INTRODUCTION

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Intrinsic optical signals (IOSs) consist of changes in the optical properties of unstained tissue measured as a change of either light transmittance or reflectance. These responses are detected over a wide waveband (Kreisman et al. 1955; Tao 2000); the more narrowband autofluorescence responses are not usually classified as IOSs. There are several types of perturbation that cause readily detectable IOS in CNS tissue, either in situ or in tissue slices, including synaptic activation, osmotic pressure changes, spreading depression (SD), hypoxic SD-like depolarization (HSD, also known as anoxic depolarization, AD), and seizures (Aitken et al. 1999; Andrew et al. 1999; Buchheim et al. 1999; Hochman 1997; Holthoff and Witte 1998; Joshi and Andrew 2001; Kreisman and LaManna 1999; Müller and Somjen 1999; Villringer and Chance 1997). For example, synaptic activation or moderate hypo-osmotic exposure lead to increased light transmittance or decreased reflectance, indicating reduced light scattering (Aitken et al. 1999; Andrew et al. 1997; Kreisman et al. 1995; Tao 2000). These perturbations are known to be associated with cell swelling, usually recorded in brain tissue as shrinkage of interstitial volume (ISV), derived from extracellular tetramethylammonium concentration [TMA⁺]_o (Dietzel et al. 1980; Holthoff and Witte 1996). Indeed, the swelling of cells in suspension has long been routinely estimated from the decrease of light scattering (reviewed by Aitken et al. 1999; Johnson et al. 2000).

However, very strong hypo-osmotic treatment is associated with a light-scattering increase (Aitken et al. 1999; Vargová et al. 1999). Also, during various forms of SD and HSD, light-scattering increases (Aitken et al. 1998, 1999; Martins-Ferreira and de Oliviera Castro 1966; Melzian et al. 1996; Müller and Somjen 1999; Snow et al. 1983; Világi et al. 2001) even though it is well known that these conditions are associated with powerful cell swelling (Hansen and Olsen 1980; Jing et al. 1994; Pérez-Pinzón et al. 1995). We also have found that when most of the Cl^- in bathing solution is replaced by an impermeant anion, either methylsulfate or gluconate, the scattering increase associated with SD or HSD is suppressed and replaced by monotonic scattering decrease even though cell swelling is not inhibited (Bahar et al. 2000; Müller and Somjen 1999). Epileptiform discharges induced by low external Mg²⁺ or by 4-aminopyridine are associated with a scattering decrease while low [Ca²⁺]_o-induced spontaneous discharges are accompanied by increased light scattering, even though all three treatments cause cell swelling (Buchheim et al. 1999).

The light-scattering increase occurring during SD has been attributed by Kreisman et al. (1995) to an artifact caused by the change in the shape of tissue slices placed at the interface of liquid and air. This particular explanation suggests that, as the slice swells, its surface bulges in the manner of a convex lens, causing less light to enter the optical detector. According to Kreisman et al. (1995), slices fully submerged in bathing medium, which present no strong change of the index of refraction at the surface, do not show the apparent scattering increase. A second possible explanation was presented by Andrew et al. (1999), who attribute the scattering increase during severe forms of SD to beading of dendrites, signaling possibly irreversible injury. However, such an increase would generally be limited to dendritic areas. But additional explanations may also underlie this phenomenon.

The present study had two purposes. First, to establish the relationship between ISV and IOS recorded at the same time from the same site in tissue slices during a variety of experimental conditions. In the majority of previous studies, either one or the other technique was used, but not both at the same time. These experiments represent the first comprehensive analysis, in hippocampal slices, of the relationship between the two signals, IOS and interstitial volume. In addition, we also compared the changes of both, IOS and relative cell volume, in both interface and submerged slices. We conclude that there are at least two mechanisms that underlie the observed IOS: one closely related to cell-volume changes and the other likely originating from intracellular organelle swelling, other subcellular mechanisms, or alterations in cell shape. The data have been presented in part as an abstract (Fayuk et al. 1999).

	METHODS

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Tissue slice preparation

Hippocampal tissue slices were prepared from male Sprague-Dawley rats (75-150 g). All animal use was approved by the Duke University Animal Care and Use Committee. The rats were decapitated under fluothane (halothane) anesthesia; the brain was rapidly removed from the skull and placed in chilled artificial cerebrospinal fluid (ACSF) for 1-2 min. One hippocampus was isolated, and transverse slices of 400 µm thickness were cut using a tissue chopper. Slices were immediately transferred to a holding chamber and maintained at 22°C, then transferred to either an interface or a submerged recording chamber, where they were perfused with oxygenated ACSF (1.5 ml/min), continuously aerated with 95% O₂-5% CO₂ humidified gas mixture and maintained at 35°C. The small, submerged chamber was designed to fit completely within the larger interface chamber (with the net removed) so that all optical pathways, including the light sources, microscope and camera, were the same between the two chamber types. The depth of submersion was 3-5 mm. All experimental procedures were started 90 min after slice preparation, to allow the tissue slices to recover and to stabilize. The number of experimental slices is shown accompanying the data.

Solutions

Standard ACSF had the following composition (in mM): 130 NaCl, 3.5 KCl, 1.25 NaH₂PO₄, 24 NaHCO₃, 1.2 CaCl₂, 1.2 MgSO₄, and 10 dextrose, pH 7.4, when saturated with a gas mixture containing 5% CO₂. To test the effects of osmotic perturbations, 20, 40, or 80 mM of NaCl was omitted from standard ACSF (to decrease osmotic pressure) or 25, 50, or 100 mM of mannitol was added to the ACSF (to increase osmotic pressure). Mannitol was chosen because it is neither metabolized nor actively transported into cells in the CNS in the time period of these experiments and thus provides a relatively inert (and nontoxic) mechanism to enhance extracellular osmolarity. In addition, both sucrose and hyperonic NaCl were also tested. The actual osmolarity values resulting from these manipulations are shown in Table 1. However, our convention in labeling the figures is to give the manipulation rather than the resulting osmolarity value. In low-Cl solutions, methylsulfate replaced all but 3.4 mM Cl [the remaining as CaCl₂ and tetramethylammonium chloride (TMA-Cl)]. TMA-Cl (0.2 or 1 mM) was added to all solutions to provide a baseline concentration when TMA⁺-sensitive electrodes were used.

Experimental procedures

HYPOXIA. The interface slices were deprived of oxygen by rapidly replacing the O₂ in the gas phase above the slices with N₂.

SYNAPTIC ACTIVATION. A bipolar stimulating electrode made of insulated platinum-iridium wire was placed in stratum radiatum of the Schaffer collaterals of CA1 region. A 30-s stimulus train (100-µs pulses at 10 Hz) was delivered. Stimulus current was adjusted, using single pulses prior to the train, to produce a field excitatory postsynaptic potential (EPSP) of 50% of the maximum amplitude.

HIGH-K⁺ MICRO-INJECTIONS. A glass electrode (5 M) filled with 1 M KCl or KCH₃SO₄ (for slices bathed in low-Cl solution) was hermetically fixed in a special holder connected with plastic tube to a Picospritzer II (General Valve, E. Hanover, NJ) and the tip inserted into the st. radiatum. A high-pressure pulse (40-80 lbs/in², 20-40 ms) was applied to inject in the tissue an amount of K⁺ sufficient to induce SD.

OSMOTIC MANIPULATIONS. In most experiments, the slices were exposed for 30 min to each of three hypo-osmotic (20, 40, or 80 mM of NaCl or NaCH₃SO₄ was omitted) or hyper-osmotic (25, 50, or 100 mM of mannitol was added) test solutions, in order of decreasing or increasing osmotic pressure (osmotic strength given in Table 1). Between administrations of the test solutions, the slices were allowed to recover in control ACSF for 30 min. This period of time was sufficient for equilibration of interstitial space with modified ACSF. These are clinically relevant changes. For example, patients may start to become confused with a Na⁺ of 130 mM (osmolarity of 260 mOsm). The Hypo 20 mM condition is in this range. With more severe changes (particularly Na⁺ <120 mM or <240 mOsm), patients may experience confusion and seizures, and treatment is required. This threshold concentration is similar to the Hypo 40 mM condition used here. Na⁺ values <110 mM are life-threatening, if untreated.

Relative ISV measurements with TMA+ ion-selective electrodes

Changes of ISV were estimated from the extracellular concentration change of a probe ion, tetra-methyl ammonium (TMA⁺). This ion enters cells very slowly and therefore becomes more concentrated in extracellular fluid if cells swell or less concentrated if cells shrink (Dietzel et al. 1980; Nicholson and Phillips 1981). The probe ion (0.2 or 1 mM) was dissolved in the ACSF. In experiments with osmotic manipulations when ISV changes were slow, TMA⁺ was also administered by micro-iontophoresis and detected by the sensing pipette. Pulses of 0.1-1.0 s, 0.1-0.5 µA amplitude, and at 0.5-1 min intervals were used to iontophorese TMA⁺ from a micropipette located ~50-100 µm from the TMA⁺-sensitive electrode, resulting in transient [TMA⁺]_o changes. The iontophoresis pipettes contained 1.0 M solution of TMA-Cl (resistance of 8-20 M). Changes in relative ISV were calculated as a percent of "control" according to the formula (Dietzel et al. 1980)
where [TMA⁺]_Control and [TMA⁺]_Experiment are the amplitudes of the transient increases of concentration (transient peak  baseline) in control and experimental conditions, respectively. However, for most of the rapid responses, including synaptic activation and the various forms of spreading depression, TMA⁺ transients were not performed because of the necessary long intervals between iontophoretic pulses, which were comparable to the overall duration of the responses. In these circumstances, only the baseline response of extracellular TMA⁺ concentration changes was measured and the calculation of relative ISV changes was made as
where [TMA⁺]_Control and [TMA⁺]_Experiment are the extracellular TMA⁺ concentration in control and experimental conditions, respectively.

The concentration of TMA⁺ in extracellular space was measured with double-barreled ion-selective microelectrodes (Jing et al. 1994; Nicholson 1993). The tip of the sensing barrel was filled with the World Precision Instruments (WPI) IE-190 ion exchanger, which is equivalent to the Corning 477317 exchanger and is about three orders of magnitude more sensitive to TMA⁺ than to K⁺ (Dietzel et al. 1980). Electrodes were calibrated before and after each experiment, and an experiment was rejected in the rare instance when the variation exceeded 10%. The sensitivity ("slope") ranged from 56 to 59 mV/decade. Ionic signals were recorded as electrode potential changes by a computer and then recalculated to millimolar scale. The reference barrel of ion-selective electrode served to record DC-coupled extracellular potential (V_o) (Jing et al. 1994).

Ion-selective electrodes respond to ion activity rather than to concentration, but calibration was performed in terms of the known concentrations of calibrating solutions, and the results are reported as concentrations (see also Nicholson 1993). Concentrations remain proportional to activities only as long as the activity coefficient does not change, and activity coefficients are influenced by total ionic strength. The standard calibrating solutions were made in ACSF by varying the [TMA⁺]_o but keeping total ion concentration constant by reciprocal changes in Na⁺ concentration ([Na⁺]_o) (Jing et al. 1994). For the present purpose, to check the influence of changing [Na⁺]_o, [Cl]_o, [K⁺]_o, and ionic strength, electrodes were calibrated in normal as well as in low-and high-osmotic, low-Cl (3.4 mM), and high-K⁺ (100 mM) solutions. The calibration curve in a range of 0.1-10 mM of [TMA⁺]_o shifted only a small amount (5%) in all solutions except for low Cl, when it did shift by 7 to 8 mV. The slope of the calibration curve was not affected by the shift, so the estimated relative interstitial volume changes (ISV) were equivalent in both low and normal Cl solutions.

Imaging

The chamber was illuminated from below using a stabilized xenon arc lamp for the transmission IOS measurements. The illumination consisted of either wideband white light or was filtered to a narrowband-pass centered at 650 nm. Slices in the interface or submerged chamber were imaged through a Nikon dissecting microscope (SMZ-U), using a Cooke Instruments Sensicam, with 640 × 480 digital spatial resolution (12-bit) by the interline charge-coupled device (cooled to 15°C). Images were taken once every 1-2 s with 50- to 100-ms exposure for each image. The images were usually acquired as 2 × 2 binned images, reducing the effective spatial resolution to 320 × 240 pixels. The digital images were directly stored on the host computer as 12-bit files. Each binned pixel corresponded to a slice region of 6-16 µm², depending on the magnification used. All measurements were of transmitted light. We have shown previously (Aitken et al. 1999) that measurement of IOS using either reflected or transmitted light is equivalent, with only the sign of the signal being affected.

Data analysis

Each series of images was analyzed using anatomically based regions of interest, generating a series of 12-bit (averaged across multiple pixels) intensity numbers for plotting, as a function of time. The 12-bit pixel values are linear and proportional to the absolute light level, though scaled. Difference images were also calculated for each series on a pixel by pixel basis, using a control image as a baseline for each further image in the series: I/I = (Image_i - Image_control)/Image_control. In these difference images, no difference was scaled as a value of 128, while a decrease showed smaller values (to black) and an increase showed larger values (to white). Data were analyzed for statistical significance using t-tests and ANOVA where needed. Data are shown as mean ± SD with the number of independent slices given.

	RESULTS

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Our results reveal the existence of two distinct and independent optical signal mechanisms, which underlie intrinsic optical signals and of which only one is related to cell volume. We describe the two mechanisms associated with IOS separately.

Light transmittance (LT) changes that are dependent on cell-volume changes

SYNAPTIC ACTIVATION. Synaptic activation (30 s) induced by 10-Hz electrical stimulation of the CA3 afferents projecting into CA1 (Schaffer collaterals) resulted in increased LT, accompanied by a decrease of ISV. The IOS and ISV changes were very similar in interface and submerged slices (Figs. 1 and 2 and Table 2). Under both conditions, the IOS showed an increased translucence in the CA1 region, which began at the stimulating electrode and faded away with distance. In the interface slices, both the IOS and the ISV responses were more pronounced within the cell body layer (st. pyramidale) than in dendritic regions, while in the submerged slices the responses were distributed more uniformly through the CA1 region, without a substantial difference between layers (Table 2).

View larger version (99K): [in this window] [in a new window]   Fig. 1. Images of intrinsic optical signals (IOSs) during and after synaptic activation. Top: transmitted light images showing slice landmarks and electrode placement. Circle: stimulating electrode. Rectangle: region of interest for both the light transmission and interstitial volume (ISV) measurements. Bottom rows: difference images showing changes in light transmission as per the scale (right margin). Image times are presented in the left margin. Thirty seconds of 10-Hz stimulation of Schaffer fiber bundle started at time 0. Left: interface slice in normal artificial cerebrospinal fluid (ACSF). Middle: interface slice in low-Cl medium. Right: submerged slice in normal ACSF. Synaptic activation induced a light transmittance increase under all three conditions, but IOS signal was reduced significantly when the slice was in low [Cl]o.

View larger version (12K): [in this window] [in a new window]   Fig. 2. Time courses of the changes in IOS () and ISV [- - -, based on continuous tetra-methyl-ammonium (TMA+) measurement] for the experiment of Fig. 1. A: interface slice in normal ACSF. B: interface slice in low-Cl medium. C: submerged slice in normal ACSF. ISV and IOS are both represented as percent of control (prestimulation) value. Time of stimulation indicated by horizontal bar. ISV decreased (indicating cell swelling) and LT increased (indicating light scattering decrease) in all 3 trials. In low-Cl medium, the amplitude of the IOS was reduced but not that of the ISV change. In all 3 conditions, ISV overshot the rest level following cessation of the stimulus. In the submerged slice, the poststimulus ISV overshoot was appropriately accompanied by an LT undershoot.

OSMOTIC MANIPULATION AND TMA⁺ PULSE RESPONSES. During osmotic manipulations, extracellular space changes were calculated according to changes of [TMA⁺]_o pulse amplitude [TMA⁺]_o (see METHODS for details). Figure 3 illustrates the original traces of [TMA⁺]_o measurements in st. radiatum of CA1 in submerged slices during exposure to both hypo-osmotic media (A and B; 40 mM NaCl) and hyper-osmotic media (C and D; +100 mM mannitol). The actual, resulting osmolarity for these solutions is given in Table 1. Both the baseline concentration and the superimposed concentration transients induced by iontophoresis ([TMA⁺]_o) increased during hypo-osmotic or decreased with hyperosmotic treatment, revealing a decrease or increase, respectively, of extracellular space. These responses show a similar waveform shape for the TMA⁺ pulse responses as indicated by the TMA⁺ pulse patterns during the rest, experimental, and recovery conditions (Fig. 3, B and D), in spite of widely different TMA⁺ amplitudes. These TMA⁺ responses were typical, including both the intermittent response to iontophoresis as well as the baseline response. Exposure to mildly or moderately hypo-osmotic medium (Hypo-20 or Hypo-40: removing either 20 or 40 mM of NaCl from normal ACSF or NaCH₃SO₄ from low-Cl solution) resulted in an increased light transmittance and reduced ISV in both interface and submerged slices (Fig. 3 and 4, and Table 2). While the effects were qualitatively similar, much larger IOSs were recorded in submerged slices (Fig. 4).

View larger version (32K): [in this window] [in a new window]   Fig. 3. Illustrations of TMA+ concentration measurements during typical hypo-osmotic (40 mM NaCl; A and B) and hyper-osmotic experiment (+100 mM mannitol, C and D). Logarithmic voltage response of TMA+-sensitive electrode was converted to linear millimolar scale by computer, but no other correction was applied. A and C: continuing traces that represent TMA+ measurements during full length of experiments. Horizontal bars indicate time of osmotic challenge. B and D: detailed views of individual TMA+ responses resulted of individual iontophoretic pulses during the control baseline, during the response to the osmotic change, and after return to the baseline solution (as marked by arrows in A and C). Note that TMA+ responses are rather uniform and their amplitudes change significantly under osmotic manipulations.

View larger version (25K): [in this window] [in a new window]   Fig. 4. Changes in IOS () and ISV ( - , with  representing times of TMA+ iontophoretic pulses) during exposure to altered osmolarity. A-C: hypo-osmotic treatment. D: hyper-osmotic treatment. The horizontal bar indicates the time of osmotic stress. A: interface slice in normal [Cl]o, 40 mM NaCl deleted (osmolarity decreased by 75 mOsm). B: interfaced slice in low-Cl medium, 40 mM NaCH3SO4 deleted. C: submerged slice in normal [Cl]o, 40 mM NaCl deleted. In all 3 conditions, the hypotonic treatment resulted in increased LT and reduced ISV, consistent with cell swelling. D: submerged slice, normal [Cl]o. 100 mM mannitol added to ACSF for 15 min. The ISV expanded and light transmission (LT) decreased indicating hypertonic cell shrinkage.

Decreased light transmittance concurrent with decreased ISV

SPONTANEOUS SD DURING HYPOTONICITY. The most striking example of a decreased light transmittance associated with decreased ISV was noted when spontaneous SD occurred during hypo-osmotic treatment. In some interface slices during 30-min exposure to Hypo-40 treatment, a single, spontaneous SD occurred (Fig. 5A). During the SD the light transmittance, which initially increased as result of the hypo-osmotic treatment, suddenly decreased while the ISV underwent a transient additional reduction. After the SD episode, both the IOS and ISV returned to pre-SD levels, LT remaining higher and ISV lower than the initial control level. Thus during the same trial, the LT increase attributable to cell swelling was interrupted by a LT decrease, apparently caused by the SD, indicating a summation or overlap of two opposite IOS signals. Unlike the biphasic IOS response, ISV showed only a persistent decrease, consistent with cell swelling, caused first by the low ambient osmolarity and then transiently and additionally by the superimposed episode of SD.

View larger version (21K): [in this window] [in a new window]   Fig. 5. Light transmittance changes during hypotonic treatment and "spontaneous" spreading depression (SD) episodes. A: interface slice in normal [Cl]o; 40 mM NaCl was deleted during the time indicated by the horizontal bar. B: interface slice in low-[Cl]o medium; 40 mM NaCH3SO4 deleted. C: submerged slice in normal [Cl]o; 80 mM NaCl deleted. The LT increase caused by the hypotonicity was interrupted by transient LT decrease when SD occurred in normal [Cl]o (A and C) and by additional LT increase in low [Cl]o (B). D: changes in IOS during severe hypotonia (80 mM NaCl removed) in interface slice in normal [Cl]o. The initial increase in LT turns into a gradual decrease even prior to SD occurrence. Superimposed on this decreasing signal are additional transient drops during repeated episodes of SD.

K⁺ MICROINJECTION INDUCED SD. In normally oxygenated interface slices in normal ACSF, microinjection of 1 M KCl into the CA1 region evoked SD (Figs. 6 and 7). In slices submerged in normal ACSF, injection of KCl did not trigger SD, but when tonicity was moderately reduced, high-K⁺ injection did induce a typical wave of SD that propagated throughout most or the entire CA1 region, accompanied by the same electrical, optical, and ISV changes as those noted in interface slices (Figs. 8 and 9). The failure to provoke SD with high-K⁺ injection in submerged slices in isotonic media is due perhaps to the more efficient washout of the K⁺ from the slice.

View larger version (62K): [in this window] [in a new window]   Fig. 6. Images following microinjection of high-K+ solution into CA1 region in an interface slice maintained in normal- (left) and low-Cl (right) medium. Top: unsubtracted images. The asterisks show the injection site, and the rectangles represent the IOS region of interest and the location of the TMA+ recording electrode inside the box. After the injection (time 0), the signals in normal and low [Cl]o are at first similar, but then by 8 s there is a divergence: in low [Cl]o (right), the CA1 region continues to brighten, whereas in normal ACSF (left), it darkens, then it brightens again. Note that in the pyramidal cell layer the phase of darkening occurs later than in st. radiatum.

View larger version (13K): [in this window] [in a new window]   Fig. 7. Time course of the IOS (top solid line), ISV (dashed line), and Vo (bottom solid line) in interface slices during SD caused by high-K+ injection (vertical arrow) in normal ACSF (A), low [Cl]o (B), and hypertonic (100 mM sucrose added) normal Cl medium (C). In normal ACSF (A), the onset of SD is marked by a small increase of LT followed by a rapid decrease, ending with a small overshoot, while the ISV shows a large decrease followed by a marked, prolonged overshoot. In low-[Cl]o condition (B), the 2nd decreasing phase of the IOS is replaced by a monotonic increase. The ISV shows only a prolonged decrease and no overshoot. Exposure to hypertonic medium (C) had a similar effect on the IOS as did low [Cl]o treatment, but the ISV change in hypertonic condition was more similar to that in normal ACSF.

View larger version (97K): [in this window] [in a new window]   Fig. 8. Images of IOS changes in submerged slices during SD provoked by high-K+ solution microinjection under moderate (Hypo-40) and severe (Hypo-80) hypotonic exposure, in normal and in low [Cl]o. Top row: the asterisk identifies the injection site and the small rectangle the area of the IOS and ISV measurements. With 40 mM NaCl deleted from normal ACSF ([Cl]o = 96.9 mM; left), SD was accompanied by darkening of the affected region. In similar degree of hypotonicity (40 mM NaCH3SO4 deleted) but low [Cl]o ([Cl]o = 3.4 mM), the darkening was replaced by brightening (middle). In severe hypotonia (80 mM NaCH3SO4 deleted) and low [Cl]o ([Cl]o = 3.4 mM), the entire IOS is attenuated but the LT undergoes a sequence of light-dark-light (right), qualitatively similar to normal [Cl]o conditions.

View larger version (30K): [in this window] [in a new window]   Fig. 9. Time courses of IOS (top solid line), ISV (dashed line), and Vo (bottom solid line) in submerged slices during SD provoked by high-K+ microinjection in normal (A and B) and low-[Cl]o (C and D) conditions under different osmolarities. Data presented in B-D correspond to the experiments imaged in Fig. 8. A and B as well as C and D represent 2 repetitive SD induced in the same slice. A: mild hypotonicity (20 mM NaCl deleted), normal [Cl]o. B: moderate hypotonicity (40 mM NaCl deleted), high [Cl]o. C: moderate hypotonicity (40 mM NaCH3SO4 deleted), low [Cl]o. D: severe hypotonicity, (80 mM NaCH3SO4 deleted), low [Cl]o. Note that in normal [Cl]o, mild and moderate hypotonicity (A and B) SD was accompanied by the usual light-dark-light sequence of LT change. In low [Cl]o moderate hypotonicity (C), the dark phase of LT was replaced by continuing brightening. In low [Cl]o severe hypotonicity (D), the IOSs reverted to the light-dark-light sequence, albeit at much reduced amplitude. Note also that in low [Cl]o conditions the post-SD overshooting of ISV and LT are missing. Insets: highlight the detailed, initial response to the KCl injection. Note that the ISV decreased prior to the simultaneous change in IOS and Vo in both instances.

HYPOXIC SD-LIKE DEPOLARIZATION (HSD). As reported earlier (Bahar et al. 2000), interface slices undergo several phases of the optical response during and after the withdrawal of oxygen (Fig. 10A). The first phase in the normal-[Cl]_o condition is a slow increase in light transmittance, which is accompanied by a gradual decreased ISV and a barely detectable negative shift of V_o. With the onset of the hypoxic SD, however, the accelerating decrease in ISV is accompanied by a sharp decrease in light transmittance (Fig. 10A, Table 2), similarly to the other forms of SD discussed in the preceding text. Following HSD, ISV markedly overshot the baseline, while LT started to increase, then it decreased again tending to return to control level at the same time as the ISV. During hypoxia in low-Cl solution, light transmittance increased somewhat before the SD as also occurred in normal [Cl]_o, but during HSD, the reduced transmittance was replaced by increased light transmittance in spite of increasing ISV shrinkage (Fig. 10B, Table 2) (see also Bahar et al. 2000; Müller and Somjen 1999). Thus in the case of HSD as in that of normoxic SD, removing Cl caused the LT decrease to be replaced by LT increase. Following HSD in low [Cl]_o, ISV overshot and LT undershot their respective rest levels, LT reaching bottom and returning to its rest level rather more slowly than ISV.

View larger version (15K): [in this window] [in a new window]   Fig. 10. Time courses of IOS (top solid line), ISV (dashed line), and Vo (bottom solid line) during hypoxia in an interface slice. The lack of oxygen is indicated by the horizontal bar. A: in normal ACSF. B: in low [Cl]o. In normal ACSF, soon after the start of hypoxia LT began to gradually increase, ISV to decrease, while Vo showed a barely detectable negative shift. Then at the onset of HSD, LT and ISV abruptly decreased and Vo showed the usual large negative shift. Following HSD, ISV and Vo overshot the baseline while LT went through a secondary slow decrease before recovering. In low-[Cl]o condition (B), the HSD-related LT darkening was replaced by a sharp LT brightening, and SD was followed by undershooting of LT.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The major conclusion emerging from these experiments is that there are at least two different mechanisms underlying reversible intrinsic optical signals in hippocampal slices. The first mechanism results in an increased light transmittance (decreased scattering) when ISV shrinks (cells swell) and an opposite change when ISV expands. The magnitude of this type of IOS is correlated with the magnitude of the cell-volume changes. The second mechanism appears to be independent of cell volume, and it produces an IOS of opposite direction compared with the first, namely decreased light transmittance (increased scattering) in spite of persistent or increased cell swelling. These two independent mechanisms can occur simultaneously and produce opposite optical signals that summate and at least partially cancel each other. Importantly, the signals are qualitatively similar in both interface and submerged conditions although they differ in degree (Table 2), perhaps because of the different resting ISV fraction in the two preparations. However, the two slice conditions clearly show similar results with both types of signals.

Mechanism of the IOS related to cell-volume changes

This first type of IOS has long been recognized as related to cell-volume changes (review by Aitken et al. 1999). In the present study, it was observed during synaptic transmission, mild to moderate osmotic stress, and the initial phase of hypoxia prior to SD onset. LT increased when osmolarity decreased, and conversely, LT decreased when osmolarity increased in concentration-dependent fashion (Table 2). This signal may therefore serve as an index of cell-volume changes under these particular circumstances. The generally accepted explanation of this first IOS is the relative change in size of the scattering particles caused by water flux, associated with the changes in cell volume (literature quoted in review by Aitken et al. 1999). The IOS related to cell-volume change is probably more directly proportional to relative cell-volume changes than to relative ISV changes, in agreement with the interpretation that it is generated by dilution of scattering particles. Presumably these scattering particles are macromolecules and/or organelles inside cells, having an effect on scattering, similar to hemoglobin in red blood cells within a medium.

Mechanism of the LT decrease (scattering increase) during severe hypotonia, SD and HSD

The mechanism of the second type of IOS has not been established. Its direction is opposite to that expected from cell-volume change, as it consists of an increase in light scattering (LT decrease) in the face of strong cell swelling. It occurs during severe hypo-osmotic treatment and during SD and HSD. As first suggested by Müller and Somjen (1999), it may be attributable to the swelling of intracellular organelles, especially mitochondria, provided that the refractive index of these organelles is noticeably different from that of the cytosol. This idea is supported by the abolition of the scattering increase (LT decrease) by low [Cl]_o, if we assume that the organelle swelling is Cl dependent. Also supporting the idea of organelle swelling is the observation that mitochondria depolarize suddenly at the onset of SD or HSD (Bahar et al. 2000; Sick and Perez-Pinzon 1999). The mitochondrial depolarization is depressed by lowering of [Cl]_o, similarly to the SD-related scattering increase. Mitochondrial depolarization is probably associated with mitochondrial swelling. Additionally, the suppression of the SD-related scattering increase by strong hypertonicity (Fig. 7C), and its facilitation by severe hypotonia are also in line with this hypothesis because the former can counteract and the latter can favor organelle swelling.

Other possible subcellular processes that could cause the light-scattering increase include the clumping of scattering cytoplasmic particles (Müller and Somjen 1999), i.e., heterogeneity of solute distribution (Tao 2000). As already mentioned in the INTRODUCTION, other explanations have earlier been suggested for the light-scattering increase associated with SD. Kreisman et al. (1995) suggested that changes in the radius of curvature of the surface of the slice could reduce the amount of light reaching a detector above the surface, particularly with reflectance measurements. Tao (2000), however, implemented a fiber-optic light collector in contact with the slice surface and still saw the polarity reversal IOS, precluding "lensing" as an explanation. Moreover, as we now report, the decrease of LT was in fact more pronounced in submerged than in interface slices, also indicating that the "lensing" of the slice surface cannot fully explain the decreased translucence.

Jarvis et al. (1999) and Andrew et al. (1999) suggested that dendritic beading or other cell shape change due to hypoxia and excitotoxicity may be the source of decreased light transmittance. Such beading (reversible or irreversible) has not been reported in response to normoxic SD, yet the IOS is similar in SD and HSD. Additionally, the IOSs associated with both normoxic SD and HSD are rapidly reversible, provided that oxygenation is restored soon enough after the onset of HSD, and neither condition causes permanent loss of synaptic function (Aitken et al. 1998, 1999; Müller and Somjen 1999). Hori and Carpenter (1994) and Polischuk et al. (1998) considered beading to be a sign of cell injury for neurons in tissue slices. While reversible dendritic beading has been reported in neurons in cell culture, recovery was a very slow process (Park et al. 1996), unlike the rapid reversibility of the SD-related LT decrease. Moreover, not only st. radiatum but also st. pyramidale darkened during SD, albeit sluggishly (Figs. 7, left, and 9, left), yet st. pyramidale contains no neuronal dendrites. We conclude that even though "lensing" of the tissue slice and beading of dendrites undoubtedly can occur, neither process can fully explain the reduction of tissue translucence that was observed during SD, HSD, and severe hypotonicity, particularly the rapid and dynamic time course and spatial localization.

Basarsky et al. (1998) reported increased LT accompanying SD induced by bath-applied ouabain in hippocampal slices. This is similar to the response in low [Cl]_o and severe hypertonicity reported here, but different from the scattering increase (LT decrease) more commonly observed during SD in normal ACSF (see also Martins-Ferreira and de Oliveira Castro 1966; Snow et al. 1983). The reason for this difference is not clear, but it may be that just as SD in low [Cl]_o, ouabain-induced SD is not associated with mitochondrial swelling. Even though they are opposite in sign, both the SD-related LT increase observed by Basarsky et al. (1998) and the LT decrease recorded by us evolved in cell body layers more slowly than in dendritic layers. It is noteworthy that Martins-Ferreira and de Oliveira Castro (1966) already reported the complex sequence of light-scattering changes that accompany SD in retina, labeling the successive four waves a -d. These are quite similar to the light-dark-light-dark sequences seen in Figs. 7A and 10A (see also Somjen 2001). Recently Világi with co-workers (2001) also reported a complex, multiphasic IOS response during SD in rat neocortical slices.

Interaction (superposition) of the two types of IOS

Comparing tracings obtained in low and in normal [Cl]_o illuminates the summation of the two types of IOS. This is best shown in the contrast between Fig. 10, A and B. When Cl was deleted from the bathing medium (Fig. 10B), LT behaved during hypoxia as expected from cell-volume changes. LT started to increase when ISV started to decrease, as expected from cell swelling, then at the onset of HSD suddenly increased even more when ISV underwent its sharp decrease, indicating additional cell swelling. Following HSD, LT undershot its rest level while ISV overshot it, indicating cell shrinkage during "rebound" from previous swelling. In normal [Cl]_o during hypoxia (Fig. 10A), we see the initial increase of LT and its final decrease (undershooting), but the middle part, coinciding with HSD, the LT trace is "turned upside down" compared with the trace in Fig. 10B. The light-scattering decrease caused by HSD-related cell swelling appears to be masked by the more powerful scattering increase caused perhaps by organelle swelling.

The sequence of LT changes in normoxic SD may similarly be interpreted. The initial, brief LT increase (Figs. 7A and 9A) is very probably due to cell swelling, and the late post-SD LT decrease (Fig. 7A) may be due to the cell shrinkage in the wake of the preceding swelling. In the slice illustrated in Fig. 6, the postswelling rebound shrinkage was absent when Cl was deleted from the bath, shown by the absence of over- and undershooting of both, the ISV and LT traces in Fig. 7B. The IOS induced by cell swelling also seems reduced under antecedent hypo-osmotic exposure. This could be because cells then have less remaining extracellular space into which they can swell.

IOS in other tissues and in brain in situ

The IOS associated with synaptically transmitted excitation has earlier been observed in the hippocampus (MacVicar and Hochman 1991) and the neocortex (Holthoff and Witte 1996), with the latter study showing a correlation between extracellular space decrease and increased light transmittance. A similar signal, manifested as a decrease of surface reflectance, has also been reported in neocortex studied in vivo, where it must be separated from the larger optical effect caused by changes in blood flow (Hochman 1997). This synaptic signal is also Cl dependent, and MacVicar and Hochman (1991) reported a complete abolition of the signal in low [Cl]_o conditions. Our findings are similar to those of MacVicar and Hochman (1991) in that there was a marked diminution of the IOS in low-[Cl]_o conditions although the signal could be restored by increased stimulation strength.

Poststimulus rebound of IOS and ISV indicate preceding regulatory volume adjustment

During in vivo studies of primate and human neocortex, the cell-swelling-induced scattering decrease is detected as decreased reflection and is similar to the LT increase described here in most respects in terms of mirroring excitatory synaptic activity. However, in some in vivo studies there is also a second negative signal, which may correspond to synaptic inhibition or to an undershooting during recovery from the preceding cell swelling (Haglund 1997; Hochman 1997). The latter explanation is supported by the presence of such a "rebound" of both IOS and ISV after synaptic excitation in the current experiments. The rebound signals have the same sign (LT decrease, ISV increase) as the ones observed during hypertonic exposure, confirming that they result from cell shrinkage following recovery from the stimulation. This undershoot has been consistently observed for many of the signals but particularly synaptic-induced responses. Although only a slight degree of overt regulatory volume decrease or increase was observed during prolonged hypo- or hypertonic exposure (Fig. 3) (see also Andrew et al. 1997), the postexposure over- and undershoots of ISV indicate that during the preceding osmotic stress, a compensatory transmembrane movement of solutes has taken place that limited the volume changes. This interpretation agrees, among others, with Cserr et al. (1991), Chebabo et al. (1995), and Nicholson and Kume-Kick (1997).

Cell swelling in the absence of extracellular chloride

The ISV changes during SD and HSD in low-[Cl]_o conditions confirm that the water movement and cell swelling persist in the absence of an inward Cl gradient (Bahar et al. 2000; Müller and Somjen 1999). This raises the question as to which anion accompanies the Na⁺ entering cells, contributing to the osmotic water flow. The most likely candidate is HCO (see also Discussion in Müller and Somjen 1999). It should be remembered that a relatively moderate swelling of cells could squeeze the interstitial space into a very small volume. Because the interstitial volume is small compared with the total cell volume, the influx into cells of a small amount of solute can deplete the extracellular fluid. However, because the ISV changes persist with the reduced [Cl]_o levels, there must remain significant fluid shifts into and out of cells particularly because cell excitability remains.

Limitations of TMA+ and imaging observations

It is important to bear in mind that extracellular indicators such as TMA⁺ cannot distinguish volume changes of glial cells from those of neurons. Moreover, the TMA⁺ measurements reflect only a relative measure of ISV fraction, whether using the changes of the baseline or the responses evoked by short iontophoretic pulses of TMA⁺ injection. The TMA⁺ response does, however, accurately reflect the direction and, particularly within an individual experiment, the relative magnitude of the alterations in extracellular space (Buchheim et al. 1999; Dietzel et al. 1980; Holthoff and Witte 1996; Jing et al. 1994; Nicholson 1993). There is a limitation, however, with the Cl substitution because the TMA⁺ electrodes are influenced by the Cl ion. This dependence led to a shift in the baseline voltage of TMA⁺-sensitive electrode without any changes in sensitivity precluding measurements during transition from normal to low-Cl solution and vice versa.

The imaging system used allows 12-bit resolution so that with the linear function of the digital CCD device, the subtractions used for estimation of changes can be calibrated back to alterations in photons (Aitken et al. 1999). Without need for averaging, individual frames can be used for image processing and calculation of numerical data, rather than the multi-frame averaging required for video-based systems. In our previous study, light transmission and reflectance were compared in the same slice in an interface chamber. Changes in reflectance and transmittance were always opposite in sign but precisely correlated in magnitude, demonstrating that the source of the IOSs is change in light scattering (Aitken et al. 1999; Müller and Somjen 1999). In the experiments presented here, we used transmitted light because transmittance appears to be less susceptible to artifact arising from imaging and incident angle compared with reflected light (Aitken et al. 1999).