Similar Propagation of SD and Hypoxic SD-Like Depolarization in Rat Hippocampus Recorded Optically and Electrically

P. G. Aitken1, G. C. Tombaugh1, D. A. Turner2, 3, 4, and G. G. Somjen1, 2

1 Department of Cell Biology, 2 Department of Neurobiology, and 3 Department of Neurosurgery (Surgery), Duke University Medical Center; and 4 Durham Veterans Affairs Medical Center, Durham, North Carolina 27710

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Aitken, P. G., G. C. Tombaugh, D. A. Turner, and G. G. Somjen. Similar propagation of SD and hypoxic SD-like depolarization in rat hippocampus recorded optically and electrically. J. Neurophysiol. 80: 1514-1521, 1998. 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 KCl 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.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

Hypoxia of cerebral tissue slices results in a well-defined sequence of intracellular and extracellular changes. Many but not all central neurons first become hyperpolarized for a few minutes (Hansen et al. 1982; Luhmann and Heinemann 1992). Then, if oxygenation is not restored, the membrane potential begins to decrease, at first gradually and then in a rapid, accelerating, seemingly regenerative manner. During this precipitous depolarization the neurons lose much of their cytoplasmic K+ and organic anions, accumulate Na+, Cl-, and Ca2+, and swell significantly because of osmotic uptake of water (Hansen 1985). This hypoxic depolarization is accompanied and probably caused by a major decrease of membrane electrical resistance (Czéh et al. 1992, 1993). Timely reoxygenation allows repolarization and full functional recovery from the depolarized state. The extracellular potential shift (Delta Vo) resulting from the massive depolarization was first discovered by Leão (1947) and compared to the similar Delta Vo that accompanies cortical spreading depression (SD) (Leão 1944). The similarities and the differences between the two phenomena, normoxic SD and hypoxic SD-like depolarization (HSD, or anoxic depolarization, AD), remained a subject of controversy ever since (Bures et al. 1974; Marranes et al. 1988; Marshall 1959; Scheller et al. 1992; Somjen et al. 1993; Tegtmeier 1993).

The change of intrinsic optical properties of tissue undergoing SD was discovered by Martins-Ferreira and Oliveira Castro (1966) in isolated retina, where the optical alteration is easily seen even at low power magnification without the aid of electronic detectors. Similar but less intense optical changes were later described in brain tissue slices (Snow et al. 1983). We recently compared the evolution of intrinsic optical signals and Delta Vo during normoxic SD and HSD (Turner et al. 1995, 1996). In the study presented here optical and electrical recordings revealed that the optical and voltage changes induced by hypoxia arise and propagate in a manner similar to that of normoxic SD, although differences in the response to pharmacological agents affecting gap junctions distinguish the two events. Some of the findings reported here appeared in abstract form (Aitken et al. 1997).

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Fischer 344 or Sprague-Dawley rats (4-6 wk, 100-125 g) were anesthetized by placing them in a bell jar containing absorbent material beneath a perforated floor onto which 10 ml of ether was poured. When deep anesthesia was reached, as indicated by lack of response to a tail pinch, the rat was decapitated. The brain was rapidly removed and placed in chilled artificial cerebrospinal fluid (ACSF). The hippocampus was dissected free and 0.4-mm slices were cut with the use of a tissue chopper (prepared by Duke University Physiology Shop to our specifications). Slices were transferred immediately to cold ACSF and from there directly to the experimental chamber ("Oslo" style interface chamber) at 34.5°C and illuminated from above (at a 45° angle to the microscope) with a stabilized halogen white light source. The ACSF flow through the chamber was regulated at 1.5 ml/min. A stimulating electrode was positioned in stratum radiatum of CA1, and two DC-coupled extracellular recording electrodes were placed in CA1 stratum radiatum to record both evoked focal excitatory postsynaptic potential (fEPSP) and DC potentials. Field potentials evoked by 60- to 150-µA, 0.1- to 0.5-ms stimulus pulses were used to verify the viability of all slices and to monitor the EPSP inhibition caused by bath application of heptanol or octanol. For the potassium injection experiments one of the recording electrodes was filled with 1.2 M KCl and connected to a Picospritzer (General Valve) pressure injection system for rapid microinjection of K+ within the hippocampal slice tissue. The ACSF composition was (in mmol/l) 130 NaCl, 3.5 KCl, 1.2 CaCl2, 1.2 MgSO4, 1.25 NaH2PO4, 24 NaHCO3, and dextrose 10, saturated with 95% O2-5% CO2, pH 7.35-7.40. The gas mixture above the slices consisted of warmed, humidified 95% O2-5% CO2 flowing at 300 ml/min. Normoxic SD experiments were performed on seven slices from five animals; hypoxic SD experiments were performed on nine slices from five animals.


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FIG. 1. Images taken during normoxic SD. Left panel: camera image showing the slice with hippocampal areas labeled. The dark line is the shadow of the stimulating electrode. A-D: sequential difference images taken at 3 (A), 6 (B), 9 (C), and 12 (D) s following KCl injection. In A the positions of the 2 recording electrodes are indicated by asterisks; the KCl injection electrode is on top. Scale bar indicates percent differences between control and experimental.


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FIG. 2. Time course of the optical (top graph) and electrical (bottom graph) signals during normoxic spreading depression (SD) from the slice shown in Fig. 1. In each graph the plots labeled 1 and 2 refer to the top and bottom electrode locations indicated in Fig. 1A. In both graphs plot 1 is the KCl injection electrode. The asterisk marks the time of KCl injection, and the labels A-D refer to the time that images A-D in Fig. 1 were taken. Note that there is a propagation delay from the injecting to the recording electrode, which is mirrored in the time of onset of the change in the optical signal.

Slices were imaged under illumination with reflected light by a Photometrics Star 1A digital charge-coupled camera (CCD; Photometrics, Tucson, AZ; 576 × 384 pixels, 12-bit resolution) attached to a Nikon SMZ dissecting microscope. Digitized images (exposure duration = 0.5 s, frame rate = 0.33 Hz) were transferred to a computer after 2 × 2 binning; each binned pixel corresponded to a region of 6.37-16.6 µm2 on the tissue, depending on magnification. For spatial mapping and temporal changes pixel-based image subtraction provided both numerical grid data (defined rectangular pixel regions in specific cytoarchitectonic areas) and detailed images of differences between each experimental frame and a control frame. The relative percent change in reflectance was calculated on a pixel by pixel basis by subtracting the control pixel value from the experimental pixel value, dividing by the control pixel value, and multiplying by 100.

For all experiments slices were allowed to equilibrate in the recording chamber for 90 min after preparation. Normoxic SD (n = 7 slices) was provoked by microinjecting 1.2 M KCl through one of the recording electrodes, and hypoxic SD (n = 9 slices) was provoked by replacing the O2 in the gas phase above the slices by N2. Oxygenation was restored as soon as the DC shift occurred.

For bath application, heptanol (n = 5) or octanol (n = 5) was prepared in a 1-M stock solution in ethanol, which was then added to ACSF and sonicated just before use. The final concentrations in the ACSF were 3 mM heptanol and 45 mM ethanol or 2 mM octanol and 30 mM ethanol. HSD was provoked (as described in preceding paragraph) in control ACSF after exposure to octanol or heptanol for 30-60 min (until evoked potentials were depressed by >90%) and again after washout and recovery of evoked potentials. Largo et al. (1997) showed that ethanol in these concentrations has no effect on SD. For focal administration octanol (1 mM with 15 mM ethanol in ACSF) was injected into s. radiatum from micropipettes of ~10-mm-tip diameter at a depth of 50-100 mm by Picospritzer, similar to KCl injections.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

Normoxic spreading depression

Figure 1 illustrates the recording setup for a typical normoxic SD experiment, with the positions of the two electrodes marked by asterisks in Fig. 1A. One electrode served to inject KCl as well as to record, and the other functioned for recording only. At the site of KCl injection, SD was evident as a negative sustained potential wave (Delta Vo) with maximum amplitude of -32.4 mV (Fig. 2, bottom panel, plot 1). This Delta Vo propagated to the second recording electrode with a delay of 9.8 s (between 50% points on the waveforms) and peak amplitude of -28.6 mV and was there preceded by a small positive deflection (Fig. 2, bottom panel, plot 2). Electrical propagation velocity was determined from the distance between the two micropipettes and the difference in the 50% maximum times of the negative shifts recorded by the two micropipettes. The average electrical conduction velocity was 8.73 ± 0.92 mm/min (mean ± SE, n = 7).

Optical recording during normoxic SD showed a patch of increased reflectance that originated at the injection electrode and spread to encompass most of the CA1 region. The advancing wave front of the region of increased reflectance was delineated by the 50% maximum optical change at the edge, compared with the peak optical subtraction difference (i.e., for a 10% maximum difference in the advancing wave front the leading edge was located at the region with a 5% optical difference). Figure 1, A-D, show a sequence of difference images taken immediately after a KCl injection. Note that the wave front moved between B and D, and the location of the wave front during each image was set to be at the 50% maximum (in this case, maximum is blue-green, and the 50% edge is the border between blue and purple). The movement of the wave front identified optically in this manner was used as the basis to calculate the optical conduction velocity, which averaged 5.84 ± 0.63 mm/min (n = 7).

The time course of the optical changes at specific locations is plotted in Fig. 2 (top panel), which shows the optical reflectance changes measured within regions of interest that were centered around the two recording electrodes. The optical changes were measured at these locations to directly compare the optical and electrical signals over time. At any locus, the onset of optical changes was always coincident with the onset of the electrical shift within the accuracy permitted by a 3-s interimage interval. However, the optical changes reached their maximum and also recovered more slowly than the electrical signal.

The optical change remained confined spatially to the CA1 region, although it did not always extend to all of CA1. The optical changes were maximal in s. radiatum, marked in s. oriens, but either minimal or undetectable in s. pyramidale. The favored direction of spread was along the s. radiatum toward CA3 if initiated near the subiculum but stopping distinctly at the CA1/CA2/CA3 border. (In these experiments we were not able to differentiate the CA2 region, and therefore we cannot say whether the boundary was actually CA1/CA2 or CA2/CA3.) This failure of SD to propagate beyond CA1 was seen in all seven (100%) slices. During recovery the optical changes retracted in reverse direction toward the injection site. Similar injections of high KCl solution directly into the CA3 region did not provoke SD (n = 3). Hypertonic (1.0 M) NaCl solution (n = 3) or weak (150 mM) KCl solution injected into CA1 (n = 3) also failed to provoke SD; however, such microinjections caused a small circular spot of optical change that dissipated without spreading.

Hypoxic spreading depression-like depolarization

Figure 3 shows images of two adjacent slices taken before, during, and after hypoxia, with the positions of the two recording electrodes in one of the slices marked in A as asterisks. Figure 3, A-D, shows sequences of images taken during HSD, which occurred 90 s after O2 withdrawal. In both slices seen in the visual field the optical change originated in s. radiatum of CA1 near the CA2 border and then propagated through most of CA1. In these and in all other experiments the optical changes did not arise diffusely, but arose focally in one or (rarely) two loci within CA1 and then spread into other parts of CA1. The optical signal abruptly stopped at the CA1/CA2/CA3 border, similar to the normoxic SD, in all nine (100%) slices. In some slices (4/9) there was an independent optical signal noted in the dentate gyrus, similar to the optical signal noted with the hypoxic SD-like signal in CA1 but smaller and usually delayed; however, the CA3 region between was quiescent in all of these slices. The 50% maximum point on the advancing optical wave front was used to calculate the optical propagation velocity, which averaged 6.79 ± 0.42 mm/min (n = 9).


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FIG. 3. Images taken during hypoxic spreading depression-like depolarization (HSD). Left panel: camera image showing the recorded slice with hippocampal areas labeled plus an adjacent slice from which electrical recordings were not made. The dark line is the stimulating electrode. A-D: sequential difference images taken at 3 (A), 6 (B), 9 (C), and 12 (D) s following onset of depolarization. In A the positions of the 2 recording electrodes are indicated. Scale bar indicates percent differences between control and experimental images. The stippled areas in the difference images are artifacts from the recording and stimulating electrodes.

Figure 4 (top panel) shows the optical signals recorded from regions of interest around the sites of the two recording electrodes in one of the slices in Fig. 3 (the other slice did not have any electrodes placed). These regions of interest were chosen to directly compare simultaneously the optical and electrical signals at individual points over time. The electrical recordings from one of the two slices of Fig. 3 are shown in the bottom panel in Fig. 4. Ninety seconds after O2 withdrawal, electrode 2 recorded a DC shift that reached a peak amplitude of -44.1 mV. This Delta Vo reached the second electrode after a delay of 7.4 s, where it reached a peak amplitude of -28.3 mV. As with normoxic SD, the approaching SD wave was preceded by a brief positive deflection of a few millivolts; such positive prodromal wavelets were registered only by electrodes located outside the focus where the optical signal began. The onset of electrical and optical changes coincided for each of the two electrode sites, although the time to peak and recovery of the optical signal was slower than that of the electrical signal. The average electrical propagation velocity of the hypoxic Delta Vo was 7.22 ± 1.60 mm/min (n = 6; measured from the 50% maximum point of the electrical signal), and average latency from O2 withdrawal to onset of depolarization was 2.94 ± 1.19 min (n = 10). Propagation velocity did not differ significantly between the normoxic and hypoxic phenomena, whether measured electrically or optically (P = 0.42 and P = 0.27, respectively; t-test).


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FIG. 4. The time course of the optical (top graph) and electrical (bottom graph) signals during hypoxic spreading depression-like depolarization from the slice shown in Fig. 3. In each graph the plots labeled 1 and 2 refer to the electrode location indicated in Fig. 3A. The labels A-D refer to the time that images A-D in Fig. 3 were taken. Note that there is a propagation delay between the recording electrodes, which is mirrored in the optical signal.

Microinjection of octanol prevents invasion by hypoxic spreading depression-like change

We administered octanol (1 mM, with 15 mM ethanol, in ACSF) by microinjection into s. radiatum of CA1 region. Usually two or three injections were delivered at 10-s intervals by 100-ms pressure pulses of 10 psi. The injections caused transient optical changes local to the region around the electrode tip. However, with the use of a control image obtained after the injection but before the hypoxia as the baseline for subtractions we were able to detect hypoxia-induced optical changes without interference from the injection artifact. Oxygen was withdrawn within 1 min of the last injection. In four of six trials in six slices the hypoxia-induced SD-like optical change, which propagated toward the injection site, failed to invade a small region around the pipette tip although often spreading past to leave a small "island" of noninvaded tissue. The optical signal completely avoided the injected area, whereas the electrical trace recorded by the injection pipette showed only a small deflection that probably was recorded through "passive" electric conduction from the surrounding invaded area (Fig. 5). After 1 h of recovery after the octanol injection, the SD-like optical signal triggered by hypoxia propagated without hindrance into the previously protected area. In the presence of bath-applied tetrodotoxin (TTX, 0.5 mM), SD also failed to invade a region injected by octanol, although the optical signal propagated as usual outside the injected area (n = 2). Control injections of vehicle (n = 8) caused transient local optical changes at the injection site but did not prevent invasion of the area surrounding the electrode tip by SD, indicating that the SD-blocking effect was specific to the octanol.


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FIG. 5. Focal octanol injection blocks the propagation of HSD. Pseudocolor subtraction images of a rat hippocampal slice recorded during HSD; the time in seconds after onset of hypoxia is indicated in right top corner of the images. Two glass recording micropipettes positioned in CA1 stratum radiatum are outlined; they are flanking a monopolar stimulating electrode (entering from the left). The traces in the right-hand column show electrical recordings from the 2 pipettes, with time in minutes on the abscissa scale; periods of hypoxia are indicated by the heavy horizontal bar. Top 2 panels show hypoxic depolarization under control conditions. After reoxygenation, 0.5 mM tetrodotoxin (TTX) was administered in the bath and after 45 min, when evoked responses disappeared, 2 mM octanol was injected through pipette "B" and vehicle through pipette "A." Image and traces of the middle panels were taken shortly after the microinjections; hypoxic SD failed to invade the site of octanol injection. After washout of octanol but in the presence of TTX hypoxic SD propagated into the previously protected area (bottom panels).

Bath application of octanol or heptanol delays hypoxia-induced spreading depression-like changes but does not block their propagation

In slices made hypoxic after being exposed for 30-60 min to 3 mM heptanol or 2 mM octanol in the bath, the latency of depolarization was increased [4.22 ± 1.53 min (n = 5) for heptanol and 4.63 ± 1.58 min (n = 5) for octanol compared with 2.94 ± 1.19 min (n = 10) in control experiments]. On washout of heptanol or octanol, depolarization latencies recovered partially [3.80 ± 1.52 min (n = 5) for heptanol and 3.61 ± 1.56 min (n = 5) for octanol]. Other measures of HSD were not changed (see Table 1). Confirming our earlier report (Largo et al. 1997), 3 mM heptanol in the bath completely prevented the propagation of SD provoked by microinjection of high KCl solution in slices previously tested by hypoxia (n = 3).

 
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TABLE 1. Effects of bath applied heptanol or octanol on hypoxic spreading depression-like depolarization

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

It is clear from these recordings that electrical and optical SD-like events triggered by severe acute hypoxia can propagate in a manner that closely resembles the spread of signals during normoxic SD. The velocity of propagation was similar for the optical as well as the electrical signals of normoxic SD and HSD, and these were also comparable to the SD waves recorded in hippocampus of intact brain (e.g., Herreras and Somjen 1993). Moreover, wave shape, duration, and amplitudes of the signals were indistinguishable between the normoxic and hypoxic processes. Electrical recordings outside the nascent focus of both processes showed the brief positive deflection preceding the main negative Delta Vo, which is the signature of an oncoming SD wave. This prodromal positivity is recognized as the signature of an oncoming SD wave (e.g., Leão, 1951). (See also the Discussion Fig. 1.1 in Bures et al. 1974, where the potential shift recorded near the trigger region of the SD does not but the potential recorded far from the trigger does show the initial transient positive wavelet.) The predilection of the CA1 region, particularly s. radiatum within CA1, and the failure to invade into the CA3 region for both the normoxic SD and the hypoxic SD-like changes, also emphasize the spatial similarities between the two.

It is well known that spontaneous, recurrent SD waves emanate from experimentally induced chronic cerebral focal ischemic lesions (Hossmann 1996; Strong et al. 1996). These SD waves begin at the margin of the infarcted core region, propagate through the penumbral region, and invade the adjacent healthy cerebral cortex. The phenomena described here is different in that it involves propagation of the SD-like depolarization within the acutely and uniformly hypoxic but not yet irreversibly injured area. However, in two cases we did note separate regions of onset within the CA1 region, which would later coalesce into a single signal. Likewise, we also noticed four slices with a separate region of optical signal within the dentate gyrus, although often at a delay compared with the start in the CA1 region. Balestrino et al. (1989) previously reported this apparent independence of dentate gyrus SD events with respect to CA1 SD events, although with electrical recording, which could not assess whether any connection was observed between the CA1 and dentate gyrus regions.

From our results it appears that a lack of oxygen first triggers SD-like change at one or a few scattered small foci, from which the process then propagates in a manner similar to normoxic SD. Even in the absence of an SD-like process, all neurons must eventually depolarize when deprived of oxygen or oxidizable substrate as ATP is depleted and the active transport of ions fails. For example, motoneurons of the cat spinal cord in situ depolarize at a rate of 3-4 mV/min without the abrupt, SD-like regenerative process (Collewijn and Van Harreveld 1966) (for SD-like events in hypoxic mouse spinal cord in vitro see Czéh and Somjen 1990), and the hypoxic depolarization in white matter (Ransom et al. 1993) similarly lacks the SD-like component. The SD-like event has the appearance of being driven by positive feedback and, in the neurons where it occurs, it greatly hastens the loss of membrane potential.

The relative resistance of the CA3 region is consistent for both the normoxic and hypoxic phenomena. It is not surprising that the hypoxia-induced optical changes would occur earliest in CA1, given the known susceptibility of this brain region to both ischemic damage in vivo (Kirino and Sano 1984; Spielmeyer 1929) and to hypoxic damage in vitro (Aitken and Schiff 1986; Balestrino et al. 1989; Kass and Lipton 1983). However, we have no explanation for the apparently complete resistance of the CA3 region to invasion by and direct initiation of normoxic SD beyond noting that this question was only a peripheral part of our study and we therefore did not determine whether we simply did not reach threshold. Successful initiation of normoxic SD in CA3 of hippocampal slices was reported by Kreisman et al. (1996). These same investigators also reported that the optical changes associated with hypoxic SD failed to invade the CA3 region, consistent with the present results.

What makes some cells or cell regions in hippocampus more readily undergo hypoxic SD and serve as an initial "pacemaker" focus is not clear. In some slices the hypoxic SD began near one of the micropipettes, suggesting that a small focus of injury can facilitate its initiation. However, the presence of an electrode is not necessary for focal onset, as is illustrated in Fig. 3, where the onset and the propagation of the optical signals were highly similar in two slices, one with and the other without electrodes.

Arguments were marshaled in favor of the view that HSD---often called AD---and normoxic SD are altogether different phenomena (Tegtmeier 1993). Indeed it was repeatedly reported that certain drugs, including antagonists of N-methyl-D-aspartate receptors, can slow or block the propagation of normoxic SD, particularly in neocortex, and raise the threshold required for its triggering, yet the same drugs have little or no effect on hypoxic depolarization (Hernández-Cáceres et al. 1987; Lauritzen and Hansen 1992). Similarly, we reported that nickel and cobalt in the bath can prevent the propagation of high K+-triggered SD in hippocampal slices without blocking the Delta Vo at the site of high K+ application, but these ions cannot prevent hypoxic SD (Jing et al. 1993).

We now found that bath application of the alkyl alcohols, heptanol and octanol, which effectively prevent the propagation of normoxic SD, retard the onset but do not block the propagation of hypoxic SD-like Delta Vo and optical change. On the other hand, local microinjection of octanol did, in most trials, prevent invasion of the injected region by hypoxic SD. The difference between focal and bath application may be quantitative; the concentration achieved by bath administration may not be as high as it is after direct injection. However, it may be that normoxic and hypoxic SD propagate by different mechanisms. Patent gap junctions may be essential for the propagation of normoxic SD but may only facilitate and not mediate the propagation of HSD. In this context it could be significant that extracellular K+ increases gradually before the onset of the rapid depolarization during hypoxia, but it remains normal until the onset of depolarization during normoxic SD (Hansen 1977; Herreras and Somjen 1993; Lehmenkühler 1990). Thus in hypoxic but not in normoxic SD the release of K+ could be the agent of progression. Remarkably, this mechanism was originally suggested by Grafstein (1956) for normoxic SD. Finally, although it is theoretically possible that the mechanism by which octanol and heptanol block SD propagation is not based on their effect on gap junctions, we do not believe this to be the case. The other known effects of these agents are depression of evoked EPSPs and of antidromic spikes (Largo et al. 1997). Yet SD propagation is not prevented when synaptic transmission is blocked by fluoroacetate (Largo et al. 1997) nor when axonal conduction is blocked with TTX (Garcia Ramos and De la Cerda 1974; Sugaya et al. 1975; Tobiasz and Nicholson 1982).

Numerous other differences were described between the events that precede normoxic and hypoxic SD. Synaptic transmission fails during hypoxia well before the depolarization starts, whereas it is preserved in normoxic SD until the start of the depolarization (Bures et al. 1974). CA1 pyramidal neurons and some but not all cortical cells undergo initial hyperpolarization and then a slow depolarization before the onset of hypoxic SD (Hansen et al. 1982; Luhmann and Heinemann 1992) but not before normoxic SD. [K+]o starts to slowly rise during the hyperpolarizing phase in hypoxia, whereas it stays constant during normoxic SD until the sudden onset of the depolarization (Herreras and Somjen 1993; Lehmenkühler 1990). This slow increase of [K+]o was attributed to an increase of K+ membrane conductance in addition to the possible failure of the ATP-fueled Na/K exchange pump. Moreover, inorganic phosphate is released from cells during hypoxia but not during normoxic SD (Scheller et al. 1992). These observations show important differences in the events that lead up to the SD-like depolarization as well as in the energy state of the cells. Synaptic failure occurs soon after oxygen withdrawal (Somjen et al. 1993) for reasons not present during normoxic SD. It is not surprising that cells fail to retain K+ and produce excess acid as well as inorganic phosphate during shortage of oxidative energy metabolism.

However, none of the reported differences address the biophysical mechanism of the rapid depolarization itself or of its propagation through tissue. No distinguishing features were as yet reported between the hypoxic and the normoxic SD-like membrane change that occurs in neurons. This similarity is more remarkable when viewed in the light of the very different energy state of the tissue in the two conditions. We emphasize that this similarity concerns only the SD-like membrane change that is peculiar to neurons in forebrain gray matter (and to a lesser degree cerebellar cortex) but is absent in other tissue elements of the CNS that depolarize more gradually during energy failure (Collewijn and Van Harreveld 1966; Ransom et al. 1993).

    ACKNOWLEDGEMENTS

  This work was supported by National Institute of Neurological Disorders and Stroke Grant RO1 NS-18670 to G. G. Somjen, a Veterans Affairs Medical Center Merit Review Award to D. A. Turner, and a fellowship from the American Heart Association to G. C. Tombaugh.

    FOOTNOTES

  Address for reprint requests: G. G. Somjen, Dept. of Cell Biology, Box 3709, Duke University Medical Center, Durham, NC 27710.

  Received 13 November 1997; accepted in final form 29 April 1998.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/98 $5.00 Copyright ©1998 The American Physiological Society