Calcium sensitive non-selective cation current promotes seizure-like discharges and spreading depression in a model neuron.

As described by others, an extracellular calcium-sensitive non-selective cation channel ([Ca(2+)](o)-sensitive NSCC) of central neurons opens when extracellular calcium level decreases. An other non-selective current is activated by rising intracellular calcium ([Ca(2+)]( i )). The [Ca(2+)](o)-sensitive NSCC is not dependent on voltage and while it is permeable by monovalent cations, it is blocked by divalent cations. We tested the hypothesis that activation of this channel can promote seizures and spreading depression (SD). We used a computer model of a neuron surrounded by interstitial space and enveloped in a glia-endothelial "buffer" system. Na(+), K(+), Ca(2+) and Cl(-) concentrations, ion fluxes and osmotically driven volume changes were computed. Conventional ion channels and the NSCC were incorporated in the neuron membrane. Activation of NSCC conductance caused the appearance of paroxysmal afterdischarges (ADs) at parameter settings that did not produce AD in the absence of NSCC. The duration of the AD depended on the amplitude of the NSCC. Similarly, NSCC also enabled the generation of SD. We conclude that NSCC can contribute to the generation of epileptiform events and to spreading depression.

Computer simulations of neuron-glia interactions mediated by ion flux.

Extracellular potassium concentration, [K(+)](o), and intracellular calcium, [Ca(2+)](i), rise during neuron excitation, seizures and spreading depression. Astrocytes probably restrain the rise of K(+) in a way that is only partly understood. To examine the effect of glial K(+) uptake, we used a model neuron equipped with Na(+), K(+), Ca(2+) and Cl(-) conductances, ion pumps and ion exchangers, surrounded by interstitial space and glia. The glial membrane was either "passive", incorporating only leak channels and an ion exchange pump, or it had rectifying K(+) channels. We computed ion fluxes, concentration changes and osmotic volume changes. Increase of [K(+)](o) stimulated the glial uptake by the glial 3Na/2K ion pump. The [K(+)](o) flux through glial leak and rectifier channels was outward as long as the driving potential was outwardly directed, but it turned inward when rising [K(+)](o)/[K(+)](i) ratio reversed the driving potential. Adjustments of glial membrane parameters influenced the neuronal firing patterns, the length of paroxysmal afterdischarge and the ignition point of spreading depression. We conclude that voltage gated K(+) currents can boost the effectiveness of the glial "potassium buffer" and that this buffer function is important even at moderate or low levels of excitation, but especially so in pathological states.

Seizure-like afterdischarges simulated in a model neuron.

To explore non-synaptic mechanisms in paroxysmal discharges, we used a computer model of a simplified hippocampal pyramidal cell, surrounded by interstitial space and a "glial-endothelial" buffer system. Ion channels for Na+, K+, Ca2+ and Cl- ion antiport 3Na/Ca, and "active" ion pumps were represented in the neuron membrane. The glia had "leak" conductances and an ion pump. Fluxes, concentration changes and cell swelling were computed. The neuron was stimulated by injecting current. Afterdischarge (AD) followed stimulation if depolarization due to rising interstitial K+ concentration ([K+]o) activated persistent Na+ current (INa.P). AD was either simple or self-regenerating; either regular (tonic) or burst-type (clonic); and always self-limiting. Self-regenerating AD required sufficient INa.P to ensure re-excitation. Burst firing depended on activation of dendritic Ca2+ currents and Ca-dependent K+ current. Varying glial buffer function influenced [K+]o accumulation and afterdischarge duration. Variations in Na+ and K+ currents influenced the threshold and the duration of AD. The data show that high [K+]o and intrinsic membrane currents can produce the feedback of self-regenerating afterdischarges without synaptic input. The simulated discharge resembles neuron behavior during paroxysmal firing in living brain tissue.

Conditions for the triggering of spreading depression studied with computer simulations.

In spite of five decades of study, the biophysics of spreading depression (SD) is incompletely understood. Earlier we have modeled seizures and SD, and we have shown that currents through ion channels normally present in neuron membranes can generate SD-like depolarization. In the present study, we define the conditions for triggering SD and the parameters that influence its course in a model of a hippocampal pyramidal cell with more complete representation of ions and channels than the previous version. "Leak" conductances for Na(+), K(+), and Cl(-) and an ion pump were present in the membrane of the entire cell; fast inactivating voltage dependent conductances for sodium and potassium in the soma; "persistent" conductances in soma and apical dendrite, and K(+)- and voltage-dependent N-methyl-D-aspartate (NMDA)-controlled conductance in the apical dendrite. The neuron was surrounded by restricted interstitial space and by a "glia-endothelium" system of extracellular ion regulation bounded by a membrane having leak conductances and an ion pump. Ion fluxes and concentration changes were continuously computed as well as osmotic cell volume changes. As long as reuptake into the neuron and "buffering" by glia kept pace with K(+) released from the neuron, stimulating current applied to the soma evoked repetitive firing that stopped when stimulation ceased. When glial uptake was reduced, K(+) released from neurons could accumulate in the interstitium and keep the neuron depolarized so that strong depolarizing pulses injected into the soma were followed either by afterdischarge or SD. SD-like depolarization was ignited when depolarization spreading into the apical dendrite, activated persistent Na(+) current and NMDA-controlled current. With membrane parameters constant, varying the injected stimulating current influenced SD onset but neither the depolarization nor the increase in extracellular K(+). Glial "leak" conductance influenced SD duration and SD ignition point. Varying maximal conductances (representing channel density) also influenced SD onset time but not the amplitude of the depolarization. Hypoxia was simulated by turning off the Na-K exchange pump, and this resulted in SD-like depolarization. The results confirm that, once ignited, SD runs an all-or-none trajectory, the level of depolarization is governed by feedback involving ion shifts and glutamate acting on ion channels and not by the number of channels open, and SD is ignited if the net persistent membrane current in the apical dendrites turns inward.

Mechanisms of spreading depression and hypoxic spreading depression-like depolarization.

Spreading depression (SD) and the related hypoxic SD-like depolarization (HSD) are characterized by rapid and nearly complete depolarization of a sizable population of brain cells with massive redistribution of ions between intracellular and extracellular compartments, that evolves as a regenerative, "all-or-none" type process, and propagates slowly as a wave in brain tissue. This article reviews the characteristics of SD and HSD and the main hypotheses that have been proposed to explain them. Both SD and HSD are composites of concurrent processes. Antagonists of N-methyl-D-aspartate (NMDA) channels or voltage-gated Na(+) or certain types of Ca(2+) channels can postpone or mitigate SD or HSD, but it takes a combination of drugs blocking all known major inward currents to effectively prevent HSD. Recent computer simulation confirmed that SD can be produced by positive feedback achieved by increase of extracellular K(+) concentration that activates persistent inward currents which then activate K(+) channels and release more K(+). Any slowly inactivating voltage and/or K(+)-dependent inward current could generate SD-like depolarization, but ordinarily, it is brought about by the cooperative action of the persistent Na(+) current I(Na,P) plus NMDA receptor-controlled current. SD is ignited when the sum of persistent inward currents exceeds persistent outward currents so that total membrane current turns inward. The degree of depolarization is not determined by the number of channels available, but by the feedback that governs the SD process. Short bouts of SD and HSD are well tolerated, but prolonged depolarization results in lasting loss of neuron function. Irreversible damage can, however, be avoided if Ca(2+) influx into neurons is prevented.

Enhancement of persistent sodium current by internal fluorescence in isolated hippocampal neurons.

Following up on an earlier chance observation, voltage-dependent whole-cell currents were recorded from isolated hippocampal neurons filled with the fluorescent dyes Fluo-3 and Fura-red, that were intermittently excited by 488 nm laser light. In the absence of any ion channel blocking drugs, in most cells depolarizing voltage steps initially evoked only the 'Hodgkin-Huxley' type early, fast inward surge followed by sustained outward current. Over 5-20 min of intermittent electrical stimulation and laser-excited fluorescence pulses, a voltage-dependent, slowly inactivating inward current also appeared and grew, while sustained outward current diminished. When K(+) currents were blocked, a small persistent inward current was usually detectable immediately, and then it increased in amplitude. This current was blocked by tetrodotoxin (TTX) and it had current-voltage (I-V) characteristics of a persistent sodium current, I(Na,P). In cells not filled with dye but illuminated by laser, and in cells with dye but not illuminated, I(Na,P) remained small. There was a more than 12-fold difference in the maximal amplitude of I(Na, P) of fluorescent compared to non-fluorescent cells. Once induced, I(Na,P) decreased very slowly. Fluorescence increased the duration but not the amplitude of the transient Na(+) current, I(Na,T). With membrane potential clamped to a constant voltage, the laser-induced fluorescence did not evoke a membrane current. It is not certain whether fluorescence-induced I(Na,P) potentiation is related to photodynamic action.

Potassium-induced enhancement of persistent inward current in hippocampal neurons in isolation and in tissue slices.

Previous work suggested a role for the voltage-dependent persistent sodium current, I(Na,P), in the generation of seizures and spreading depression (SD). Ordinarily, I(Na,P) is small in hippocampal neurons. We investigated the effect of raising external K(+) concentration, [K(+)](o), on whole-cell persistent inward current in freshly isolated hippocampal CA1 pyramidal neurons. I(Na,P) was identified by TTX-sensitivity and dependence on external Na(+) concentration. When none of the ion channels were blocked, I(Na,P) was not usually detectable, probably because competing K(+) current masked it, but after raising [K(+)](o) I(Na,P) appeared, while K(+) currents diminished. With K(+) channels blocked, I(Na,P) could usually be evoked in control solution and raising [K(+)](o) caused its reversible increase in most cells. The increase did not depend on external calcium [Ca(2+)](o). In CA1 pyramidal neurons in hippocampal slices a TTX-sensitive persistent inward current was always recorded and when [K(+)](o) was raised, it was reversibly enhanced. Strong depolarization evoked irregular current fluctuations, which were also augmented in high [K(+)](o). The findings support a role of potassium-mediated positive feedback in the generation of seizures and spreading depression.

Na(+) dependence and the role of glutamate receptors and Na(+) channels in ion fluxes during hypoxia of rat hippocampal slices.

Spreading depression (SD) as well as hypoxia-induced SD-like depolarization in forebrain gray matter are characterized by near complete depolarization of neurons. The biophysical mechanism of the depolarization is not known. Earlier we reported that simultaneous pharmacological blockade of all known major Na(+) and Ca(2+) channels prevents hypoxic SD. We now recorded extracellular voltage, Na(+), and K(+) concentrations and the intracellular potential of individual CA1 pyramidal neurons during hypoxia of rat hippocampal tissue slices after substituting Na(+) in the bath by an impermeant cation, or in the presence of channel blocking drugs applied individually and in combination. Reducing extracellular Na(+) concentration [Na(+)](o) to 90 mM postponed the hypoxia-induced extracellular DC-potential deflection (DeltaV(o)) and reduced its amplitude, and it also postponed the SD-like depolarization of neurons. After lowering [Na(+)](o) to 25 mM, SD-like DeltaV(o) became very small, indicating that an influx of Na(+) is required for SD; influx of Ca(2+) ions alone is not sufficient. We then asked whether the SD-related Na(+) current flows through glutamate-controlled and/or through voltage-gated Na(+) channels. Administration of either the non-N-methyl-D-aspartate (NMDA) receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX), or the NMDA receptor antagonist (+/-)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) postponed the hypoxic DeltaV(o) and depressed its amplitude but the effect of the combined administration of these two drugs was not greater than that of either alone. During the early phase of hypoxia, before SD onset, [K(+)](o) increased faster and reached a much higher level in the presence of glutamate antagonists than in their absence. The [K(+)](o) level reached at the height of hypoxic SD was, however, not affected. When TTX was added to DNQX and CPP, SD was prevented in half the trials. When SD did occur, it was greatly delayed, yet eventually neurons depolarized to the same extent as in normal solution. The SD-related sudden drop in [Na(+)](o) was depressed by only 19% in the presence of the three drugs, indicating that Na(+) can flow into cells through pathways other than ionotropic glutamate receptors and TTX-sensitive Na(+) channels. We conclude that, when they are functional, glutamate-receptor-mediated and voltage-gated Na(+) currents are the major generators of the self-regenerative rapid depolarization, but in their absence other pathways can sometimes take their place. The final level of SD-like depolarization is determined by positive feedback and not by the number of channels available. A schematic flow chart of the events generating hypoxic SD is discussed.

Mitochondrial and intrinsic optical signals imaged during hypoxia and spreading depression in rat hippocampal slices.

During hypoxia in the CA1 region of the rat hippocampus, spreading-depression-like depolarization (hypoxic spreading depression or HSD) is accompanied by both a negative shift of the extracellular DC potential (DeltaV(o)), and a sharp decrease in light transmittance (intrinsic optical signal or IOS). To investigate alterations in mitochondrial function during HSD and normoxic spreading depression (SD), we simultaneously imaged mitochondrial depolarization, using rhodamine-123 (R123) fluorescence, and IOS while monitoring extracellular voltage. Three major phases of the R123 signal were observed during hypoxia: a gradual, diffuse fluorescence increase, a sharp increase in fluorescence coincident with the HSD-related DeltaV(o), primarily in the CA1 region, and a plateau-like phase if reoxygenation is delayed after HSD onset, persisting until reoxygenation occurs. Two phases occurred following re-oxygenation: an abrupt and then slow decrease in fluorescence to near baseline and a slow secondary increase to slightly above baseline and a late recovery. Parallel phases of the IOS response during hypoxia were also observed though delayed compared with the R123 responses: an initial increase, a large decrease coincident with the HSD-related DeltaV(o), and a trough following HSD. After reoxygenation, there occurred a delayed increase in transmittance and then a slow decrease, returning to near baseline. When Ca(2+) was removed from the external medium, resulting in complete synaptic blockade, the mitochondrial response to hypoxia did not significantly differ from control (normal Ca(2+)) conditions. In slices maintained in low-chloride (2.4 mM) medium, a dramatic reversal in the direction of the IOS signal associated with HSD occurred, and the R123 signal during HSD was severely attenuated. Normoxic SD induced by micro-injection of KCl was also associated with a decrease in light transmittance and a sharp increase in R123 fluorescence but both responses were less pronounced than during HSD. Our results show two mitochondrial responses to hypoxia: an initial depolarization that appears to be caused by depressed electron transport due to lack of oxygen and a later, sudden, sharp depolarization linked to HSD. The depression of the second, sharp depolarization and the inversion of the IOS in low-chloride media suggest a role of Cl(-)-dependent mitochondrial swelling. Lack of effect of Ca(2+)-free medium on the R123 and IOS responses suggests that the protection against hypoxic damage by low Ca(2+) is not due to the prevention of mitochondrial depolarization.

Simulated seizures and spreading depression in a neuron model incorporating interstitial space and ion concentrations.

Sustained inward currents in neuronal membranes underlie tonic-clonic seizure discharges and spreading depression (SD). It is not known whether these currents flow through abnormally operating physiological ion channels or through pathological pathways that are not normally present. We have now used the NEURON simulating environment of Hines, Moore, and Carnevale to model seizure discharges and SD. The geometry and electrotonic properties of the model neuron conformed to a hippocampal pyramidal cell. Voltage-controlled transient and persistent sodium currents (I(Na,T) and I(Na,P)), potassium currents (I(K,DR) and I(K,A)), and N-methyl-D-aspartate (NMDA) receptor-controlled currents (I(NMDA)), were inserted in the appropriate regions of the model cell. The neuron was surrounded by an interstitial space where extracellular potassium and sodium concentration ([K(+)](o) and [Na(+)](o)) could rise or fall. Changes in intra- and extracellular ion concentrations and the resulting shifts in the driving force for ionic currents were continuously computed based on the amount of current flowing through the membrane. A Na-K exchange pump operated to restore ion balances. In addition, extracellular potassium concentration, [K(+)](o), was also controlled by a "glial" uptake function. Parameters were chosen to resemble experimental data. As long as [K(+)](o) was kept within limits by the activity of the Na-K pump and the "glial" uptake, a depolarizing current pulse applied to the cell soma evoked repetitive firing that ceased when the stimulating current stopped. If, however, [K(+)](o) was allowed to rise, then a brief pulse provoked firing that outlasted the stimulus. At the termination of such a burst, the cell hyperpolarized and then slowly depolarized and another burst erupted without outside intervention. Such "clonic" bursting could continue indefinitely maintained by an interplay of the rise and fall of potassium and sodium concentrations with membrane currents and threshold levels. SD-like depolarization could be produced in two ways, 1) by a dendritic NMDA-controlled current. Glutamate was assumed to be released in response to rising [K(+)](o). And 2) by the persistent (i.e., slowly inactivating) Na-current, I(Na,P). When both I(NMDA) and I(Na,P) were present, the two acted synergistically. We conclude that epileptiform neuronal behavior and SD-like depolarization can be generated by the feedback of ion currents that change ion concentrations, which, in turn, influence ion currents and membrane potentials. The normal stability of brain function must depend on the efficient control of ion activities, especially that of [K(+)](o).

Na(+) and K(+) concentrations, extra- and intracellular voltages, and the effect of TTX in hypoxic rat hippocampal slices.

Severe hypoxia causes rapid depolarization of CA1 neurons and glial cells that resembles spreading depression (SD). In brain slices in vitro, the SD-like depolarization and the associated irreversible loss of function can be postponed, but not prevented, by blockade of Na(+) currents by tetrodotoxin (TTX). To investigate the role of Na(+) flux, we made recordings from the CA1 region in hippocampal slices in the presence and absence of TTX. We measured membrane changes in single CA1 pyramidal neurons simultaneously with extracellular DC potential (V(o)) and either extracellular [K(+)] or [Na(+)]; alternatively, we simultaneously recorded [Na(+)](o), [K(+)](o), and V(o). Confirming previous reports, early during hypoxia, before SD onset, [K(+)](o) began to rise, whereas [Na(+)](o) still remained normal and V(o) showed a slight, gradual, negative shift; neurons first hyperpolarized and then began to gradually depolarize. The SD-like abrupt negative DeltaV(o) corresponded to a near complete depolarization of pyramidal neurons and an 89% decrease in input resistance. [K(+)](o) increased by 47 mM and [Na(+)](o) dropped by 91 mM. Changes in intracellular Na(+) and K(+) concentrations, estimated on the basis of the measured extracellular ion levels and the relative volume fractions of the neuronal, glial, and extracellular compartment, were much more moderate. Because [Na(+)](o) dropped more than [K(+)](o) increased, simple exchange of Na(+) for K(+) cannot account for these ionic changes. The apparent imbalance of charge could be made up by Cl(-) influx into neurons paralleling Na(+) flux and release of Mg(2+) from cells. The hypoxia-induced changes in interneurons resembled those observed in pyramidal neurons. Astrocytes responded with an initial slow depolarization as [K(+)](o) rose. It was followed by a rapid but incomplete depolarization as soon as SD occurred, which could be accounted for by the reduced ratio, [K(+)](i)/[K(+)](o). TTX (1 microM) markedly postponed SD, but the SD-related changes in [K(+)](o) and [Na(+)](o) were only reduced by 23 and 12%, respectively. In TTX-treated pyramidal neurons, the delayed SD-like depolarization took off from a more positive level, but the final depolarized intracellular potential and input resistance were not different from control. We conclude that TTX-sensitive channels mediate only a fraction of the Na(+) influx, and that some of the K(+) is released in exchange for Na(+). Even though TTX-sensitive Na(+) currents are not essential for the self-regenerative membrane changes during hypoxic SD, in control solutions their activation may trigger the transition from gradual to rapid depolarization of neurons, thereby synchronizing the SD-like event.

Two mechanisms that raise free intracellular calcium in rat hippocampal neurons during hypoosmotic and low NaCl treatment.

Previous studies have shown that exposing hippocampal slices to low osmolarity (pi(o)) or to low extracellular NaCl concentration ([NaCl](o)) enhances synaptic transmission and also causes interstitial calcium ([Ca(2+)](o)) to decrease. Reduction of [Ca(2+)](o) suggests cellular uptake and could explain the potentiation of synaptic transmission. We measured intracellular calcium activity ([Ca(2+)](i)) using fluorescent indicator dyes. In CA1 hippocampal pyramidal neurons in tissue slices, lowering pi(o) by approximately 70 mOsm caused "resting" [Ca(2+)](i) as well as synaptically or directly stimulated transient increases of calcium activity (Delta[Ca(2+)](i)) to transiently decrease and then to increase. In dissociated cells, lowering pi(o) by approximately 70 mOsm caused [Ca(2+)](i) to almost double on average from 83 to 155 nM. The increase of [Ca(2+)](i) was not significantly correlated with hypotonic cell swelling. Isoosmotic (mannitol- or sucrose-substituted) lowering of [NaCl](o), which did not cause cell swelling, also raised [Ca(2+)](i). Substituting NaCl with choline-Cl or Na-methyl-sulfate did not affect [Ca(2+)](i). In neurons bathed in calcium-free medium, lowering pi(o) caused a milder increase of [Ca(2+)](i), which was correlated with cell swelling, but in the absence of external Ca(2+), isotonic lowering of [NaCl](o) triggered only a brief, transient response. We conclude that decrease of extracellular ionic strength (i.e., in both low pi(o) and low [NaCl](o)) causes a net influx of Ca(2+) from the extracellular medium whereas cell swelling, or the increase in membrane tension, is a signal for the release of Ca(2+) from intracellular stores.

Intrinsic optical signals in rat hippocampal slices during hypoxia-induced spreading depression-like depolarization.

In interfaced rat hippocampal slices spreading depression (SD) and hypoxia-induced SD-like depolarization are associated with increased light reflectance and decreased light transmittance, indicating increased light scattering. By contrast, mild hypotonicity or electrical stimulation decrease light scattering, which is usually taken to be caused by cell swelling. This difference has been attributed to experimental conditions, but in our laboratory moderate osmotic challenge and SD produced opposite intrinsic optical signals (IOSs) in the same slice under identical conditions. To decide whether the SD-induced IOS is related to cell swelling, we investigated the effects of Cl(-) transport inhibitors and Cl(-) withdrawal on both light reflectance and transmittance, as well as on changes in interstitial volume and tissue electrical resistance. In normal [Cl(-)](o), early during hypoxia, there was a slight decrease in light reflectance paired with increase in transmittance. At the onset of hypoxic SD, coincident with the onset of cell swelling (restriction of TMA(+) space), the IOS signals suddenly inverted, indicating sharply increased scattering. The SD-related IOSs started in a single spot and spread out over the entire CA1 region without invading CA3. Application of 2 mM furosemide decreased IOS intensity. When [Cl(-)](o) was substituted by methylsulfate or gluconate, the SD-related reflectance increase and transmittance decrease were suppressed and replaced by opposite signals, indicating scattering decrease. Yet Cl(-) withdrawal did not prevent cell swelling measured as shrinkage of TMA(+) space. The SD-related increase of tissue electrical resistance was reduced when bath Cl(-) was replaced by methylsulfate and almost eliminated when replaced by gluconate. The TMA(+) signal is judged to be a more reliable indicator of interstitial space than tissue resistance. Neither application of cyclosporin A nor raising [Mg(2+)](o) depressed the SD-related reflectance increase, suggesting that Cl(-) flux through mitochondrial "megachannels" may not be a major factor in its generation. Fluoroacetate poisoning of glial cells (5 mM) accelerated SD onset and enhanced the SD-induced reflectance increase threefold. This suggests, first, that glial cells normally moderate the SD process and, second, that neurons are the predominant generators of the light-scattering increase. We conclude that light scattering by cerebral tissue can be changed by at least two different physical processes. Cell swelling decreases light scattering, whereas a second process increases scattering. During hypoxic SD the scattering increase masks the swelling-induced scattering decrease, but the latter is revealed when Cl(-) is removed. The scattering increase is Cl(-) dependent, nevertheless it is apparently not related to cell volume changes. Its underlying mechanism is as yet not clear; possible factors are discussed.

Use of intrinsic optical signals to monitor physiological changes in brain tissue slices.

Optical imaging techniques have the potential to bring a combination of high spatial and temporal resolution to studies of brain function. Many optical techniques require the addition of a dye or fluorescent marker to the tissue, and such methods have proven extremely valuable. It is also known that the intrinsic optical properties of neural tissue are affected by certain physiological changes and that these intrinsic optical signals can provide information not available by other means. Most authors attribute the intrinsic optical change to alterations in cell volume and concomitant change in the concentration of the cytosol. In this article we review the literature on intrinsic optical signals, covering both the mechanisms of the optical change and its use in various branches of neurophysiology. We also discuss technical aspects of the technique as used with hippocampal slices, including illumination methods, cameras, experimental methods, and data collection and analysis procedures. Finally we present data from investigations in which we used intrinsic optical signals in hippocampal slices to study the extent of spread of synaptic activation, propagation of spreading depression, extent and severity of the response to hypoxia, and tissue response to osmotic challenges. We conclude that (1) at least two processes generate intrinsic optical signals in hippocampal slices, one of which causes light scattering to change inversely with cell volume and is related to dilution of the cytoplasm, while the other, opposite in sign, may be due to mitochondrial swelling; and (2) the intrinsic optical signal can be a useful tool for spatial mapping of relatively slow events, but is not suitable for study of fast physiological processes.

Low external NaCl concentration and low osmolarity enhance voltage-gated Ca currents but depress K currents in freshly isolated rat hippocampal neurons.

In previous experiments, reducing bath osmolarity (pi o) or external NaCl concentration ([NaCl]o) caused an increase in synaptic currents recorded in whole-cell configuration from pyramidal cells of CA1 region of hippocampal slices. Slow inward current surges, assumed to be calcium currents, were also enhanced. This contrasted with the strong, reversible, generalized depression of voltage-dependent ion currents in isolated neurons, caused by sudden, brief exposure to very low pi o. I have now recorded voltage-dependent whole-cell Na-, K- and Ca-currents from freshly isolated hippocampal CA1 pyramidal neurons during more gradual lowering of pi o or [NaCl]o. Changes in cell size were determined from image areas and changes in intracellular Ca2+ activity were measured as the ratio of the fluorescences of fluo-3/fura-red by confocal microscopy. Iso-osmotic substitution of 40 or 60 mM NaCl by mannitol or sucrose for 5-7 min, or reducing pi o by deleting NaCl from the bath (osmolarity decreased by 69 or 108 mosM/kg) depressed K currents. Na currents were also strongly depressed, but this is in part attributable to reduced driving potential and ionic conductance. The depression of IK varied widely and it was inversely correlated with the degree of hypotonic swelling, suggesting that reduced ion channel conductance also reduces permeability to water. Reducing [NaCl]o by 60 mM, or pi o by 105 mosM/kg consistently and reversibly increased Ca currents. Intracellular Ca2+ level also increased, but the changes of [Ca2+]i and ICa were not correlated. Facilitation of the Ca influx into presynaptic terminals could explain the increase of synaptic currents. Depression of outward currents could also contribute to the irritability of the central nervous system typical of clinical hyponatremia.

Report of the worldwide survey on teaching physiology.

This report by George G. Somjen, for the Commission on Teaching Physiology, International Union of Physiological Sciences, presents a summary of answers received to a questionnaire concerning the state of Physiology Teaching. One hundred seventeen responses have been received from fifty countries. The results have been tabulated and contain information about the teaching methods and resources as well as the commitment in time and effort by the teaching staff. Free-ranging, sometimes pithy, comments made by the respondents have been excerpted and are included.

Inhibition of major cationic inward currents prevents spreading depression-like hypoxic depolarization in rat hippocampal tissue slices.

Hypoxia-induced spreading depression-like depolarization (hypoxic SD, or anoxic depolarization) is accompanied by the near-loss of membrane potential, severe reduction of membrane resistance, and influx of Na+, Ca2+, Cl- and water into neurons. The biophysical nature of these membrane changes is incompletely understood. In the present study we applied a pharmacological mixture (10 microM DNQX, 10 microM CPP, 1 microM TTX, and 2 mM Ni2+) to rat hippocampal tissue slices to inhibit major Na+ and Ca2+ inward currents. This inhibitory cocktail slightly depolarized CA1 pyramidal neurons and completely blocked all evoked potentials. In its presence severe hypoxia of up to 20 min duration failed to induce hypoxic SD and the accompanying intrinsic optical signal. Instead, only moderate, very slow negative shifts of the extracellular DC potential were observed. Following 10 min hypoxia and 1 hour wash-out of the inhibitors antidromic and orthodromic responses were still blocked but hypoxic SD with markedly delayed onset could be induced in most slices. In current-clamped CA1 pyramidal cells hypoxia induced a rapid, near-complete depolarization and decreased the input resistance by 89%. In the presence of the cocktail, however, hypoxia caused a gradual, partial depolarization, to about -40 mV; the membrane resistance decreased by only 37%. We conclude that simultaneous blockade of the known major Na+ and Ca2+ channels consistently prevents hypoxic SD. The hypothesis that SD initiation is the consequence of general loss of selective permeability or general membrane breakdown becomes unlikely. Instead, influx of Na+ and Ca2+ might play a crucial role in the generation of the rapid SD-like depolarization.

Volume changes induced by osmotic stress in freshly isolated rat hippocampal neurons.

The degree to which osmotic stress changes the volume of mammalian central neurons has not previously been determined. We isolated CA1 pyramidal cells and measured cell volume in four different ways. Extracellular osmolarity (pio) was lowered by omitting varying amounts of NaCl and raised by adding mannitol; the extremes of pio tested ranged from 134 to 396 mosm/kg. When pio was reduced, cell swelling varied widely. We distinguished three types of cells according to their response: "yielding cells" whose volume began to increase immediately; "delayed response cells" which swelled after a latent period of 2 min or more; and "resistant cells" whose volume did not change during exposure to hypo-osmotic solution. When pio was raised, most cells shrank slowly, reaching minimal volume in 15-20 min. We observed neither a regulatory volume decrease nor an increase. We conclude that the water permeability of the membrane of hippocampal CA1 pyramidal neurons is low compared to that of other cell types. The mechanical support of the plasma membrane given by the cytoskeleton may contribute to the resistance to swelling and protect neurons against swelling-induced damage.

Similar propagation of SD and hypoxic SD-like depolarization in rat hippocampus recorded optically and electrically.

Neuron membrane changes and ion redistribution during normoxic spreading depression (SD) induced, for example, by potassium injection, closely resemble those that occur during hypoxic SD-like depolarization (HSD) induced by oxygen withdrawal, but the degree to which the two phenomena are related is controversial. We used extracellular electrical recording and imaging of intrinsic optical signals in hippocampal tissue slices to compare 1) initiation and spread of these two phenomena and 2) the effects of putative gap junction blocking agents, heptanol and octanol. Both events arose focally, after which a clear advancing wave front of increased reflectance and DC shift spread along the CA1 stratum radiatum and s. oriens. The rate of spread was similar: conduction velocity of normoxic SD was 8.73 +/- 0.92 mm/min (mean +/- SE) measured electrically and 5.84 +/- 0.63 mm/min measured optically, whereas HSD showed values of 7.22 +/- 1.60 mm/min (electrical) and 6.79 +/- 0.42 mm/min (optical). When initiated in CA1, normoxic SD consistently failed to enter the CA3 region (7/7 slices) and could not be initiated by direct KC1 injection in the CA3 region (n = 3). Likewise, the hypoxic SD-like optical signal showed onset in the CA1 region and halted at the CA1/CA3 boundary (9/9 slices), but in some (4/9) slices the dentate gyrus region showed a separate onset of signal changes. Microinjection into CA1 stratum radiatum of octanol (1 mM), which when bath applied arrests the spread of normoxic SD, created a small focus that appeared to be protected from hypoxic depolarization. However, bath application of heptanol (3 mM) or octanol (2 mM) did not prevent the spread of HSD, although the onset was delayed. This suggests that, although gap junctions may be essential for the spread of normoxic SD, they may play a less important role in the spread of HSD.