Functional and pharmacological analysis of agmatine administration in different cerebral ischemia animal models.

Agmatine (AgM, 100 mg/kg i.p.) effect was tested in parallel at two animal models of cerebral ischemia - rat MCAO model (60'/24 h, 60'/48 h, 90'/24 h, 90'/48 h) and gerbil global ischemia (10') model, administrated 5 min after reperfusion. Aim was to evaluate AgM effect on functional outcome 24 and 48 h after MCAO on neurological and sensor-motor function, and coordination in rats. AgM administration significantly reduced infarct volume, improved neurological score and improved post-ischemic oxidative status. Results of behavioral tests (cylinder test, beam walking test, and adhesive removal test) have shown very effective functional recovery after AgM administration. Efficiency of AgM administration in gerbils was observed in forebrain cortex, striatum, hippocampus, and cerebellum at the level of each examined oxidative stress parameter (nitric oxide level, superoxide production, superoxide dismutase activity, and index of lipid peroxidation) measured in four different time points starting at 3 h up to 48 h after reperfusion. The highest levels were obtained 6 h after the insult. The most sensitive oxidative stress parameter to AgM was nitric oxide. Additionally, we performed pharmacological analysis of AgM on rat isolated common carotid arteries. The findings imply that mixed population of potassium channels located on the smooth muscle cells was involved in common carotid artery response to AgM, with predominance of inward rectifying K+ channels. In our comparative experimental approach, judged by behavioral, biochemical, as well as pharmacological data, the AgM administration showed an effective reduction of ischemic neurological damage and oxidative stress, hence indicating a direction towards improving post-stroke recovery.

Enriched environment alters the behavioral profile of tenascin-C deficient mice.

Tenascin-C (TnC) is an extracellular matrix glycoprotein implicated in a variety of processes ranging from brain development to synaptic plasticity in the adult vertebrates. Although the role of the TnC gene in regulation of behavior has been investigated, it remained elusive how TnC deficiency interacts with the environment in shaping the behavioral phenotype. To address this, 3-week-old TnC+/+ and TnC-/- male mice were housed over an 8-week period in standard conditions (SC), or enriched environment (EE). A comprehensive battery of tests was used in behavioral phenotyping. When housed in SC, TnC-/- mice showed spontaneous nocturnal hyperactivity, as well as poor sensorimotor coordination and low swimming velocity. However, housing of TnC-/- mice in EE abolished hyperlocomotion, led to faster habituation to novel environment, strengthened the grasp of fore limbs and partially improved movement coordination, while the swimming ability remained deficient. Conversely, TnC deficiency attenuated both the beneficial effects of EE on learning/memory capacity and the anxiolytic effect of EE in reducing the level of acrophobia. This study expands the existing knowledge about the phenotype associated with TnC deficiency, and reveals that the effect of genetic background on the behavioral response could be altered by post-weaning housing in a highly stimulating environment.

GABAB receptors as a common target for hypothermia and spike and wave seizures: intersecting mechanisms of thermoregulation and absence epilepsy.

In the current study the link among the γ-hydroxybutyrate (GHB)/pentylenetetrazole (PTZ)-induced absence-like seizures and concomitant decreases in the core temperature, as well as electroencephalographic (EEG) activity during rewarming from deep hypothermia produced by a drug-free protocol were investigated. During the rewarming period after deep cooling, most Wistar rats suffered from bilaterally synchronous spike and waves with no or mild behavioral correlates. Spike and wave seizures were temperature-dependent and were initially registered when body temperature (Tb) reached 25-27°C, but mostly during the mild hypothermia of 0.3-1.3°C (Tb of 36.3-37.3°C). In chemical absence models, spike and wave discharges were also closely accompanied by mild systemic hypothermia, as both PTZ- and GHB-induced temperature decreases ranged from about 1-1.4°C respectively, together with EEG markers of absence activity. Thus, throughout the different experimental designs, the occurrence of spike and wave discharges was always related to a mild (0.3-1.4°C) decrease of Tb. Benzodiazepine diazepam as the GABAA-positive allosteric modulator and CGP 62349 as the selective antagonist of GABAB receptors were used to determine if their well-known anticonvulsant properties also affect hypothermia elicited by these drugs. Finally, during the course of spontaneous rewarming from deep hypothermia, another selective GABAB-blocking agent, CGP 35348, was used to elucidate if GABAB inhibitory system could be critically implicated in the generation of hypothermia-dependent spike and waves. Diazepam prevented both the PTZ-induced hypothermia and electrographic absence seizures, but these two beneficial effects did not occur in the GHB model. Even though diazepam delayed GHB-induced maximal temperature decrease, the GHB effects remained highly significant. The GABAB antagonist CGP 62349 completely prevented hypothermia as well as absence seizures in both chemical models. Likewise, spike and wave discharges, registered during the spontaneous rewarming from deep hypothermia, were completely prevented by CGP 35348. These findings show that systemic hypothermia should definitely be regarded as a marker of GABAB receptor activation. Moreover, the results of this study clearly show that initial mild temperature decrease should be considered as strong absence-provoking factor. Hypothermia-induced nonconvulsive seizures also highlight the importance of continuous EEG monitoring in children undergoing therapeutic hypothermia after cardiac arrest. Since every change in peripheral or systemic temperature ultimately must be perceived by preoptic region of the anterior hypothalamus as the primary thermoregulatory and sleep-inducing center, the preoptic thermosensitive neurons in general and warm-sensitive neurons in particular, simply have to be regarded as the most probable candidate for connected thermoregulatory and absence generating mechanisms. Therefore, additional studies are needed to confirm their potential role in the generation and propagation of absence seizures.

Amyotrophic lateral sclerosis immunoglobulins G enhance the mobility of Lysotracker-labelled vesicles in cultured rat astrocytes.

AIM: We examined the effect of purified immunoglobulins G (IgG) from patients with amyotrophic lateral sclerosis (ALS) on the mobility and exocytotic release from Lysotracker-stained vesicles in cultured rat astrocytes. METHODS: Time-lapse confocal images were acquired, and vesicle mobility was analysed before and after the application of ALS IgG. The vesicle counts were obtained to assess cargo exocytosis from stained organelles. RESULTS: At rest, when mobility was monitored for 2 min in bath with Ca(2+), two vesicle populations were discovered: (1) non-mobile vesicles (6.1%) with total track length (TL) < 1 µm, averaging at 0.33 ± 0.01 µm (n = 1305) and (2) mobile vesicles (93.9%) with TL > 1 µm, averaging at 3.03 ± 0.01 µm (n = 20,200). ALS IgG (0.1 mg mL(-1)) from 12 of 13 patients increased the TL of mobile vesicles by approx. 24% and maximal displacement (MD) by approx. 26% within 4 min, while the IgG from control group did not alter the vesicle mobility. The mobility enhancement by ALS IgG was reduced in extracellular solution devoid of Ca(2+), indicating that ALS IgG vesicle mobility enhancement involves changes in Ca(2+) homeostasis. To examine whether enhanced mobility relates to elevated Ca(2+) activity, cells were stimulated by 1 mm ATP, a cytosolic Ca(2+) increasing agent, in the presence (2 mm) and in the absence of extracellular Ca(2+). ATP stimulation triggered an increase in TL by approx. 7% and 12% and a decrease in MD by approx. 11% and 1%, within 4 min respectively. Interestingly, none of the stimuli triggered the release of vesicle cargo. CONCLUSION: Amyotrophic lateral sclerosis-IgG-enhanced vesicle mobility in astrocytes engages changes in calcium homeostasis.

Molecular imaging of brain lipid environment of lymphocytes in amyotrophic lateral sclerosis using magnetic resonance imaging and SECARS microscopy.

This paper highlights some of the key technologies of using two innovative molecular imaging modalites, magnetic resonance imaging (MRI) and nonlinear optical microscopy, for imaging intravenously injected ultra small paramagnetic iron oxide nanoparticles cross linked with antibodies (CLUSPIO) in the amyotrophic lateral sclerosis (ALS) experimental model in vivo or ex vivo, respectively. Intensive efforts have been made in investigating the causes of abnormalities in lipid metabolism, monitored in some neurodegenerative disorders systems. It has been shown that an abnormal accumulation of some common lipids in motor nerve cells may play a critical role in the development of amyotrophic lateral sclerosis. The presented experiments were performed on brain specimens from the transgenic rat model expressing multiple copies of mutated (G93A) human SOD-1 gene, after CD4+ lymphocytes were magnetically labeled with i.v.i. CLUSPIO antibodies. In vivo MRI revealed marked signal intensity enhancements in specific pathological regions of the ALS rat brain as compared to the wild type. Surface-enhanced coherent anti-Stokes Raman scattering (SECARS) microscopy indicated cellular interactions based on lipids association to anti-CD4 CLUSPIO.

Ivan Djaja (Jean Giaja) and the Belgrade School of Physiology.

The founder of physiology studies in the Balkans and the pioneer of research on hypothermia, Ivan Djaja (Jean Giaja) was born 1884 in L'Havre. Giaja gained his PhD at the Sorbonne in 1909. In 1910 he established the first Chair of Physiology in the Balkans and organized the first Serbian Institute for Physiology at the School of Philosophy of the University of Belgrade. He led this Institute for more than 40 subsequent years. His most notable papers were in the field of thermoregulation and bioenergetics. Djaja became member of the Serbian and Croatian academies of science and doctor honoris causa of Sorbonne. In 1952 for the seminal work on the behavior of deep cooled warm blooded animals he became associate member of the National Medical Academy in Paris. In 1955 the French Academy of Sciences elected him as associate member in place of deceased Sir Alexander Fleming. Djaja died in 1957 during a congress held in his honor. He left more than 200 scientific and other papers and the golden DaVincian credo "Nulla dies sine experimento". His legacy was continued by several generations of researchers, the most prominent among them being Stefan Gelineo, Radoslav Andjus and Vojislav Petrovic.

Survivin expression in odontogenic keratocysts and correlation with cytomegalovirus infection.

OBJECTIVES: The aim of this study was to investigate the expression of survivin, an inhibitor of apoptosis, in odontogenic keratocysts and to compare it to the findings in non-neoplastic jaw cysts - periapical cysts, as well as to establish a possible relationship between survivin expression and human cytomegalovirus presence within these cysts. MATERIALS AND METHODS: Samples of 10 odontogenic keratocysts (five positive and five negative for the presence of cytomegalovirus, as determined by polymerase chain reaction) and 10 periapical cysts (five positive and five negative for the cytomegalovirus presence) were analysed. The expression of survivin was assessed by immunohistochemical methods, using monoclonal antibody that selectively recognizes the cytoplasmic form of survivin. RESULTS: All 10 odontogenic keratocysts showed immunostaining for survivin, while all 10 periapical cysts were negative for its presence. There was no correlation between cytomegalovirus presence and expression of survivin within odontogenic keratocysts. CONCLUSION: Survivin may contribute to the aggressive behavior of odontogenic keratocysts, and thus support the emerging opinion of their neoplastic nature.

The d-wave in fish and the state of light adaptation.

Comparative electroretinographic studies of the d-wave evoked with long duration photo stimuli in dark- and light-adapted fish species (three marine and three freshwater) were performed. At the end of prolonged photo-stimulation in scotopic conditions a negative d-wave appears in electroretinograms of dogfish shark, eel and goldfish diminishing and eventually changing with intensity of photo-stimulation, while in rudd it only increases. Dark-adapted electroretinograms of two percids (perch and painted comber) exhibit a positive d-wave that approaches the b-wave amplitude under bright photopic conditions. Judging from the d-wave, only the rod pathway is active in dark-adapted dogfish shark, eel, and goldfish. Under the light adaptation, cone pathways are active in eel and goldfish, whereas the positive response to the end of light stimuli in dogfish shark could be explained by independent ON and OFF pathways from outer to inner retina via bipolar cells. In the case of two percids, dark adaptation has no influence on cone pathways. The d-wave of rudd behaves like cone-driven d-waves but in opposite sign. The data thus show that the d-wave form, amplitude and sign depend on interconnection of ON and OFF pathways as determined by the state of adaptation and/or type of photoreceptor.

A change in the pattern of activity affects the developmental regression of the Purkinje cell polyinnervation by climbing fibers in the rat cerebellum.

Pattern of activity during development is important for the refinement of the final architecture of the brain. In the cerebellar cortex, the regression from multiple to single climbing fiber innervation of the Purkinje cell occurs during development between postnatal days (P) 5 and 15. However, the regression is hampered by altering in various ways the morpho-functional integrity of the parallel fiber input. In rats we disrupted the normal activity pattern of the climbing fiber, the terminal arbor of the inferior olive neurons, by administering harmaline for 4 days from P9 to P12. At all studied ages (P15-87) after harmaline treatment multiple (double only) climbing fiber EPSC-steps persist in 28% of cells as compared with none in the control. The ratio between the amplitudes of the larger and the smaller climbing fiber-evoked EPSC increases in parallel with the decline of the polyinnervation factor, indicating a gradual enlargement of the synaptic contribution of the winning climbing fiber synapse at the expense of the losing one. Harmaline treatment had no later effects on the climbing fiber EPSC kinetics and I/V relation in Purkinje cells (P15-36). However, there was a rise in the paired-pulse depression indicating a potentiation of the presynaptic mechanisms. In the same period, after harmaline treatment, parallel fiber-Purkinje cell electrophysiology was unaffected. The distribution of parallel fiber synaptic boutons was also not changed. Thus, a change in the pattern of activity during a narrow developmental period may affect climbing fiber-Purkinje cell synapse competition resulting in occurrence of multiple innervation at least up to 3 months of age. Our results extend the current view on the role of the pattern of activity in the refinement of neuronal connections during development. They suggest that many similar results obtained by different gene or receptor manipulations might be simply the consequence of disrupting the pattern of activity.

Temperature sensitivity of the tonoplast Ca(2+)-activated K+ channel in Chara: the influence of reversing the sign of membrane potential.

A detailed temperature dependence study of a well-defined plant ion channel, the Ca(2+)-activated K+ channel of Chara corallina, was performed over the temperature range of their habitats, 5-36 degrees C, at 1 degree C resolution. The temperature dependence of the channel unitary conductance at 50 mV shows discontinuities at 15 and 30 degrees C. These temperatures limit the range within which ion diffusion is characterized by the lowest activation energy (Ea = 8.0 +/- 1.6 kJ/mol) as compared to the regions below 15 degrees C and above 30 degrees C. Upon reversing membrane voltage polarity from 50 to -50 mV the pattern of temperature dependence switched from discontinuous to linear with Ea = 13.6 +/- 0.5 kJ/mol. The temperature dependence of the effective number of open channels at 50 mV showed a decrease with increasing temperature, with a local minimum at 28 degrees C. The mean open time exhibited a similar behavior. Changing the sign of membrane potential from 50 to -50 mV abolished the minima in both temperature dependencies. These data are discussed in the light of higher order phase transitions of the Characean membrane lipids and corresponding change in the lipid-protein interaction, and their modulation by transmembrane voltage.

Discontinuities in the temperature function of transmembrane water transport in Chara: relation to ion transport.

The NMR (nuclear magnetic resonance) method of Conlon and Outhred (1972) was used to measure diffusional water permeability of the nodal cells of the green alga Chara gymnophylla. Two local minima at 15 and 30 degreesC of diffusional water permeability (Pd) were observed delimiting a region of low activation energy (Ea around 20 kJ/mol) indicative of an optimal temperature region for membrane transport processes. Above and below this region water transport was of a different type with high Ea (about 70 kJ/mol). The triphasic temperature dependence of the water transport suggested a channel-mediated transport at 15-30 degreesC and lipid matrix-mediated transport beyond this region. The K+ channel inhibitor, tetraethylammonium as well as the Cl- channel inhibitor, ethacrynic acid, diminished Pd in the intermediate temperature region by 54 and 40%, respectively. The sulfhydryl agent p-(chloromercuri-benzensulfonate) the water transport inhibitor in erythrocytes also known to affect K+ transport in Chara, only increased Pd below 15 degreesC. In high external potassium ('K-state') water transport minima were pronounced. The role of K+ channels as sensors of the optimal temperature limits was further emphasized by showing a similar triphasic temperature dependence of the conductance of a single K+ channel also known to cotransport water, which originated from cytoplasmic droplets (putatively tonoplast) of C. gymnophylla. The minimum of K+ single channel conductance at around 15 degreesC, unlike the one at 30 degreesC, was sensitive to changes of growth temperature underlining membrane lipid involvement. The additional role of intracellular (membrane?) water in the generation of discontinuities in the above thermal functions was suggested by an Arrhenius plot of the cellular water relaxation rate which showed breaks at 13 and 29 degreesC.

Electroretinographic evaluation of spectral sensitivity in yellow and silver eels (Anguilla anguilla).

Although differences in visual pigments between developmental stages of the European eel are well known, the expected differences in spectral sensitivity have not been demonstrated at the electrophysiological level. In fact, one past electroretinographic study led to the conclusion that in eels there is no change in scotopic sensitivity, with increasing sexual maturity. In the present experiments, electroretinograms (ERGs) were recorded from in situ eyecups of immobilized eels Anguilla anguilla (L.) caught in coastal running waters. It was shown that the ERG b-wave is as good an indicator of spectral sensitivity as the unmasked late receptor potential (LRP) which directly reflects the responsiveness of photoreceptors. Complete spectral-sensitivity curves, based on b-wave thresholds and on thresholds of LRP subsequently isolated by means of sodium iodate, have been obtained in the same eel. Using fitted amplitude-log intensity functions for threshold calculation, and two models for computer-assisted fitting of spectral-sensitivity curves, significant differences in lambda max were found between yellow and silver developmental stages of the eel, identified by ocular index measurements.

Immunoglobulins from motoneurone disease patients enhance glutamate release from rat hippocampal neurones in culture.

1. The whole-cell configuration of the patch-clamp technique was used to study the effects of immunoglobulins (IgGs) from patients affected by amyotrophic lateral sclerosis (ALS) on spontaneous glutamatergic currents in rat hippocampal cells in culture. 2. Focal application of ALS IgGs (100 micrograms ml-1) to hippocampal cells induced a rise in frequency but not in amplitude of spontaneous excitatory postsynaptic currents (SEPSC) which outlasted the period of IgG application. The mean frequency ratio (ALS over control) was 3.2 +/- 0.6 (n = 19). No changes in frequency or amplitude of SEPSCs were observed after treatment with IgGs obtained from healthy donors (n = 5) or from patients with Alzheimer's disease (n = 4). 3. ALS IgGs also increased the frequency (by a factor of 2.0 +/- 0.3) but not the amplitude of miniature excitatory postsynaptic currents (mEPSC) recorded in the presence of TTX (n = 19). A rise in frequency of mEPSC was also seen in cells superfused with a calcium-free solution (n = 4). 4. In the presence of TTX, ALS IgGs did not modify the amplitude or the shape of currents evoked by AMPA (100 microM), recorded at a holding potential of -50 mV. 5. It is concluded that ALS IgGs enhance both SEPSCs and mEPSCs through a presynaptic type of action. The excessive release of glutamate from nerve endings may be the cause of motoneurone death in ALS patients.

ALS IgGs suppress [Ca2+]i rise through P/Q-type calcium channels in central neurones in culture.

Confocal laser scanning microscopy (with the fluorescent calcium dye fluo-3) was used to test the effect of IgG obtained from patients with amyotrophic lateral sclerosis (ALS) on the KCl-induced [Ca2+] rise in rat hippocampal neurones in culture. In the presence of tetrodotoxin and ionotropic glutamate receptor antagonists, ALS IgGs depressed (by 30-40%) Ca2+ transients evoked by influx of Ca2+ through voltage-activated channels; such an effect did not occur with IgG obtained from healthy donors. The depressant action of ALS IgG was selectively prevented by the inhibitor of P/Q-type Ca2+ channels, omega-agatoxin IVA (which alone reduced Ca2+ transients by 40%). The reduced Ca2+ transients might impair Ca(2+)-dependent glutamate receptor desensitization and thus facilitate excitotoxic damage.

Changes in intracellular calcium induced by NMDA in cultured rat hippocampal neurons require exogenous glycine.

Confocal laser scanning microscopy was used to study changes in intracellular free calcium concentration ([Ca2+]i) at the level of the soma of cultured hippocampal neurones following pressure application of glutamate or N-methyl-D-aspartate (NMDA). [Ca2+]i was imaged in the presence of tetrodotoxin after loading cells with the fluorescent dye indicator fluo-3/AM. Responses to glutamate were potently antagonized by 6-cyano-7 nitroquinoxaline-2,3-dione (CNQX: 20 microM). They were also strongly and reversibly depressed by 3-((+/-)-2-carboxypiperazine-4-yl)-propyl-1-phosphonic acid (CPP; 20 microM), leaving a small CNQX-sensitive component. Responses to NMDA were also blocked by CNQX. In the presence of saturating concentrations of glycine (100 microM), the depression of glutamate or NMDA responses by CNQX was greatly reduced. Exogenously applied glycine also potentiated the NMDA response. These data indicate that the glycine binding site of the NMDA receptor channel is not saturated in cultured hippocampal neurones and thus is susceptible to the action of agonists or antagonists.

D2O-induced ion channel activation in Characeae at low ionic strength.

Effects of D2O were studied on internodal cells of the freshwater alga Nitellopsis obtusa under plasmalemma perfusion (tonoplast-free cells) with voltage clamp, and on Ca2+ channels isolated from the alga and reconstituted in bilayer lipid membranes (BLM). External application of artificial pond water (APW) with D2O as the solvent to the perfused plasmalemma preparation led to an abrupt drop of membrane resistance (Rm = 0.12 +/- 0.03 k omega.cm2), thus preventing further voltage clamping. APW with 25% D2O caused a two-step reduction of Rm: first, down to 2.0 +/- 0.8 k omega.cm2, and then further to 200 omega.cm2, in 2 min. It was shown that in the first stage, Ca2+ channels are activated, and then, Ca2+ ions entering through them activate the Cl- channels. The Ca2+ channels are activated irreversibly. If 100 mM CsCl was substituted for 200 mM sucrose (introduced for iso-osmoticity), no effect of D2O on Rm was observed. Intracellular H2O/D2O substitution also did not change Rm. In experiments on single Ca2+ channels in BLM H2O/D2O substitution in a solution containing 100 mM KCl (trans side) produced no effect on channel activity, while in 10 mM KCl, at negative voltage, the open channel probability sharply increased. This effect was irreversible. The single channel conductance was not altered after the H2O/D2O substitution. The discussion of the possible mechanism of D2O action on Ca2+ and Cl- channels was based on an osmotic-like stress effect and the phenomenon of higher D-bond energy compared to the H-bond.

Depolarization-Activated K+ Channel in Chara Droplets.

A novel potassium channel was characterized in the droplet membrane of Chara gymnophylla. This channel has a conductance of about 90 pS (in symmetrical 0.15 M KCl), which is lower compared to the 170-pS K+ channel predominant in this preparation. In contrast to the large conductance K+ channel, the novel channel opened with a delay at depolarization and closed at hyperpolarization and did not require cytosolic Ca2+ for its opening. It also showed comparatively weak selectivity for K+ over other monovalent cations, although its cation to anion selectivity was high. Externally or internally applied Cs+ blocked the channel in a voltage-dependent manner, similarly to the 170-pS channel. The sensitivity of the 90-pS channel to external tetraethylammonium chloride (half-blocking concentration approximately 1.5 mM) was 20-fold higher compared to the large conductance channel. With respect to its voltage-gating kinetics, the 90-pS channel was identified as a "slow delayed rectifier."

Effects of D2O on permeation and gating in the Ca(2+)-activated potassium channel from Chara.

We studied the effects of H2O/D2O substitution on the permeation and gating of the large conductance Ca(2+)-activated K+ channels in Chara gymnophylla droplet membrane using the patch-clamp technique. The selectivity sequence of the channel was: K+ > Rb+ >> Li+, Na+, Cs+ and Cl-. The conductance of this channel in symmetric 100 mM KCl was found to be 130 pS. The single channel conductance was decreased by 15% in D2O as compared to H2O. The blockade of channel conductance by cytosolic Ca2+ weakened in D2O as a result of a decrease in zero voltage Ca2+ binding affinity by a factor of 1.4. Voltage-dependent channel gating was affected by D2O primarily due to the change in Ca2+ binding to the channel during the activation step. The Hill coefficient for Ca2+ binding was 3 in D2O and around 1 in H2O. The values of the Ca2+ binding constant in the open channel conformation were 0.6 and 6 microM in H2O and D2O, respectively, while the binding in the closed conformation was much less affected by D2O. The H2O/D2O substitution did not produce a significant change in the slope of channel voltage dependence but caused a shift as large as 60 mV with 1 mM internal Ca2+.

D2O-induced cell excitation.

The effects of deuterium oxide (D2O) on giant internodal cells of the fresh water alga Chara gymnophylla, were investigated. D2O causes membrane excitation followed by potassium leakage. The primary effect consists of an almost instantaneous membrane depolarization resembling an action potential with incomplete repolarization. A hypothesis was proposed which deals with an "osmotic stress" effect of D2O on membrane ion channels followed by the suppression of the electrogenic pump activity. The initial changes (potential spike and rapid K+ efflux) may represent the previously undetected link between the D2O-induced temporary arrest of protoplasmic streaming and the early events triggered at the plasma membrane level as the primary site of D2O action.