Depolarization Events Research Articles

Resolving the biophysics of axon transmembrane polarization in a single closed-form description

When a depolarizing event occurs across a cell membrane there is a remarkable change in its electrical properties. A complete depolarization event produces a considerably rapid increase in voltage that propagates longitudinally along the axon and is accompanied by changes in axial conductance. A dynamically changing magnetic field is associated with the passage of the action potential down the axon. Over 75 years of research has gone into the quantification of this phenomenon. To date, no unified model exist that resolves transmembrane polarization in a closed-form description. Here, a simple but formative description of propagated signaling phenomena in the membrane of an axon is presented in closed-form. The focus is on using both biophysics and mathematical methods for elucidating the fundamental mechanisms governing transmembrane polarization. The results presented demonstrate how to resolve electromagnetic and thermodynamic factors that govern transmembrane potential. Computational results are supported by well-established quantitative descriptions of propagated signaling phenomena in the membrane of an axon. The findings demonstrate how intracellular conductance, the thermodynamics of magnetization, and current modulation function together in generating an action potential in a unified closed-form description. The work presented in this paper provides compelling evidence that three basic factors contribute to the propagated signaling in the membrane of an axon. It is anticipated this work will compel those in biophysics, physical biology, and in the computational neurosciences to probe deeper into the classical and quantum features of membrane magnetization and signaling. It is hoped that subsequent investigations of this sort will be advanced by the computational features of this model without having to resort to numerical methods of analysis.

High-density mapping of atrial fibrillation in a chronic substrate: Evidence for distinct modes of repetitive wavefront propagation

BackgroundTemporal dynamics of electrical wave propagation during AF is unknown. There are reports of transient linking of atrial activation. We aim to characterize temporal dynamics of wave propagation patterns during AF in an established chronically remodeled substrate. MethodsBi-atrial epicardial mapping of AF (mean duration 62±61s) was performed in 13 sheep with induced hypertension using custom-designed plaques. Wave propagation patterns were classified into periods of repetitive activity termed modes. ResultsIn total, we identified 9241 distinct depolarization events which were classified as: passing wave (69% occurrence, 68.6% of total time), point source (20.4%, 13.1%), wave collision (4%, 2.8%), re-entrant wave (0.7%, 6.3%), half-rotation (2.9%, 4.4%), wave splitting (2.7%, 4.3%), conduction block (0.05%, 0.03%) and figure of eight reentry (0.05%, 0.05%). Episodes of re-entrant activity had mean length 701±1012ms. A total of 435 modes of distinct periods of repetitive activity were detected (121 in LA and 314 in RA). Looking at temporal changes between modes, we found a preferential transition: change between train of waves propagating from direction of coronary sinus and reentrant activity. High density mapping of the hypertensive fibrillating atria observed 20% point sources and 0.7% of reentrant activation which may have served as drivers of AF. Remaining activations were peripheral waves. Majority of the activation was organized into events of transient linking with existence of preferential types of transitions. ConclusionsThese findings support the importance of substrate based regions of anatomically or functionally determined preferential conduction in the maintenance of AF.

Copper-induced activation of TRP channels promotes extracellular calcium entry, activation of CaMs and CDPKs, copper entry and membrane depolarization in Ulva compressa.

In order to identify channels involved in membrane depolarization, Ulva compressa was incubated with agonists of TRP channels C5, A1 and V1, and the level of intracellular calcium was detected. Agonists of TRPC5, A1 and V1 induced increases in intracellular calcium at 4, 9, and 11 min of exposure, respectively, and antagonists of TRPC5, A1, and V1 corresponding to SKF-96365 (SKF), HC-030031 (HC), and capsazepin (CPZ), respectively, inhibited calcium increases indicating that functional TRPs exist in U. compressa. In addition, copper excess induced increases in intracellular calcium at 4, 9, and 12 min which were inhibited by SKF, HC, and CPZ, respectively, indicating that copper activate TRPC5, A1, and V1 channels. Moreover, copper-induced calcium increases were inhibited by EGTA, a non-permeable calcium chelating agent, but not by thapsigargin, an inhibitor of endoplasmic reticulum (ER) calcium ATPase, indicating that activation of TRPs leads to extracellular calcium entry. Furthermore, copper-induced calcium increases were not inhibited by W-7, an inhibitor of CaMs, and staurosporine, an inhibitor of CDPKs, indicating that extracellular calcium entry did not require activation of CaMs and CDPKs. In addition, copper induced membrane depolarization events at 4, 8, and 11 min and these events were inhibited by SKF, HC, CPZ, and bathocuproine, a specific copper chelating agent, indicating that copper entry through TRP channels leads to membrane depolarization. Moreover, membrane depolarization events were inhibited by W-7 and staurosporine, indicating that activation of CaMs and CDPKs is required to allow copper entry through TRPs. Interestingly, copper-induced calcium increases and depolarization events were light-dependent and were inhibited by DCMU, an inhibitor of photosystem II, and ATP-γ-S, a non-hydrolizable analog of ATP, suggesting that ATP derived from photosynthesis is required to activate TRPs. Thus, light-dependent copper-induced activation TRPC5, A1 and V1 promotes extracellular calcium entry leading to activation of CaMs and CDPKs which, in turn, promotes copper entry through TRP channels and membrane depolarization.

Modeling of stochastic behavior of pacemaker potential in interstitial cells of Cajal

It is widely accepted that interstitial cells of Cajal (ICCs) generate pacemaker potentials to propagate slow waves along the whole gastrointestinal tract. Previously, we constructed a biophysically based model of ICCs in mouse small intestine to explain the pacemaker mechanism. Our previous model, however, could not explain non-uniformity of pacemaker potentials and random occurrence of unitary potentials, thus we updated our model. The inositol 1,4,5-trisphosphate (IP3)-mediated Ca2+ mobilization is a key event to drive the cycle of pacemaker activity and was updated to reproduce its stochastic behavior. The stochasticity was embodied by simulating random opening and closing of individual IP3-mediated Ca2+ channel. The updated model reproduces the stochastic features of pacemaker potentials in ICCs. Reproduced pacemaker potentials are not uniform in duration and interval. The resting and peak potentials are −75.5 ± 1.1 mV and −0.8 ± 0.5 mV, respectively (n = 55). Frequency of pacemaker potential is 14.3 ± 0.4 min−1 (n = 10). Width at half-maximal amplitude of pacemaker potential is 902 ± 6 ms (n = 55). There are random events of unitary potential-like depolarization. Finally, we compared our updated model with a recently published model to speculate which ion channel is the best candidate to drive pacemaker depolarization. In conclusion, our updated mathematical model could now reproduce stochastic features of pacemaker activity in ICCs.

O-hexadecyl-dextran entrapped berberine nanoparticles abrogate high glucose stress induced apoptosis in primary rat hepatocytes.

Nanotized phytochemicals are being explored by researchers for promoting their uptake and effectiveness at lower concentrations. In this study, O-hexadecyl-dextran entrapped berberine chloride nanoparticles (BC-HDD NPs) were prepared, and evaluated for their cytoprotective efficacy in high glucose stressed primary hepatocytes and the results obtained compared with bulk berberine chloride (BBR) treatment. The nanotized formulation treated primary hepatocytes that were exposed to high glucose (40 mM), showed increased viability compared to the bulk BBR treated cells. BC-HDD NPs reduced the ROS generation by ∼3.5 fold during co-treatment, prevented GSH depletion by ∼1.6 fold, reduced NO formation by ∼5 fold and significantly prevented decline in SOD activity in stressed cells. Lipid peroxidation was also prevented by ∼1.9 fold in the presence of these NPs confirming the antioxidant capacity of the formulation. High glucose stress increased Bax/Bcl2 ratio followed by mitochondrial depolarization and activation of caspase-9/−3 confirming involvement of mitochondrial pathway of apoptosis in the exposed cells. Co- and post-treatment of BC-HDD NPs prevented depolarization of mitochondrial membrane, reduced Bax/Bcl2 ratio and prevented externalization of phosphatidyl-serine confirming their anti-apoptotic capacity in those cells. Sub-G1 phase apparent in high glucose stressed cells was not seen in BC-HDD NPs treated cells. The present study reveals that BC-HDD NPs at ∼20 fold lower concentration are as effective as BBR in preventing high glucose induced oxidative stress, mitochondrial depolarization and downstream events of apoptotic cell death.

Optogenetic Excitation of the Pancreatic β-Cell

The β-cell is a subunit of the Islet of Langerhans, a micro-organ of the pancreas which maintains glucose homeostasis. This endocrine cell senses changes in blood glucose levels and releases the hormone insulin. An electrophysiological process governing insulin release occurs through initial uptake of blood glucose and generation of ATP which binds the K-ATP channel causing inhibition and subsequent membrane depolarization. Influx of calcium from voltage-gated channels then interact with insulin secretory vesicles to promote exocystosis into the blood stream. In order to directly influence calcium dynamics, insulin release, and to study the electrophysical properties of the β-cell we used Channelrhodopsin-2 (ChR2), a light activated cation channel capable of creating membrane depolarization with light stimulation. Transient transfection of the MIN6 β-cell line using ChR2-YFP was carried out followed by calcium imaging using Fura2-AM. Following ChR2 stimulation at 490nm we observed local depolarization events in positively transfected cells. We demonstrated the creation of new oscillatory behavior at high glucose levels that can be set at specified frequencies and demonstrate that ChR2 can be used to overcome disruption in the K-ATP channel. Next, we isolated intact islets from a mouse model with β-cell specific expression of ChR2-YFP. We demonstrated global depolarization events upon ChR2 stimulation and performed insulin secretion assays to demonstrate enhanced insulin secretion. In addition, a computational multi-cellular islet model including ChR2 was used for comparison between in-vitro and in-silico experiments. This work demonstrates the ability of cell-specific electrophysiological control in the islet of Langerhans which will give further insight into β-cell behavior. Future work will include analyzing local depolarization on global islet behavior, examining the effect of ChR2 on islet-islet coupling, and developing an in-vivo model; which will include testing the feasibility for reversing diabetes.

Detection of Spreading Depolarization with Intraparenchymal Electrodes in the Injured Human Brain

Spreading depolarization events following ischemic and traumatic brain injury are associated with poor patient outcome. Currently, monitoring these events is limited to patients in whom subdural electrodes can be placed at open craniotomy. This study examined whether these events can be detected using intra-cortical electrodes, opening the way for electrode insertion via burr hole. Animal work was carried out on adult Sprague-Dawley rats in a laboratory setting to investigate the feasibility of recording depolarization events. Subsequently, 8 human patients requiring craniotomy for traumatic brain injury or aneurysmal subarachnoid hemorrhage were monitored for depolarization events in an intensive care setting with concurrent strip (subdural) and depth (intra-parenchymal) electrode recordings. (1) Depolarization events can be reliably detected from intra-cortically placed electrodes. (2) A reproducible slow potential change (SPC) waveform morphology was identified from intra-cortical electrodes on the depth array. (3) The depression of cortical activity known to follow depolarization events was identified consistently from both intra-cortical and sub-cortical electrodes on the depth array. Intra-parenchymally sited electrodes can be used to consistently identify depolarization events in humans. This technique greatly extends the capability of monitoring for spreading depolarization events in injured patients, as electrodes can be sited without the need for craniotomy. The method provides a new investigative tool for the evaluation of the contribution of these events to secondary brain injury in human patients.

OPA1 promotes pH flashes that spread between contiguous mitochondria without matrix protein exchange

The chemical nature and functional significance of mitochondrial flashes associated with fluctuations in mitochondrial membrane potential is unclear. Using a ratiometric pH probe insensitive to superoxide, we show that flashes reflect matrix alkalinization transients of ∼0.4 pH units that persist in cells permeabilized in ion-free solutions and can be evoked by imposed mitochondrial depolarization. Ablation of the pro-fusion protein Optic atrophy 1 specifically abrogated pH flashes and reduced the propagation of matrix photoactivated GFP (paGFP). Ablation or invalidation of the pro-fission Dynamin-related protein 1 greatly enhanced flash propagation between contiguous mitochondria but marginally increased paGFP matrix diffusion, indicating that flashes propagate without matrix content exchange. The pH flashes were associated with synchronous depolarization and hyperpolarization events that promoted the membrane potential equilibration of juxtaposed mitochondria. We propose that flashes are energy conservation events triggered by the opening of a fusion pore between two contiguous mitochondria of different membrane potentials, propagating without matrix fusion to equilibrate the energetic state of connected mitochondria.

Characterization of homogenous depolarizing media based on Mueller matrix differential decomposition

In a depolarizing medium in which the optical properties are uniformly distributed, the logarithm of the Mueller matrix can be used to calculate the differential Mueller matrix. From the differential Mueller matrix, the 10 optical properties of a homogeneous depolarizing medium are recovered. A modified calculation is introduced for media showing small time-irreversal depolarization events. The benefits of this method are illustrated in the determination of circular dichroism and circular birefringence of a nickel sulfate hexahydrate crystal from spectroscopic Mueller matrix measurements.

Abstract 136: Deleterious Effects of Synaptic Zn2+ in a Murine Distal Middle Cerebral Artery Occlusion Model

Background: Previous work has implicated accumulation of Zn2+ as a contributor to ischemic brain injury, however the sources of toxic Zn2+ accumulation are not fully understood. Previous data report that ICV delivery of a Zn2+ chelator limited mild focal stroke (Neuroscience 115 (2002) 871-8). We have recently demonstrated substantial synaptic Zn2+ release following spreading depolarization (JCBFM 31 (2011) 1073-84), and thus it is possible that repetitive spreading depolarization_ known to occur following stroke_ could be a major source of toxic Zn2+. In the present study, we examined whether synaptic Zn2+ release contributes to neuronal injury in a murine focal stroke model. Methods The effect of synaptic Zn2+ was assessed using mice lacking synaptic Zn2+, due to genetic deletion of the synaptic vesicle transporter ZnT3 (ZnT3 KO). Wild type C57Bl/6 mice were age and sex matched as controls. Mice were anesthetized with isofluorane and the cortical branch of the right middle cerebral artery was cauterized via a temporal burrhole. A single surgeon performed the occlusions. Body temperature was maintained at 37±0.5oC during the procedure and recovery. Animals were allowed to recover for 1 week before euthanasia. Stroke volume was assessed by Fluoro-Jade staining. Statistical analysis with 2-way ANOVA was used to determine the effects of sex and genotype on stroke volume. Results: There was no peri- or post-procedural mortality. Infarct was confirmed histologically in all animals. Stroke volume was reduced in the ZnT3 KO mice compared to wild type (mean volume 3.54mm3 versus 8.42mm3 respectively, n=7 for each arm). Only genotype (not sex) had a significant effect on stroke volume by 2-way ANOVA (p=0.0042). Conclusion: Synaptic Zn2+ release leads to increased stroke volume, likely due to toxic Zn2+accumulation. Therapies targeting synaptic release of Zn2+ or repetitive spreading depolarization events (which cause Zn2+ release) may decrease delayed ischemic injuries.

Alkalinization Transients in Individual Mitochondria

The pH within the mitochondrial matrix (pHmito) is an important bioenergetic parameter well studied in isolated mitochondria but poorly characterized in intact cells, and all in situ studies available report spatially averaged measurements of pHmito. We used a new pH-sensitive GFP-based fluorescent probe targeted to the mitochondrial matrix, mito-SypHer, to study pHmito homeostasis at the level of single mitochondria in intact living cells. We observed that individual mitochondria undergo spontaneous alkalinization transients. The pH transients occurred randomly in time and space and had a characteristic profile, with a rapid onset (time to peak 1.6±0.1 sec), a slower decay (τ=8.5±0.6 sec), and an average amplitude of 0.38±0.05 pH units. The pH transients were absent in Rho-0 cells that lack a functional respiratory chain, were abrogated by protonophores, occurred concomitantly with transient mitochondrial depolarization events measured with TMRE, and their frequency was strongly decreased by respiratory chain inhibitors. These kinetics and functional properties resemble the “superoxide flashes” previously reported in single mitochondria with a pericam-derived probe. However, the fluorescence of purified mito-SypHer was not altered by all the ROS tested including superoxide, and in live cells increasing the pH buffering power of mitochondria with NH4Cl decreased the amplitude and slowed the kinetics of the transients, confirming that these were caused by protons. The pH spikes were not spatially restricted to single mitochondria but were also observed in clusters of interconnected mitochondria and their spatial extent was altered by enforced fusion or fission of mitochondria. Our data indicate that in live cells individual mitochondria undergo spontaneous basification transients that require functional OXPHOS machinery. The synchronicity between the spontaneous pH spikes and depolarization events suggests that H+ extrusion by the respiratory chain complexes or by the reverse mode of the ATP-synthase might be transiently enhanced during bouts of depolarization.

Astrocytes Display Complex and Localized Calcium Responses to Single-Neuron Stimulation in the Hippocampus

Astrocytes show a complex structural and physiological interplay with neurons and respond to neuronal activation in vitro and in vivo with intracellular calcium elevations. These calcium changes enable astrocytes to modulate synaptic transmission and plasticity through various mechanisms. However, the response pattern of astrocytes to single neuronal depolarization events still remains unresolved. This information is critical for fully understanding the coordinated network of neuron-glial signaling in the brain. To address this, we developed a system to map astrocyte calcium responses along apical dendrites of CA1 pyramidal neurons in hippocampal slices using single-neuron stimulation with channelrhodopsin-2. This technique allowed selective neuronal depolarization without invasive manipulations known to alter calcium levels in astrocytes. Light-evoked neuronal depolarization was elicited and calcium events in surrounding astrocytes were monitored using the calcium-sensitive dye Calcium Orange. Stimulation of single neurons caused calcium responses in populations of astrocytes along the apical axis of CA1 cell dendrites. Calcium responses included single events that were synchronized with neuronal stimulation and poststimulus changes in calcium event frequency, both of which were modulated by glutamatergic and purinergic signaling. Individual astrocytes near CA1 cells showed low ability to respond to repeated neuronal depolarization events. However, the response of the surrounding astrocyte population was remarkably accurate. Interestingly, the reliability of responses was graded with respect to astrocyte location along the CA1 cell dendrite, with astrocytes residing in the primary dendrite subregion being most responsive. This study provides a new perspective on the dynamic response property of astrocyte ensembles to neuronal activity.

Tensile stress induced depolarization in [001]-poled transverse mode Pb(Zn1/3Nb2/3)O3 -(6-7)%PbTiO3 single crystals

This paper investigates the effects of electrically induced and direct tensile stress on the deformation and dielectric properties of Pb(Zn1/3Nb2/3)O3-(6-7)%PbTiO3 single crystals of [110]L× [001]T cut by using a unimorph sample and a four-point-bend (FPB) sample, respectively. The results show a dip in tip displacement for the unimorph sample at sufficiently high electric field parallel to the poling field direction and a sudden rise in capacitance for the FPB sample at sufficiently high tensile stress in the [110] crystal direction, respectively. These phenomena are attributed to the tensile stress induced rhombohedral-to-orthorhombic phase transition and associated depolarization events in the crystal. For the said crystal cut, the obtained tensile depoling stress is in the range of 15-20 MPa. The present work furthermore shows that the occurrence of tensile stress-induced depolarization is possible even when the direction of the applied electric field is parallel to the poling field direction, as in the unimorph sample examined.

Automated haematology analysis to diagnose malaria

For more than a decade, flow cytometry-based automated haematology analysers have been studied for malaria diagnosis. Although current haematology analysers are not specifically designed to detect malaria-related abnormalities, most studies have found sensitivities that comply with WHO malaria-diagnostic guidelines, i.e. ≥ 95% in samples with > 100 parasites/μl. Establishing a correct and early malaria diagnosis is a prerequisite for an adequate treatment and to minimizing adverse outcomes. Expert light microscopy remains the 'gold standard' for malaria diagnosis in most clinical settings. However, it requires an explicit request from clinicians and has variable accuracy. Malaria diagnosis with flow cytometry-based haematology analysers could become an important adjuvant diagnostic tool in the routine laboratory work-up of febrile patients in or returning from malaria-endemic regions. Haematology analysers so far studied for malaria diagnosis are the Cell-Dyn®, Coulter® GEN·S and LH 750, and the Sysmex XE-2100® analysers. For Cell-Dyn analysers, abnormal depolarization events mainly in the lobularity/granularity and other scatter-plots, and various reticulocyte abnormalities have shown overall sensitivities and specificities of 49% to 97% and 61% to 100%, respectively. For the Coulter analysers, a 'malaria factor' using the monocyte and lymphocyte size standard deviations obtained by impedance detection has shown overall sensitivities and specificities of 82% to 98% and 72% to 94%, respectively. For the XE-2100, abnormal patterns in the DIFF, WBC/BASO, and RET-EXT scatter-plots, and pseudoeosinophilia and other abnormal haematological variables have been described, and multivariate diagnostic models have been designed with overall sensitivities and specificities of 86% to 97% and 81% to 98%, respectively. The accuracy for malaria diagnosis may vary according to species, parasite load, immunity and clinical context where the method is applied. Future developments in new haematology analysers such as considerably simplified, robust and inexpensive devices for malaria detection fitted with an automatically generated alert could improve the detection capacity of these instruments and potentially expand their clinical utility in malaria diagnosis.

Sensitivity to perturbations in vivo implies high noise and suggests rate coding in cortex

It is well known that neural activity exhibits variability, in the sense that identical sensory stimuli produce different responses, but it has been difficult to determine what this variability means. Is it noise, or does it carry important information – about, for example, the internal state of the organism? We address this issue from the bottom up, by asking whether small perturbations to activity in cortical networks are amplified. Based on in vivo whole-cell recordings in rat barrel cortex, we find that a perturbation consisting of a single extra spike in one neuron produces ~28 additional spikes in its postsynaptic targets, and we show, using simultaneous intra- and extra-cellular recordings, that a single spike produces a detectable increase in firing rate in the local network. Theoretical analysis indicates that this amplification leads to intrinsic, stimulus-independent variations in membrane potential on the order of ±2.2 - 4.5 mV – variations that are pure noise, and so carry no information at all. Therefore, for the brain to perform reliable computations, it must either use a rate code, or generate very large, fast depolarizing events, such as those proposed by the theory of synfire chains – yet in our in vivo recordings, we found that such events were very rare. Our findings are consistent with the idea that cortex is likely to use primarily a rate code.

Retinophilin Is a Light-Regulated Phosphoprotein Required to Suppress Photoreceptor Dark Noise in Drosophila

Photoreceptor cells achieve high sensitivity, reliably detecting single photons, while limiting the spontaneous activation events responsible for dark noise. We used proteomic, genetic, and electrophysiological approaches to characterize Retinophilin (RTP) (CG10233) in Drosophila photoreceptors and establish its involvement in dark-noise suppression. RTP possesses membrane occupation and recognition nexus (MORN) motifs, a structure shared with mammalian junctophilins and other membrane-associated proteins found within excitable cells. We show the MORN repeats, and both the N- and C-terminal domains, are required for RTP localization in the microvillar light-gathering organelle, the rhabdomere. RTP exists in multiple phosphorylated isoforms under dark conditions and is dephosphorylated by light exposure. An RTP deletion mutant exhibits a high rate of spontaneous membrane depolarization events in dark conditions but retains the normal kinetics of the light response. Photoreceptors lacking neither inactivation nor afterpotential C (NINAC) myosin III, a motor protein/kinase, also display a similar dark-noise phenotype as the RTP deletion. We show that NINAC mutants are depleted for RTP. These results suggest the increase in dark noise in NINAC mutants is attributable to lack of RTP and, furthermore, defines a novel role for NINAC in the rhabdomere. We propose that RTP is a light-regulated phosphoprotein that organizes rhabdomeric components to suppress random activation of the phototransduction cascade and thus increases the signaling fidelity of dark-adapted photoreceptors.

Questions about STDP as a General Model of Synaptic Plasticity

According to spike-timing-dependent plasticity (STDP), the timing of the Na+ spike relative to the EPSP determines whether LTP or LTD will occur. Here, we review our reservations about STDP. Most investigations of this process have been done under conditions in which the spike is evoked by postsynaptic current injection. Under more realistic conditions, in which the spike is evoked by the EPSP, the results do not generally support STDP. For instance, low-frequency stimulation of a group of synapses can cause LTD, not the LTP predicted by the pre-before-post sequence in STDP; this is true regardless of whether or not the EPSP is large enough to produce a Na+ spike. With stronger or more frequent stimulation, LTP can be induced by the same pre-before-post timing, but in this case block of Na+ spikes does not necessarily prevent LTP induction. Thus, Na+ spikes may facilitate LTP and/or LTD under some conditions, but they are not necessary, a finding consistent with their small size relative to the EPSP in many parts of pyramidal cell dendrites. The nature of the dendritic depolarizing events that control bidirectional plasticity is of central importance to understanding neural function. There are several candidates, including backpropagating action potentials, but also dendritic Ca2+ spikes, the AMPA receptor-mediated EPSP, and NMDA receptor-mediated EPSPs or spikes. These often appear to be more important than the Na+ spike in providing the depolarization necessary for plasticity. We thus feel that it is premature to accept STDP-like processes as the major determinant of LTP/LTD.

Long-Lasting Hyperexcitability Induced by Depolarization in the Absence of Detectable Ca2+Signals

Learning and memory depend on neuronal alterations induced by electrical activity. Most examples of activity-dependent plasticity, as well as adaptive responses to neuronal injury, have been linked explicitly or implicitly to induction by Ca(2+) signals produced by depolarization. Indeed, transient Ca(2+) signals are commonly assumed to be the only effective transducers of depolarization into adaptive neuronal responses. Nevertheless, Ca(2+)-independent depolarization-induced signals might also trigger plastic changes. Establishing the existence of such signals is a challenge because procedures that eliminate Ca(2+) transients also impair neuronal viability and tolerance to cellular stress. We have taken advantage of nociceptive sensory neurons in the marine snail Aplysia, which exhibit unusual tolerance to extreme reduction of extracellular and intracellular free Ca(2+) levels. The axons of these neurons exhibit a depolarization-induced memory-like hyperexcitability that lasts a day or longer and depends on local protein synthesis for induction. Here we show that transient localized depolarization of these axons in an excised nerve-ganglion preparation or in dissociated cell culture can induce short- and intermediate-term axonal hyperexcitability as well as long-term protein synthesis-dependent hyperexcitability under conditions in which Ca(2+) entry is prevented (by bathing in nominally Ca(2+) -free solutions containing EGTA) and detectable Ca(2+) transients are eliminated (by adding BAPTA-AM). Disruption of Ca(2+) release from intracellular stores by pretreatment with thapsigargin also failed to affect induction of axonal hyperexcitability. These findings suggest that unrecognized Ca(2+)-independent signals exist that can transduce intense depolarization into adaptive cellular responses during neuronal injury, prolonged high-frequency activity, or other sustained depolarizing events.

The Voltage-Gated Ca2+Channel Is the Ca2+Sensor Protein of Secretion

Neurotransmitter release involves two consecutive Ca(2+)-dependent steps, an initial Ca(2+) binding to the selectivity filter of voltage-gated Ca(2+) channels (VGCC) followed by Ca(2+) binding to synaptic vesicle protein. The unique Ca(2+)-binding site of the VGCC is located within the alpha(1) subunit of the Ca(2+) channel. The structure of the selectivity filter allows for the binding of Ca(2+), Sr(2+), Ba(2+), and La(3+). Despite its cell impermeability, La(3+) supports secretion, which is in contradistinction to the commonly accepted mechanism in which elevation of cytosolic ion concentrations ([Ca(2+)](i)) and binding to synaptotagmin(s) trigger release. Here we show that a Cav1.2-mutated alpha(1)1.2/L775P subunit which does not conduct Ca(2+) currents supports depolarization-evoked release by means of Ca(2+) binding to the pore. Bovine chromaffin cells, which secrete catecholamine almost exclusively via nifedipine-sensitive Cav1.2, were infected with the Semliki Forest Virus, pSFV alpha(1)1.2/L775P. This construct also harbored a second mutation that rendered the channel insensitive to nifedipine. Depolarization of cells infected with alpha(1)1.2/L775P triggered release in the presence of nifedipine. Thus, the initial Ca(2+) binding at the pore of the channel appeared to be sufficient to trigger secretion, indicating that the VGCC could be the primary Ca(2+) sensor protein. The 25% lower efficiency, however, implied that additional ancillary effects of elevated [Ca(2+)](i) were essential for optimizing the overall release process. Our findings suggest that the rearrangement of Ca(2+) ions within the pore of the channel during membrane depolarization triggers secretion prior to Ca(2+) entry. This allows for a tight temporal coupling between the depolarization event and exocytosis of vesicles tethered to the channel.

Persisting Depletion of Brain Glucose following Cortical Spreading Depression, despite Apparent Hyperaemia: Evidence for Risk of an Adverse Effect of Leão's Spreading Depression

Rapid sampling microdialysis (rsMD) directed towards the cerebral cortex has allowed identification of a combined time-series signature for glucose and lactate that characterizes peri-infarct depolarization in experimental focal ischaemia, but no comparable data exist for 'classical' cortical spreading depression (CSD) associated with hyperaemia in the normally perfused brain. Here, we examined the rsMD responses of dialysate glucose and lactate to five hyperaemic spreading depressions induced with intracortical microinjections, typically of 1 mol/L KCl, in open-skull preparations in five cats under chloralose anaesthesia. Depolarization was verified with microelectrodes, and laser speckle flowmetry was used to examine propagation of the events and perfusion responses near the MD probe. Ten minutes after depolarization, dialysate glucose fell and lactate rose by 28% and 58% respectively. There was no recovery of dialysate glucose 30 mins after depolarization. Mean baseline indicative cerebral blood flow was 25.5+/-4.1 mL/100 g/min and mean maximum hyperaemic increase was by 29.6+/-6 mL/100 g/min; hyperaemia remained present 30 mins after CSD. As CSD events are repetitive, frequent, and often clustered temporally in human acute brain injury, these results indicate a high risk of depletion of extracellular glucose in association with depolarization events of a pattern previously thought to be largely benign.