quiescence 2+ spike activity (n = 3). This suggests that it is the Na+/K+ pump that generates the quiescent periods in the Purkinje response to TTX. This experiment is well recapitulated by the model (In the presence of TTX (1 µM), the Purkinje cell expresses a repeating bimodal pattern of Caactivity A .in vitro studies .This investigation valuably reconciles the divergent Purkinje behaviours observed in different in vitro – it may be an in vitro artifact - and the length of its constituent modes  is reported as fixed for a particular Purkinje cell +/K+ pumping might act to regulate the length of modes as a signalling element to the deep cerebellar nuclei (DCN). The model shows how modifying the KNa and KK pump parameters, which might be modulated by signalling cascades in vivoTo date, the trimodal firing pattern has only been observed + concentration. Neighboring Purkinje cells share an immediate extracellular milieu, which raises the possibility that adjacent or nearby Purkinje cells can signal to each other through the extracellular K+ concentration variable. This communication could be modulated by glial cells, which buffer K+The model shows that the trimodal pattern of firing is regulated by the extracellular K+/K+ pumping, must match this timescale. Na+/K+ pump activity is stimulated by intracellular Na+ and for it to persist at sufficient intensity during the quiescence, to perpetuate the quiescence, there must be a maintained intracellular Na+ accumulation. This persistence is non-intuitive as pump activity would be expected to quickly erode the Na+ accumulation during quiescence (by continuing to pump Na+ out of the cell in the absence of any voltage-gated Na+ entry), which would down-regulate pump activity. However, intracellular Na+ accumulation has actually been experimentally observed to persist minutes after the end of voltage-gated Na+ entry; a phenomenon that has been ascribed to a non-uniform cytoplasmic distribution of ions +, the fuzzy space and the bulk cytoplasm. A steep Na+ gradient has been experimentally measured between these two spaces +/K+ pump is located in the fuzzy space whilst the voltage-gated Na+ channels might be localised to a “third functional domain” +] “hot spots” alternating with areas of lower [Na+] + channel and pump appear geographically distinct. In the model, the lag parameter (τ) accounts for the duration of sodium's diffusion from channel to pump. This duration can be on the order of seconds to minutes because diffusion is significantly restricted in the fuzzy space by an, as yet, uncharacterized physiochemical process + influx stops it takes time to feed through and reduce the Na+ concentration underneath the pump, which is the critical concentration for pump activity. This lag could account for why the pump current decays on the “order of seconds to minutes”, as reported by Su et al. + entry τ parameter, because an explicit account would be too computationally expensive and ill constrained by the literature. Intracellular Na+ and Ca2+ dynamics are linked  through the action of the Na+/Ca2+ exchanger and intracellular Ca2+ dynamics may be an additional complexity involved.The trimodal pattern's quiescent mode can span seconds or minutes +/K+ pump block might eradicate quiescence in the trimodal and bimodal firing patterns, not because the Na+/K+ pump generates quiescence (per se), but because this block causes depolarization which compromises the activity of the true entity responsible. This is actually a problem inherent to many pharmacological block experiments that seek to parse the function of individual neuronal currents. By knocking out a current, the voltage trajectory is changed and in interpretation one is then unsure as to whether functional changes are due to that loss or due to changes in another current(s), which is regulated by the membrane potential. We did control experiments where we injected hyperpolarizing current to counter the ouabain induced depolarization (n = 3) and in which we still observed the loss of the quiescent mode (data not shown). However, this injection was at the soma and it probably didn't prevent depolarization in the entirety of the elaborate dendritic tree. So, it is with this caveat that we propose the Na+/K+ pump as the generative mechanism to trimodal quiescence.The reader must be aware that Na+/K+ pump block. With hypoglossal motoneurons of the rat, “bath application of a 4–20 µM ouabain solution produced a partial block of Na+/K+ pump activity” and produced “no significant change in either the initial, early, or late phases of spike-frequency adaptation” i.e. 4–20 µM ouabain application did not cease or significantly alter the firing rate of rat motorneuons +/K+ activity in rat brain”. They reported that “ouabain concentrations >20 µM generally resulted in broad, short spikes and eventual cessation of repetitive discharge” (data not shown in their paper). By contrast, in our study with cerebellar Purkinje neurons of the rat, just a 1.5 µM ouabain solution can lead to the cessation of repetitive discharge. And this cessation occurs after a stereotypical sequence of changes, before any significant depolarization, that is not described in (and we presume is not applicable to) the motorneuron study of Different cell types can have different response profiles to a Na+/K+ pump has been shown to promote firing and prevent the generation of quiescent periods in canine coronary sinus fibers. This result is interpreted as evidence that the Na+/K+ pump is the generative mechanism to these quiescent periods +/K+ pump block experiments and our tentative conclusions from them. Although, incidentally, with coronary sinus fibers there is another layer of complexity that is not observed with cerebellar Purkinje cells - with Na+/K+ pumps blocked, quiescent periods can still occur in coronary sinus fibers, in some instances, all be it delayed. These are interpreted to occur because Na+/K+ pump block compromises the maintenance of the Na+ electrochemical gradient, which compromises Na+/Ca2+ exchanger activity, increasing [Ca2+]i and raising Ca2+-activated K+ current flow, which hyperpolarizes the cell - overcomes firing - and generates quiescence. So, again, one can ascertain from this account that different cell types can have very different response profiles to a Na+/K+ pump block.Pharmacological block of the Na+/K+ pump as the drive to trimodal quiescence, we entertained the possibility that a Ca2+-activated K+ current might be responsible. However, we discounted the SK K+ current because the trimodal pattern's quiescent mode can still occur when this current is blocked by apamin + current because the trimodal pattern's quiescent mode can still occur when this current is pharmacologically blocked by IBTX +/K+ pump block experiment could indicate that Ca2+-activated K+ currents are not involved - Na+/K+ pump block has been shown to cause intracellular Ca2+ accumulation in a number of cell types because it compromises the Na+ electrochemical gradient, which the Na+/Ca2+ exchanger requires to pump Ca2+ out of the cell +/K+ pump, but instead by a Ca2+-activated hyperpolarizing current, we might expect Na+/K+ pump block to promote quiescence rather than shorten and eradicate it. Indeed, as aforementioned, Na+/K+ pump block has been shown to cause quiescent periods via Ca2+-activated K+ currents in canine coronary sinus fibers Before we considered the Na+ channels  and its derivatives (the 41 compartment and 5 compartment models), serves to illustrate the reproducibility (semi-quantitative) of the computational experiments performed with the full model.+ has been experimentally observed to accumulate during sustained neural activity +/K+ pump (regulated by the intracellular Na+ concentration) has been shown experimentally to generate quiescent periods in canine coronary sinus fibers The mechanisms that we propose to drive the Purkinje cell's trimodal pattern of firing have been observed in other classes of neuron. Extracellular KIn vitro, repetitive climbing fiber (CF) input  switches Purkinje cells out of the trimodal firing pattern and into a nonbursting pattern of activity in vivo. However, in these experiments, although CF input blocks the trimodal pattern's bursting mode, quiescent periods can still be observed.in vitroin vivo+/K+ pump generates these quiescent periods. However, there are much longer quiescent periods observed, where CF input cannot evoke a complex spike or a state transition to tonic firing , which has symptoms to indicate that it is a pathology of cerebellar computation +/K+ pumps might subserve information processing roles in neurons CF input has been observed to toggle the cell between a tonic firing state and a quiescent state. So, state transitions occur at the frequency of CF input (∼1 Hz) and tonic firing periods of ∼1 s alternate with quiescent periods of ∼1 s (c firing , and we


Motor dysfunction is a consistently reported but understudied aspect of schizophrenia. Postural sway area was examined in individuals with schizophrenia under four conditions with different amounts of visual and proprioceptive feedback: eyes open or closed and feet together or shoulder width apart. The nonlinear complexity of postural sway was assessed by detrended fluctuation analysis (DFA). The schizophrenia group (n = 27) exhibited greater sway area compared to controls (n = 37). Participants with schizophrenia showed increased sway area following the removal of visual input, while this pattern was absent in controls. Examination of DFA revealed decreased complexity of postural sway and abnormal changes in complexity upon removal of visual input in individuals with schizophrenia. Additionally, less complex postural sway was associated with increased symptom severity in participants with schizophrenia. Given the critical involvement of the cerebellum and related circuits in postural stability and sensorimotor integration, these results are consistent with growing evidence of motor, cerebellar, and sensory integration dysfunction in the disorder, and with theoretical models that implicate cerebellar deficits and more general disconnection of function in schizophrenia. Motor abnormalities were noted in some of the first descriptions of schizophrenia and incorporated into early characterizations of this complex disorder Schizophrenia is associated with increased involuntary movements While NSS are traditionally considered nonlocalizing, there is evidence for a specific role of the cerebellum in NSS. For example, there is evidence that cerebellar volume and NSS severity are inversely related Additionally, there are neuroanatomical and behavioral findings that indicate cerebellar dysfunction in schizophrenia, including structural abnormalities in the cerebellum Cerebellar impairments in schizophrenia are particularly important when considered in the context of the theory of cognitive dysmetria. According to this theory, the reciprocal connections between the cerebellum and the frontal cortex  coordinate and sequence motor and cognitive information in a well-timed, fluid manner. This critical circuitry is referred to as the cortico-cerebellar-thalamic-cortical circuit (CCTCC), and there is much evidence of abnormalities in the nodes of the CCTCC in schizophrenia  abnormalities in schizophrenia. However, certain confounds exist that have historically tempered enthusiasm for these important lines of research. Various authors have acknowledged skepticism of the presence of motor deficits in schizophrenia in light of the documented effects of dopamine-blocking antipsychotics on the motor system pattern of motor behavior. This approach expands the breadth of information on motor abnormalities in schizophrenia, and may provide unique insight into the mechanism of the dysfunction.Studies of motor dysfunction in schizophrenia have relied primarily on conventional  statistical methods. In addition to studying group differences in the magnitude and variance of motor behavior, the present study also examined differences in the time-dependent Nonlinear dynamical systems approaches offer such benefits by illuminating the emergent properties of a system When components of a nonlinear system begin to falter due to disease or dysfunction, both the input of individual components and some of the interactions between them begin to disappear from the system. As a result, the process becomes less complex and a stereotyped pattern emerges due to the fact that there are now fewer components and interactions available to generate the complex output There are several novel contributions that will arise from the application of a nonlinear analysis to motor behavior in schizophrenia. Specifically, the investigation of the time-dependent pattern of motor behavior allows for the investigation of dependent variables such as complexity and self-similarity that have yet to be explored in the domain of motor dysfunction in schizophrenia. Given that these variables are emerging as important indicators of dysfunction in the medical field cf. , it is oThe present study expands on the important but understudied area of motor dysfunction in schizophrenia through the application of a nonlinear analysis to postural sway. Postural sway is the area over which one’s center of pressure moves during the process of maintaining upright posture. Unlike the more gross assessments of upright balance found in NSS batteries, very sensitive instruments are required to quantitatively measure the small-scale alterations in center of pressure characteristic of postural sway, which occurs on a scale of millimeters. This fine motor behavior requires cerebellar integration of visual, proprioceptive, and vestibular information to correctly orient the body in space and maintain an upright standing position In the only such study to date, Marvel and colleagues negative symptoms are most consistently associated with NSS in schizophrenia. The current study investigated whether this relationship between motor abnormalities and symptom severity would be present in the domain of postural control by assessing the strength of the correlations between postural sway dependent variables and subscales of the Positive and Negative Syndrome Scale (PANSS) The present study also investigated the relationship of postural sway abnormalities to symptom severity. In a review of NSS in schizophrenia, Bombin and colleagues Given the empirical evidence of cerebellar deficits in schizophrenia as well as the theoretical importance of the cerebellum as a node in the CCTCC, it was hypothesized that abnormal postural sway would be present in individuals with schizophrenia, manifested both as increased postural sway  with schizophrenia (n = 14) or schizoaffective disorder (n = 13) comprised the schizophrenia sample and were compared with 37 non-psychiatric comparison subjects  . All parDiagnoses were determined through the Structured Clinical Interview for DSM-IV Axis I disorders (SCID-I) Exclusion criteria included current drug or alcohol abuse or dependence, and report of loss of consciousness. Control participants were excluded for past drug or alcohol dependence. A one-way ANOVA revealed no significant interactions between diagnosis (schizophrenia versus control) and age, height, weight or body mass index (BMI), and Pearson’s chi-square test revealed no systematic relationship between gender and diagnosis. Medication regimens in the clinical sample were as follows: atypical antipsychotics (n = 6); typical antipsychotics (n = 6); mood stabilizers or elevators (n = 14); and unmedicated (n = 6).Participants were asked to stand as still as possible with their arms resting comfortably at their sides on an AMTI Accusway  force platform under four conditions with different vision (eyes open vs. closed) and base (feet together vs. shoulder-width apart) conditions. Two minute recordings of center of pressure (COP) were made for each of the following conditions for each participant in this order: eyes open, closed base (EOCB); eyes closed, closed base (ECCB); eyes closed, open base (ECOB); and eyes open, open base (EOOB), sampled at 50 Hz along the anteroposterior and mediolateral axes.th order Butterworth lowpass filter with a 25 Hz cutoff frequency was applied to recordings to isolate the low-frequency postural sway process. COP and its 95% confidence area encompassed by COP movement was measured using principal component analysis (PCA) A 9The COP data were also subjected to detrended fluctuation analysis (DFA) to quantify the complexity of the postural sway time series. DFA was applied to the mediolateral and anteroposterior dimensions independently. These dimensions are known to be driven by different muscles and joint action and are differently affected by the loss of sensory inputs. Due to these fundamental differences in input and output, movement along the mediolateral and anteroposterior dimensions possesses distinct characteristic dynamics DFA indexes the relative distribution of variance within the data across a range of different time scales. This produces a profile of the time series in terms of the correlation/anticorrelation properties of the signal, quantified by the rate of growth in fluctuation of variance as a function of increasing time scales. The growth in fluctuation magnitude across time scales is indexed by the slope of this function in double-logarithmic space  . á-valueA major advantage of applying DFA is the sensitivity of this analysis to the differential contributions of sensory inputs that occur on different timescales. The current study manipulates both visual and proprioceptive input. Visual information is relatively slow sensory input, occurring at lower frequencies and on slower time scales Repeated measures ANOVAs were performed on sway area and DFA in the mediolateral (ML) and anteroposterior (AP) directions. Eyes (open or closed) and base (open or closed) were within-subjects factors and group was the between subjects factor.To explore the potential confounding effects of alcohol on postural sway in schizophrenia, all ANOVAs were repeated excluding schizophrenia participants dually diagnosed for past alcohol dependence (n = 14). All ANOVAs also were repeated comparing singly- (n = 13) and dually-diagnosed (n = 14) individuals with schizophrenia. To address the potential effect of dopamine-blocking antipsychotic medication, chlorpromazine equivalent dosages were computed for all participants taking medications for which accepted chlorpromazine conversions had been established (n = 17) M = 75.44 mm2, SE = 11.43 mm2) compared to controls   = 8.42, p = 0.005, ηp2 = 0.12), indicating abnormalities in postural control. There were main effects of eyes  = 13.47, p = 0.001, ηp2 = 0.18), and base  = 5.27, p = 0.025, ηp2 = 0.08), indicating that sway area was greater when eyes were closed  and when feet were adjacent in the closed base condition , respectively.Analysis of sway area revealed a main effect of group, with mean sway area larger in schizophrenia participants (p2 = 0.07)  = 4.431, p = 0.039, η = 0.07) . Post-ho = 0.07) . These rp2 = 0.085) and base  = 78.725, p<0.001, ηp2 = 0.559). As expected M = 1.371, SE = 0.013) compared to open . When vision, a slower sensory input that provides lower-frequency information on longer timescales is removed, faster, higher frequency sensory processes such as proprioception dominate the control of posture and therefore generate a more complex output. Conversely, average DFA values were larger  in the closed  compared to the open base  conditions. This is also consistent with expectations, as proprioceptive information is transmitted at a very high frequency p2 = 0.045), with the schizophrenia group exhibiting less complex postural sway.Analysis of DFA-ML values revealed a significant main effect of eyes  = 5.79, p = 0.010, ηp2 = 0.23), a main effect of base  = 5.00, p = 0.029, ηp2 = 0.08), and a significant eyes X base interaction  = 47.23, p<0.001, ηp2 = 0.43)  = 18.33, p<0.001, η.43) see . There wWhen individuals with schizophrenia who also had a history of alcohol dependence were excluded, results were unchanged for the analysis of sway area.p2 = 0.080), with schizophrenia participants without a history of alcohol dependence exhibiting higher DFA-ML values  compared to controls , and therefore less complex postural sway along this axis. This decrease in postural sway complexity in the schizophrenia group indicates that there are potentially fewer and/or slower sensory components contributing to the process of postural control.Main effects of eyes and base of the same direction remained significant for the analysis of DFA-ML values. However, a significant main effect of group emerged  = 4.151, p = 0.047, ηp2 = 0.076), while the main effect of base and eyes X base interaction remained significant  = 3.924, p = 0.053, ηcant see .p2 = 0.081), indicating decreased complexity of postural sway in singly-diagnosed individuals with schizophrenia  compared to controls . There was also a significant eyes X group interaction  = 8.888, p = 0.004, ηp2 = 0.156), indicating that only controls demonstrated the expected increase in complexity upon the removal of visual input (p2 = 0.185), in which only dually diagnosed individuals demonstrated the expected increase in complexity when eyes were closed compared to open  = 4.204, p = 0.046, ηal input   and off (n = 6) antipsychotic medication revealed no main effects of group or group interactions for any postural sway dependent variable.There was a significant correlation between sway area in the EOOB condition and the general psychopathology (r(22) = 0.466, p = 0.022) PANSS factor. However, this correlation did not survive á-level Bonferroni correction for the 4 sway conditions (p>0.0125). There were also significant correlations between DFA-ML values in the ECCB condition and the negative (r(23) = 0.404, p = 0.045) and general psychopathology (r(23) = 0.531, p = 0.006) PANSS factors, with the correlation between DFA-ML values and the general psychopathology PANSS factor surviving Bonferroni correction (p<0.0125) .The application of traditional and nonlinear analyses to postural sway indicated that participants with schizophrenia exhibited increased but less complex postural sway compared to controls. Schizophrenia was associated with inefficient adaptation to a loss of visual information  during maintenance of posture. These abnormalities are likely due to abnormal integration of sensory information from a variety of modalities critical to the process of postural control . Importantly, the findings could not be explained by medication effects or a history of alcohol dependence in the schizophrenia sample. Finally, postural sway deficits were associated with increased symptom severity. Given the importance of the cerebellum in postural control, the observed sway abnormalities in schizophrenia are consistent with evidence of cerebellar and sensory integration dysfunction in schizophrenia.Analyses of sway area replicated previous findings structure of the postural sway process. Specifically, DFA indexes the complexity of postural sway and can be informative as to how many and what type of component processes underlie the maintenance of posture. The finding of decreased postural sway complexity in schizophrenia in both the mediolateral and anteroposterior directions is consistent with findings of decreased complexity in a variety of pathological physiological processes DFA provides a more complete characterization of postural sway than sway area alone because it reveals information about the In addition, an eyes by group interaction emerged for DFA-AP values when dually diagnosed schizophrenia participants were excluded. This interaction was driven by greater complexity in the eyes closed compared to the eyes open condition in controls, but not in schizophrenia participants. Removal of visual input should result in increased sway complexity because visual input normally provides low-frequency feedback for postural sway on longer timescales Results from the analysis of sway area were unchanged when schizophrenia participants with a history of alcohol dependence were excluded. However, additional group effects emerged for DFA upon exclusion of these individuals. The only difference that emerged when singly and dually diagnosed schizophrenia participants were compared on all sway dependent variables was an eyes by group interaction for DFA-AP, in which the dually diagnosed group demonstrated the predicted change in complexity between the eyes conditions. These results indicate that the current findings were not driven by the effects of past alcohol dependence-related cerebellar damage in the schizophrenia sample.No results appear to be driven by the effects of antipsychotics on the motor system, as there were no significant correlations between chlorpromazine equivalent dosage values and any of the postural sway dependent variables. Furthermore, there were no group effects or interactions for any of the postural sway dependent variables when the individuals with schizophrenia taking antipsychotic medications were compared to those who were not.The general psychopathology subscale of the PANSS correlated with mediolateral DFA in the most difficult condition (ECCB). This finding of symptom correlates of postural sway is consistent with the existing literature Abnormalities in postural sway in schizophrenia manifested through both larger and less complex postural sway are likely (if not exclusively) indicative of cerebellar dysfunction in schizophrenia. Such dysfunction is consistent with the cognitive dysmetria theory of schizophrenia, in which the cerebellum acts as a general coordinative organ in the CCTCC for both motor and cognitive functions Several limitations of the current study must be addressed. First, although a concerted effort was made to rule out potential confounds of medication regimens, possible effects of the dopamine-blocking properties of antipsychotics on the motor system could not be completely eliminated. While the AIMS is sensitive to general movement abnormalities, other measures such as the Simpson-Angus Extrapyramidal Side Effects Scale Second, because there is strong evidence for the involvement of the cerebellum in integrating visual, proprioceptive, and vestibular information for the maintenance of posture Third, there is evidence to suggest that individuals with schizophrenia age more rapidly than non-psychiatric controls, particularly later in life Finally, the results of the current study are consistent with models of schizophrenia that point to cerebellar dysfunction as well as theories postulating functional and structural disconnection. As a result, future studies of motor abnormalities in schizophrenia should aim to examine more integrative functions rather than sensory or motor functions alone.Supporting Information S1Sway area eyes X group interaction post-hoc pair-wise comparisons.(DOC)Click here for additional data file.Supporting Information S2DFA-AP eyes X base interaction post-hoc pair-wise comparisons.(DOC)Click here for additional data file.Supporting Information S3DFA-AP eyes X base interaction  post-hoc pair-wise comparisons.(DOC)Click here for additional data file.Supporting Information S4DFA-AP eyes X group interaction  post-hoc pair-wise comparisons.(DOC)Click here for additional data file.Supporting Information S5DFA-AP eyes X group interaction  post-hoc pair-wise comparisons.(DOC)Click here for additional data file.


Multivariate regression analysis revealed that PNPLA3 148 M was significantly associated with serum AFP level (p = 0.032), other than body mass index, platelet count, and alanine aminotransferase , and that serum AFP level was significantly associated with PNPLA3 148 M (p = 0.017). Serum AFP level is an important factor in predicting HCC development after the antiviral therapy for Japanese patients with chronic hepatitis C, the mechanism of which might involve its significant associations with the SNP genotype of PNPLA3.The impact of single-nucleotide polymorphisms (SNP) of patatin-like phospholipase domain-containing protein 3 (PNPLA3) on development of hepatocellular carcinoma (HCC) is not clarified for Japanese patients with chronic hepatitis C. The present study investigated the associations of rs738409 PNPLA3 with HCC development after the antiviral therapy with peg-interferon and ribavirin for Japanese patients with hepatitis C virus serotype 1 and high viral load. Of the 271 patients enrolled in the study, 20 patients developed HCC, during a median follow-up period of 4.6 years. Multivariate analysis in the proportional hazards models revealed that sex, body mass index, platelet counts, and alpha feroprotein (AFP) had significant associations with HCC development ( Hepatitis C virus (HCV) infection causes chronic hepatitis, and may progress to liver cirrhosis and hepatocellular carcinoma (HCC). More than 170 million people worldwide are infected with HCV, creating a serious global health problem Kato . CombinaRecently, the single-nucleotide polymorphism (SNP) of rs738409 patatin-like phospholipase domain-containing protein 3 (PNPLA3) has been identified for its significant associations with liver steatosis and fibrosis in patients with fatty liver disease and alcoholic liver disease  or 2b (1.5 μg/body weight/week) with ribavirin (600-1000 mg/day) at either Okayama University Hospital or Kagawa Prefectural Central Hospital. The outcomes of the interferon therapy were evaluated, according to the practice guidelines by the American association for the study of liver diseases, and the Japan society of hepatology . The SNPs of rs8099917 IL28B, and rs738409 PNPLA3 were genotyped using the TaqMan predesigned SNP genotyping assays, as recommended by the manufacturer . The SNP genotypes of all the samples could be obtained with these systems.For the 241 patients in the study, variations of the amino acid 70 of HCV core (arginine or glutamine) were determined with direct sequence method. The amplifications of HCV core in the serum were performed as previously reported .Data are expressed as means ± standard deviations. Proportional hazards models were utilized to identify the factors associated with HCC development during the follow-up period after interferon therapy. The incidences of HCC development were also estimated using the Kaplan–Meier method and compared with the log-rank test. Factors associated with the SNP genotype of PNPLA3 and AFP were analyzed by stepwise logistic regression analysis. A value of p = 0.0067, Table p = 0.048). The incidences of HCC development were compared among patient groups formed according to interferon therapy outcomes . The SVR and relapse patients had similar risks of HCC development during the first 2 years after interferon therapy, while relapse patients had increased risk thereafter.The characteristics of patients enrolled in the study are shown in Table  Figure A. The NVp = 0.0062, Hazards ratio 1.2, p = 0.0086, Hazards ratio 3.8, p <0.0001, Hazards ratio 0.77, p <0.0001, and Hazards ratio 1.1, p = 0.0002, respectively). The incidence of HCC development was significantly lower in the patients with PNPLA3 148 M than in those with PNPLA3 148I . Liver steatosis and amino acid 70 of HCV core affected HCC development slightly , while the SNP genotype of IL28B was not associated with the risk of HCC development . Stepwise multivariate analysis revealed that male gender, high BMI, low platelet counts, and high levels of AFP had significant associations with HCC development .We compared the patient characteristics related to HCC development in proportional hazards models Table . Male gep = 0.0058, the log-rank test, Figure p = 0.021). We further examined the patient characteristics prior to the interferon therapy regarding associations with serum AFP by stepwise logistic regression analysis . The SNP genotype of PNPLA3 was also an independent factor associated with serum AFP (p = 0.032). Therefore, we studied the patient characteristics associated with the PNPLA3 148 M genotype (Table p = 0.017). Advanced liver fibrosis was also associated with PNPLA3 148 M in univariate analysis (p = 0.028), although this association did not show statistical significance in multivariate analysis (p = 0.20).Serum AFP levels prior to the interferon therapy were significantly associated with HCC development after interferon therapy (s Table . The resThe impact of the SNP of PNPLA3 on interferon therapy and HCC development after interferon therapy is still undetermined for Japanese patients with chronic hepatitis C. The present study investigated the factors associated with HCC development after interferon therapy for Japanese patients with chronic hepatitis C, and is the first to clarify significant associations of the SNP of PNPLA3 with HCC development after interferon therapy.The incidence of HCC development was significantly lower in the patients with PNPLA3 148 M than in those with PNPLA3 148I Table , which mSerum AFP was only the factor we found to be significantly associated with the PNPLA3 genotype in the present study; patients with PNPLA3 148 M had lower levels of serum AFP than those with PNPLA3 148I. Meanwhile, multiple factors such as advanced liver fibrosis, inflammation, and body composition were significantly associated with serum AFP. Only the SNP genotype of PNPLA3 is the significant factor that is genetically determined. The associations of serum AFP with HCC development and the mechanisms by which the SNP genotypes of PNPLA3 contribute to serum AFP are still unknown, and need to be clarified in a future study.Recent studies on Caucasian patients have shown that PNPLA3 148 M was significantly associated with liver steatosis (Poynard et al. In conclusion, serum AFP level is an important factor in predicting HCC development after the antiviral therapy for Japanese patients with chronic hepatitis C, the mechanism of which might involve its significant associations with the SNP genotype of PNPLA3.


PKCε−/− MEFs exhibited reduced insulin uptake which was associated with decreased insulin receptor phosphorylation, while downstream signalling through IRS-1 and Akt was unaffected. Cellular fractionation demonstrated that PKCε deletion changed the localization of the insulin receptor, a greater proportion of which co-fractionated with flotillin-1, a marker of membrane microdomains. Insulin stimulation resulted in redistribution of the receptor in WT cells, while this was markedly reduced in PKCε−/− cells. These alterations in insulin receptor trafficking were associated with reduced expression of CEACAM1, a receptor substrate previously shown to modulate insulin clearance. Virally-mediated reconstitution of PKCε in MEFs increased CEACAM1 expression and partly restored the sensitivity of the receptor to insulin-stimulated redistribution. These data indicate that PKCε can affect insulin uptake in MEFs through promotion of receptor-mediated endocytosis, and that this may be mediated by regulation of CEACAM1 expression.We have previously shown that deletion of protein kinase C epsilon (PKCε) in mice results in protection against glucose intolerance caused by a high fat diet. This was in part due to reduced insulin uptake by hepatocytes and insulin clearance, which enhanced insulin availability. Here we employed mouse embryonic fibroblasts (MEFs) derived from wildtype (WT) and PKCε-deficient (PKCε Mice expressing a dominant negative CEACAM1 mutant in liver developed hyperinsulinemia resulting from reduced insulin clearance due to impaired insulin receptor internalization −/− mice. We found that the absence of PKCε in these cells was associated with alterations in insulin receptor trafficking, and that this may be linked to the expression levels of CEACAM1.We have examined this further in mouse embryonic fibroblasts (MEFs) generated from wild type (WT) and PKCεGeneral reagents of analytical grade were obtained from Sigma-Aldrich Pty. Ltd. , Calbiochem  and Invitrogen  unless otherwise stated. The human insulin analogue Actrapid™ was from Novo Nordisk  and fluorescein isothiocyanate (FITC)-labelled insulin was from Molecular Probes . Complete Protease Inhibitor and PhosSTOP were from Roche Pty. Ltd. . Details of antibodies used are given in −/− mice were generated and genotyped as described previously +/− mice on a pure C57Bl/6 background were mated and WT and PKCε−/− MEFs were isolated from 10-day-old embryos by digestion with trypsin, after removal of visible organs. Cells were maintained in Dulbecco’s Modified Eagle Medium with 5 mM glucose, containing 10% (v/v) fetal calf serum and 1% (v/v) antibiotic/antimycotic solution . Three independently generated pools of WT and PKCε−/− MEFs were selected after genotyping.Ethical approval for mouse studies was granted by the Garvan/St Vincent’s Hospital Animal Ethics Committee . PKCεInsulin uptake by MEFs was determined either by fluorescence microscopy or fluorescence-activated cell sorting (FACS). In each case, cells were serum-starved for 2 hours prior to insulin stimulation for 10 min using 100 nM FITC-labelled insulin, and surface bound insulin removed by acid washes. Full details are given in Gross membrane structure of MEFs was examined by transmission electron microscopy (TEM) after cells were fixed and incubated with osmium tetroxide 2(g) for 15 min in a 50 mL chamber . Lysates were collected and unbroken cells removed by centrifugation at 500 g for 10 min. The protein content of the supernatants was measured by Bradford assay (Biorad), and equal amounts of protein (500 µg) were loaded onto 15% (v/v) iodixanol  and centrifuged at 100,000 g for 4 h to form a continuous gradient. Fractions were removed starting from the top of the gradient for analysis by immunoblotting.After serum starvation for 2 h, cells grown in 15 cm dishes were treated with or without 100 nM insulin for the indicated time. Media was aspirated and cells placed on ice and washed three times with ice-cold PBS. Subsequent procedures were carried out at 4°C. Cells were harvested in 1 ml fractionation buffer . Cells were disrupted by nitrogen cavitation  alone or together with PKCε were generated and characterised using the pAdEasy system as described previously Rat PKCε cDNA, a gift from G. Baier, University of Innsbruck, Austria −/− mice displayed decreased insulin clearance in vivo and that primary hepatocytes isolated from PKCε−/− mice exhibited reduced insulin uptake −/− animals. The presence or absence of PKCε expression was confirmed by PCR (not shown) and immunoblot analysis , which affected the intracellular routing of the receptor to regulate sorting between degradative and retroendocytotic pathways 125-labelled insulin uptake by primary PKCε−/− hepatocytes −/− MEFs was observed in fractions enriched for flotillin-1, suggesting localization within membrane microdomains. In addition, insulin stimulation had little effect on receptor distribution, in contrast to the effects of the hormone on the receptor in fractions from WT MEFs. This was associated with markedly reduced levels of receptor autophosphorylation. Because the phosphorylation of downstream signalling components was not defective, and phosphatase treatment of MEFs was able to restore receptor phosphorylation, these findings may indicate rapid dephosphorylation specifically of the insulin receptor after initial activation of its intrinsic tyrosine kinase activity and phosphorylation of IRS-1. Signalling may also be enhanced by the prior location of the receptor to flotillin-enriched lipid rafts −/− cells, although this did not appear to be transmitted to Akt activation. A discrepancy between insulin receptor and IRS-1 tyrosine phosphorylation has also been observed in the absence of Grb14, under which conditions dephosphorylation of the receptor is promoted while downstream signalling is in fact enhanced The data obtained using FITC-labelled insulin is in good agreement with our previous work showing a reduced rate of I−/− MEFs expressed CEACAM1 at far lower levels than WT cells, and that two different approaches resulting in reconstitution of PKCε restored CEACAM1 protein, implicating PKCε as a regulator of CEACAM1 expression. The fact that longer term retrovirally-mediated PKCε reconstitution, even at lower levels than observed with acute adenovirus treatment, was able to restore CEACAM1 more fully may indicate that this effect is indirect, and may be an adaptation to the presence of PKCε which confers a survival or proliferative advantage on the MEFs.The difference in insulin receptor redistribution led us to examine CEACAM1 protein levels in MEFs. CEACAM1 plays a role in insulin receptor endocytosis −/− MEFs, and the failure of insulin to promote insulin receptor redistribution, these findings suggest that the effect of PKCε deletion on insulin receptor trafficking could be mediated by a loss of CEACAM1. We speculate that this reduces the ability of the receptor to be recruited from lipid microdomains into clathrin coated pits for endocytosis, in agreement with the proposed role of CEACAM1 Taken together with the altered localization of insulin receptor in unstimulated PKCεInhibition of insulin receptor internalization has previously been shown to selectively attenuate insulin signalling pathways. Consistent with the data presented here, a reduction in clathrin-mediated endocytosis of the receptor, mediated by overexpression of a dominant-interfering dynamin mutant, did not significantly affect IRS-1 tyrosine phosphorylation or Akt activation −/− mice, rather than primary hepatocytes, which would be more closely relevant to the study of insulin internalization. The mechanisms involved in insulin receptor trafficking in these different cell types could be different. However, insulin uptake is defective in both cell types, indicating that the defect in insulin receptor trafficking is associated with the deletion of PKC independent of the cell type. Interestingly, mice with a null mutation of CEACAM1 exhibit increased triglyceride storage in the liver, which is similar to short term effects observed in PKCε−/− mice fed a high fat diet It is important to note that the experiments reported here concerned different clones of MEFs derived from PKCεIn summary, the data presented here support the hypothesis that ablation of PKCε alters insulin receptor trafficking, and that this could be mediated through alterations in the expression of the insulin receptor substrate, CEACAM1.Methods S1Methodological details of antibodies used, fluorescence microscopy, fluorescence-activated cell sorting, transmission electron microscopy, and 2-photon microscopy are given.(DOCX)Click here for additional data file.


Background: Over 20 genetic risk factors have been confirmed to associate with elevated risk for Alzheimer’s disease (AD), but the identification of environmental and/or acquired risk factors has been more elusive. At present, recognized acquired risks for AD include traumatic brain injury, hypercholesterolemia, obesity, hypertension, and type 2 diabetes.Methods: Based on reports associating various inhalants with AD pathology, we investigated the possibility that air pollution might contribute to AD risk by exposing wild-type mice to a standard air pollution modeling system employing nickel nanoparticle-enriched atmosphere for 3 hr.Results: Mice exposed to air pollution showed 72-129% increases in brain levels of both amyloid-β peptides Aβ40 and Aβ42, as well as Aβ42/40 (p <0.01).Conclusions: These effects on elevation of brain Aβ exceed those associated with trisomy 21, a known risk for early onset AD pathology, raising the possibility that clinical importance might be attached. Further work is required to establish the molecular and physiological basis for these phenomena. The rapid, dramatic effect, if verified, would suggest that inhalant exposures should be evaluated for their possible roles in contributing to the environmental risk for common forms of AD. Similarly, inhaled aluminum dust has been associated with neurotoxic effects and pre-clinical cognitive impairment3. Certain inhalation anesthetics have also been implicated in elevating AD risk, possibly by exacerbating the neurotoxic oligomerization of the amyloid-β (Aβ) peptide4. The early involvement of the olfactory cortex in AD has caused longtime speculation that some inhaled agent might play a role in AD risk5.One common neurodegenerative disease, Parkinson’s disease, has been linked to exposure to MPTP  and to inhaled manganese7. Furthermore, impaired cognition has been recently attributed to air pollution exposure in some populations8. These converging lines of evidence led us to analyze brain levels of Aβ40 and Aβ42 in mice exposed to an inhaled particulate matter  model of air pollution.Recently, AD pathology was identified in young people living in areas with high levels of air pollution3, which is the current Occupational Safety and Health Administration’s Permissible Exposure Limit for nickel hydroxide [http://www.osha.gov/pls/oshaweb/owadisp.show_document?p_table=standards&p_id=9992]) (n = 16 per group) or control filtered air (n = 5 per group) for 3 hours in a nose-only exposure chamber. This protocol has been established as a model for air pollution toxicity in pulmonary disease9, atherosclerosis10, and insulin resistance11. Twenty-four hours post exposure, mice were given pentobarbital, bled out via the vena cava, and then their brains were harvested, snap frozen and stored at -80ºC until assay. For measurement of endogenous mouse brain Aβ40 and Aβ42, we employed the Schmidt method12 and human/rat Aβ 1–40/1–42 ELISA kits . Statistical analysis was performed via Mann-Whitney test. #8 Ni NP is excluded from the analysis due to being more than 2 SD's away from mean or closest value.All procedures involving animals were conducted in compliance with guidelines for ethical animal research and approved by the New York University School of Medicine Animal Care and Use Committee. Two-month-old male and female FVBN mice  were randomly assigned to Ni NP inhalation , and Aβ42 was increased by 2.29-fold . Aβ42/40 ratio was also increased in the Ni NP-exposed group compared to the filtered air control group . Both male and female mice responded similarly to Ni NP exposure .Both endogenous Aβ40 and Aβ42 were elevated in the brains of mice following Ni NP exposure . Aβ40 wa3) (n = 16 per group) versus filtered air (n = 5 per group) for 3 hours in a nose-only exposure chamber. *Mouse 8 was excluded from the data analysis as these results were 2 SD's away from mean or closest value.Raw data of endogenous mouse brain Aβ40 and Aβ42 levels (pmol/L) in mice exposed to nickel nanoparticles  might increase the risk for AD by elevating levels of brain Aβ. We have not identified whether this accumulation occurs at the level(s) of transcription, translation, or post-translational processing. It is tempting to speculate that the well-known links between inhaled toxins and brain inflammation, and other links between brain inflammation and AD established by Griffin and colleagues13 may underlie these phenomena.These data add credence to the proposalThe changes that we observed were dramatic, rapid, and unexpected. Human Aβ is more aggregatable than murine Aβ, making it conceivable that the effect on Aβ levels in human brain could be even greater. While elucidating the genesis and molecular underpinnings will be an important next step, an even more important step will be a rigorous application of environmental toxicology and epidemiology to determine whether the elevated brain Aβ caused in mice by this air pollution model corresponds to any situation of authentic human inhalation exposure that is linked to an increased risk for AD. This is an intriguing observation that might potentially have important relevance to the etiopathogenesis of Alzheimer’s Disease.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Linkage of environmental air toxicity from nanoparticles leading to Alzheimer-like changes in the brain of mice opens a new avenue to understanding the development of brain diseases. These findings are consistent with prior work showing particulate air pollution can affect the brains of human as well as dogs living in polluted cities.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Neurodegenerative consequences are a looming possibility with the current increased burden of air-borne pollutants. Many studies have suggested an association between anesthesia administered to older adults and cognitive decline and development or progression of Alzheimer’s disease (AD). A number of studies have already established links between air-borne pollutants and risk for development of systemic disorders several of which, like AD are inflammatory in nature. Gandy and his colleagues have taken a purposeful step toward more directly connecting air-pollution to increased risk for development of AD in a study of wild type mice exposed to a toxin at the OSHA permissible level for an 8h human exposure. The results are very convincing, showing that a one-time 3h exposure to nickel hydroxide nanoparticles at the “permissible” level doubled brain levels of Aβ40 and Aβ42 within 24h—increases that are similar to levels reported in younger adults with Down’s syndrome. Interestingly, as noted by the authors, and in accord with systemic inflammatory consequences reported following exposure to air-borne pollutants, neuroinflammatory changes that drive Alzheimer neuropathological change may also be elicited by such exposure. This may be particularly important as we necessarily inhale air that appears to be “clean,” but which contains permissible levels of agents that may have adverse effects on the brain, especially in persons with genetic risk factors and or co-morbid conditions that already predispose them for development of AD.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.