Effects of Osmotic Stress on Dextran Diffusion in Rat Neocortex Studied With Integrative Optical Imaging

Lian Tao

Department of Physiology and Neuroscience, New York University Medical Center, New York, New York 10016


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Tao, Lian Effects of osmotic stress on dextran diffusion in rat neocortex studied with integrative optical imaging. This study investigated how dextran (Mr = 3,000) diffused in rat cortical slices when the osmolarity of the bathing artificial cerebrospinal fluid was altered by varying the NaCl content. The apparent diffusion coefficient, D*, was measured in the neocortex region using fluorescent molecules and the integrative optical imaging (IOI) method. The main results were: 1) the value of D* in rat neocortex in the isotonic (300 mOsm) artificial cerebrospinal fluid at 34°C was D* = 0.68 ± 0.01 × 10-6 cm2 s-1 (mean ± SE, n = 78) and it could be changed within minutes by varying the extracellular osmolarity. 2) Hypotonic stresses up to -100 mOsm decreased D* by 35% and were fully reversible when the slices were returned to the isotonic medium. Further hypotonic stress to -150 mOsm caused further decrease in D* but after removal of the stress, D* overshot its control value. 3) Hypertonic stress of +50 mOsm increased D*, but the maximum reversible increase in D* was only 15%. Further hypertonic stress (to +200 mOsm) did not cause any further increase in D* and, after removal of the stress, D* undershot the control value. The changes in D* are thought to be related to volume changes of cells in tissue: hypotonic solutions caused cell swelling, resulting in reduced extracellular space and compressed extracellular matrix so that the dextran diffusion was more hindered. Hypertonic solutions had the opposite effect. Recordings of extracellular field potentials in the hippocampal CA1 region demonstrated that, on return to the isotonic solution after exposure to an extreme hypotonic or hypertonic stress, the neurons retained their ability to generate synaptic responses.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES

The diffusion of molecules through the extracellular space (ECS) of the brain plays a critical role in fundamental biological processes such as the movement of metabolic substrates (Fellows et al. 1992) and volume transmission. Volume transmission also is called extrasynaptic or nonsynaptic transmission, in contrast to classical synaptic transmission, and has been proposed to be another mechanism of intercellular communication via substance diffusion in the ECS. (Agnati et al. 1995; Bach-y-Rita 1994; Fuxe and Agnati 1991; Syková 1997). The diffusion of molecules in ECS also plays an important role in clinical applications such as the delivery of drugs (Krewson et al. 1995) and diffusion-weighted magnetic resonance imaging (Latour et al. 1994; Szafer et al. 1995). One of the most important parameters that characterize the diffusion properties of molecules in ECS is the apparent diffusion coefficient (ADC), denoted by D*. The ADC is defined by the diffusion equation partial C*/partial t = D*nabla 2C*, where C* is the local average concentration of the diffusing molecules in brain tissue (Nicholson and Phillips 1981; Nicholson and Syková 1998). It is known that for small ions, such as tetramethylammonium (TMA+, Mr = 74), D* is relatively constant across a spectrum of brain tissues (Nicholson 1993; Nicholson and Syková 1998; Syková 1997). It also is known that in the same brain region, different macromolecules, such as dextrans (Nicholson and Tao 1993), albumins (Tao and Nicholson 1996), and N-(2-hydroxypropyl) methacrylamide polymer (HPMA) (Vargová et al. 1998), may have different values of D*, even when their molecular weights are similar. This indicates that the ADC of a molecule depends not only on its molecular weight but also on other properties, including shape, radius of gyration, electric charge, etc. These molecular attributes, together with the physico-chemical properties of the ECS, determine the interactions between the molecule and the structure of the ECS. Consequently, if the diffusing molecule is kept the same and the ECS properties are varied, we may anticipate that the ECS constraints encountered by the diffusing molecule will change, thereby resulting in changes in the ADC. This raises the following questions. For a given molecule, especially a macromolecule, in a given tissue, can the ADC be changed experimentally? If yes, how quickly or how effectively can such changes be made? What are the limits of the changes? The answers to these questions are not only helpful to understanding the characteristics of the interactions between the molecule and the ECS structure but also useful in many clinical applications.

To explore these issues, the ADC of fluorescent dextran molecules (Mr = 3,000) was measured in rat cortical slices using the integrative optical imaging (IOI) method. The slices were subjected to different osmotic stresses induced by changing the NaCl content of the bathing medium.


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INTRODUCTION
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Brain slices were obtained from Sprague Dawley rats of either sex (~150 g). The rats were anesthetized deeply with pentobarbital sodium (65 mg/kg) and decapitated, and the cerebrum was removed. Coronal slices with a thickness of 400 µm were cut at the interaural 5- to 6-mm planes (Paxinos and Watson 1986). After incubation in artificial cerebrospinal fluid (ACSF, defined later) at room temperature, the slice was transferred to the measurement chamber kept at 34 ± 1°C. The chamber was perfused with ACSF and the slice was submerged 3 mm below the fluid surface. The flow of ACSF was maintained at 1 ml/ min using a peristaltic pump. Diffusion measurements were made in cortical layers III, V, and VI, but no differences were noted in different layers. A total of 26 rats were used with between two and four slices from each rat. Several measurement sequences were made on each slice, and the value of n cited in statistics represents the number of measurement sequences.

The IOI system as well as the related theory and data analysis techniques have been detailed elsewhere (Nicholson and Tao 1993; Tao and Nicholson 1995), so here is only a brief description. The measurement chamber was mounted on the stage of a compound epi-fluorescent microscope. Dextran (Mr = 3,000), tagged with the fluorescent dye Texas Red, (D-3329, Molecular Probes, OR) was dissolved in 154 mM NaCl saline to form a 1-mM solution. The fluorescent molecules were ejected into the brain slice with a brief pressure-pulse from a micropipette, which was inserted 200 µm below the slice surface. The diffusing molecules were imaged with a ×10 water-immersion objective. Before each dextran ejection, an image was taken and saved as the background image. After each ejection, a sequence of 10 images was recorded at an interval of 10 s using a cooled CCD camera and transferred directly to a personal computer, where the background image was subtracted from the diffusion images. Then intensity profile was taken through the ejection point in each image and fitted with the theoretical expression for a diffusion image using the nonlinear Levenberg-Marquardt algorithm to extract the apparent diffusion coefficient D* (Nicholson and Tao 1993).

The normal isotonic ACSF had the following composition (in mM): 115 NaCl, 5 KCl, 35 NaHCO3, 1.25 NaH2PO4, 1.3 MgCl2, 1.5 CaCl2, and 10 D-glucose, resulting in an osmolarity of 300 mOsm. The osmolarities of all the ACSFs were determined with a freezing point depression osmometer (Osmette, Precision Systems, MA), and the errors in the osmolarities were within ±5 mOsm. The ACSFs were gassed continuously with 95% O2-5% CO2 to maintain a pH of 7.5. Three hypotonic ACSF solutions were used representing decreases of -50, -100, and -150 mOsm from the isotonic ACSF. They were obtained by reducing the NaCl content of the isotonic solution. Three hypertonic ACSF were used representing increases of +50, +100, and +200 mOsm from the isotonic ACSF, and they were obtained by increasing NaCl content.

The measurement chamber initially was perfused with the isotonic ACSF, then a brain slice was transferred into the chamber and allowed to equilibrate with the bath for ~10 min. Two or three sequences of diffusion images were recorded to obtain the control value of D*. To study the effect of an osmotic stress, the isotonic ACSF was replaced with either a hypotonic or a hypertonic solution. Test experiments showed that the ADC began to change in ~5 min after a change of solution and reached a steady value within 10 min. Therefore the recording of diffusion images under the osmotic stress was started after 10 min and several sequences of images were taken. Then the isotonic ACSF was switched back, and 10 min later more image sequences were recorded to study the recovery behavior. As an example, Fig. 1 shows the diffusion images of the fluorescent dextran in a cortical slice recorded with the IOI method. In the figure, each of the three rows shows an image sequence taken after an ejection. The three sequences were taken from the same slice but in different solutions. The middle row was the image sequence in the normal isotonic solution (300 mOsm). The top row was the sequence in a mild hypotonic solution (-50 mOsm), whereas the bottom row was the sequence in a mild hypertonic solution (+50 mOsm).



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Fig. 1. Diffusion images of fluorescent dextran (Mr = 3000) in neocortex recorded with IOI method. At time 0 (0 s), approximately a nanoliter of the dextran labeled with the fluorescent dye Texas Red were briefly pressure ejected from a micropipette and a sequence of images were recorded. Each row in the figure shows a sequence of images taken after an ejection with the dye concentration depicted in pseudo color (red highest concentration, blue lowest). A background image taken before each ejection has been subtracted from the subsequent images. Appropriate analysis of the intensity curves of the images enables apparent diffusion coefficient (ADC) to be obtained (Nicholson and Tao 1993). The three sequences were taken from the same slice but in different solutions. Middle: sequence in the normal isotonic solution (300 mOsm). Top: sequence in a mild hypotonic solution (-50 mOsm). Note that the intensity changed more slowly than that in isotonic solution. Bottom: sequence in a mild hypertonic solution (+50 mOsm); the intensity changed slightly more rapidly than that in the isotonic solution. Vertical dimension of each image is 564 µm.

The viability of the slices was assessed by recording extracellular field potentials from slices in the same chamber, under superfusion conditions identical to those for the imaging experiments. The recordings were performed in the hippocampal CA1 region instead of neocortical region because the pathway formation is well defined in the hippocampus and allows a reliable recording of monosynaptic transmission and monitoring the tissue excitability. The field potentials were evoked by stimulating the Schaffer collaterals with brief current pulses (25 mA, 30 µs) from a bipolar twisted wire electrode and recorded from the pyramidal layer with a glass micropipette filled with 2 M NaCl. As an example, Fig. 2A shows field potentials recorded for a hypotonic stress (-100 mOsm), whereas B shows the potentials for a hypertonic stress (+200 mOsm). The top row in the figure shows the initial control recordings, which have a single population spike typical for a healthy unchallenged slice (Andrew and Macvicar 1994; Ballyk et al. 1991; Chebabo et al. 1995a; Saly and Andrew 1993). When the hypotonic stress was applied, multiple population spikes appeared with the amplitude several times greater than the initial control signal (Fig. 2A, middle). This phenomenon has been reported previously (Andrew and Macvicar 1994; Ballyk et al. 1991; Chebabo et al. 1995a; Saly and Andrew 1993). Direct intracellular recordings from the CA1 and CA3 neurons in rat hippocampal slices have shown that the evoked excitatory postsynaptic potential (EPSP) was not affected by hypotonic stress and, therefore, the greater amplitude of population spike has been attributed to enhanced synchronization of neuron discharge due to reduction of ECS. (Ballyk et al. 1991; Saly and Andrew 1993). As argued later in the present paper, the size of ECS was reduced markedly under an extreme hypotonic stress. After removal of the hypotonic stress, the single population spike was restored but with a smaller amplitude (Fig. 2A, bottom), indicating that the neurons had become less excitable. In contrast, the hypertonic stress eliminated the population spike completely (Fig. 2B, middle); however, after removal of the stress, multiple population spikes appeared with greater amplitudes (Fig. 2B, bottom), indicating the neurons had become hyperexcitable. Each trace in the figure is an average of four field recordings taken 30 min after a change of solution, which was comparable with the length of time for the image experiments. From the field potential recording, it was evident that after exposure to an extreme hypotonic or hypertonic stress, the neurons did not come back their initial condition completely. However, synaptic transmission clearly was restored.



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Fig. 2. Field potentials recorded in hippocampal CA1 region to show the slice viability. Field potentials were evoked by stimulating the Schaffer collaterals and recorded from the pyramidal layer. A: fields recorded for a hypotonic stress (-100 mOsm). B: fields for a hypertonic stress (+200 mOsm). Top: initial control recordings, which have a single population spike typical for a healthy unchallenged slice. When the hypotonic stress was applied, multiple population spikes appeared with the amplitude several times greater than the initial control signal (A, middle). After removal of the hypotonic stress, the single population spike was restored but with a smaller amplitude (column A, bottom). In contrast, the hypertonic stress eliminated the population spike completely (column B, middle) and after removal of the stress multiple population spikes appeared with greater amplitudes (column B, bottom). Each trace in the figure is an average of 4 field recordings taken 30 min after a change of solution. Potentials were recorded with positive potentials up. From the field potential recording, it was evident that after exposure to an extreme hypotonic or hypertonic stress, the neurons cannot restore to their initial condition completely. However, the neuron population always restored its synaptic responses.


    RESULTS
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INTRODUCTION
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Normal isotonic condition

The apparent diffusion coefficient D* of the dextran in the neocortex under the normal isotonic condition served as the control value for the experiment. Measurements were made in each cortical slice under isotonic ACSF flow. These data are shown on each quantile plot in Figs. 3 and 4. A quantile plot shows the complete distribution of a set of data. Each point in the plot represents a measured D*. The ordinate of the point is just the value of the D* while the abscissa represents the fraction of data that have values lower than this value (Chambers et al. 1983). The mean value of the ADC for all the experiments was D* = 0.68 ± 0.01 × 10-6 cm2 s-1 (n = 78) at 34°C. The values for D* here and elsewhere in RESULTS are quoted as means ± SE.



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Fig. 3. Quantile plots of ADC in hypotonic solutions. Quantile plot shows the complete distribution of a set of data. Each point in the plot represents a measured D*. Ordinate of the point is just the value of the D*, whereas the abscissa represents the fraction of data that have values lower than this value (Chambers et al. 1983). In each panel, the control data (open circle ) in normal isotonic solution (300 mOsm) is repeated as a reference. In A, the effect on D* of reducing the NaCl content of the ACSF to obtain a solution 50 mOsm lower than control is shown (black-down-triangle ). It is seen that the value of D* was reduced. On return to normal ACSF (), D* returned to the control values. B: effect of a reduction in osmolarity by 100 mOsm; the reduction in D* was greater than shown in A, but the slice was still able to recover to control values of D*. C: effect of a reduction of 150 mOsm is shown. Value of D* was further reduced, compared with B, but on return to control solution, the value of D* overshot the original value.



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Fig. 4. Quantile plots of ADC in hypertonic solutions. In each figure, the control data (open circle ) in normal isotonic solution (300 mOsm) is repeated as a reference. A: effect on D* of increasing the NaCl content of the ACSF to obtain a solution 50 mOsm higher than control is shown (black-triangle). It is seen that the value of D* was increased. On return to normal ACSF (), D* returned to control values. B: effect of an increase in osmolarity by 100 mOsm; the increase in D* was slightly greater than shown in A but the slice undershot the control values of D* on return to normal ACSF. C: effect of an increase of 200 mOsm is shown. Value of D* did not increase further, compared with B, but on return to control solution, the value of D* again undershot the original value.

Hypotonic conditions

To study the effect of hypotonic stress, the isotonic ACSF was replaced with a hypotonic solution (Fig. 3). The value of D* began to decrease after ~5 min and reached a steady value in ~10 min. For some slices, the measurement of D* in the hypotonic solution was repeated during a period of 90 min, but no further changes were observed after the first 10 min. Consequently, the data presented here were acquired during the period of 10-30 min after initiating the flow of hypotonic ACSF.

At the conclusion of the hypotonic stress, the isotonic solution was restored (Fig. 3). The value of D* began to recover in ~5 min and reached a steady value in ~10 min. Again, prolonged measurements showed that no significant changes occurred after the first 10 min after the restitution of the normal solution, and so the recovery data presented here were acquired during the first 10-30 min after the isotonic solution was restored.

Figure 3A shows the quantile plot of the data acquired under the mild hypotonic stress of -50 mOsm. The mean value of D* was 0.53 ± 0.02 × 10-6 cm2 s-1 (n = 23), representing a 22% decrease from the normal D* value. Figure 3A also shows the data acquired after the slices had recovered from the hypotonic stress; under these conditions, D* = 0.69 ± 0.02 × 10-6 cm2 s-1 (n = 17), which was very close to the normal value, showing that the changes in D* caused by this hypotonic stress were reversible.

When the hypotonic stress was increased, by reducing osmolarity by -100 mOsm, D* decreased further (Fig. 3B). Under these conditions, D* = 0.44 ± 0.02 × 10-6 cm2 s-1 (n = 34), representing a 35% decrease from the control D* value. After recovery from the hypotonic stress (Fig. 3B), D* = 0.67 ± 0.02 × 10-6 cm2 s-1 (n = 28), which was still close to the control value, indicating that the changes in D* caused by the stronger hypotonic stress were still reversible.

As more hypotonic stress was applied, D* decreased further. Figure 3C shows the data acquired under the most extreme hypotonic stress that was used, -150 mOsm. In this case, D* = 0.38 ± 0.02 × 10-6 cm2 s-1 (n = 18), which represented a 45% decrease from control. However, after such a strong hypotonic stress was removed, D* no longer returned to the initial value (Fig. 3C) but instead exhibited an overshoot so that a value of D* = 0.78 ± 0.03 × 10-6 cm2 s-1 (n = 17) was measured. For some slices, D* repeatedly was measured for 2 h, but the overshoot was unchanged.

Hypertonic conditions

The time courses of the changes under hypertonic conditions were similar to those under the hypotonic ones so the data were acquired during the period of 10-30 min after initiating the hypertonic ACSF flow.

Figure 4A shows the quantile plot of the data acquired under the weak hypertonic stress of +50 mOsm. The mean value of the ADC was D* = 0.78 ± 0.02 × 10-6 cm2 s-1 (n = 19), representing a 15% increase over control. After recovery from the hypertonic stress, D* = 0.69 ± 0.03 × 10-6 cm2 s-1 (n = 13), which was very close to the initial value, indicating that the change in D* was reversible.

As more hypertonic stress was applied, D* did not increase further. Figure 4, B and C, shows the data acquired under hypertonic stresses of +100 and +200 mOsm, respectively. The values of D* were 0.77 ± 0.03 × 10-6 cm2 s-1 (n = 9) and 0.78 ± 0.02 × 10-6 cm2 s-1 (n = 27), respectively. They both represented about a 15% increase in D* compared with control, indicating that the increase in D* caused by hypertonic stress has an upper limit, at least over the range tested here. However, after the stronger hypertonic stresses were removed, D* no longer returned to the control value. Figure 4, B and C, shows the data acquired after the isotonic ACSF flow was restored, revealing an undershoot in D* in each case. The values of the undershoots were D* = 0.62 ± 0.02 × 10-6 cm2 s-1 (n = 13) and D* = 0.58 ± 0.02 × 10-6 cm2 s-1 (n = 21), respectively. These values were significantly lower than the control value. For some slices, D* was repeatedly measured for 2 h, but the undershoot was sustained.

The data obtained under hypotonic stresses (Fig. 3) and under hypertonic stresses (Fig. 4) are summarized in Fig. 5. Note in the figure that while the ADC could decrease dramatically with osmolarity, it could only increase a very limited amount from its control value no matter how much hypertonic stress was applied. Also note that the maximum reversible change under hypotonic stress was also much greater than that under hypertonic stress.



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Fig. 5. Changes in ADC with osmolarity. Results shown in Figs. 3 and 4 are summarized. , effects of the osmotic stress; open circle , recovery values (D* after return to control ACSF). Error bars are SE, and exact values are given in text. Note that while the ADC could decrease dramatically with osmolarity, it could only increase a very limited amount from its control value no matter how much hypertonic stress was applied. Also note that the maximum reversible change under hypotonic stress was also much greater than that under hypertonic stress.

The goal of this study was to investigate how a moderately large molecule diffused in brain tissue when the ECS osmolarity was changed. The main results were: 1) the ADC, D*, of dextran (Mr = 3,000) in brain tissue could be changed within minutes by varying the extracellular osmolarity. 2) Hypotonic stress, brought about by reduced NaCl content, decreased D*. Decreases of 35% in D* (-100 mOsm) were fully reversible when the tissue was returned to control medium. Further hypotonic stress caused further decrease in D*, but after removal of the stress, D* overshot its control value. 3) Hypertonic stress, induced by excess NaCl content, increased D*. The maximum reversible increase in D* was 15%, (+50 mOsm). Further hypertonic stress did not cause any further increase in D*, and after removal of the stress D* undershot the control value.


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To understand the experimental results, I suggest the following explanations. Many studies have demonstrated that varying the extracellular osmolarity can alter the volume of cells in tissue. One recent study (Chebabo et al. 1995b), using ion-selective microelectrodes to measure extracellular concentration change of a probe ion TMA+ or tetraethylammonium (TEA+), estimated that when hippocampal tissue slices were exposed to a severe hypotonic stress (-180 mOsm), the extracellular volume was reduced to 25% of its control value, corresponding to a mean cell volume increase of >= 11%. Another recent study (Krizaj et al. 1996), using ion-selective microelectrodes to measure TMA+ diffusion in the isolated turtle cerebellum combined with wet- and dry-weight measurements of tissue, showed that the volume fraction of the ECS (defined as the ratio of the volume of the ECS to volume of whole tissue in a small representative region) changes when extracellular osmolarity is varied by altering the NaCl content of the ACSF. That study further demonstrated that such changes in ECS volume fraction primarily are brought about by water moving between the extra- and intracellular compartments. Tosteson and Hoffman (Tosteson 1964; Tosteson and Hoffman 1960) proposed a theoretical model for regulation of cell volume when external NaCl is varied. The model was based on the interaction of electrical and osmotic effects. According to this model, when the rate of the Na-K pump is fixed, the cell volume is inversely proportional to the total extracellular ion concentration. Moreover, when the extracellular ion ratio [Na+]o/[K+]o is reduced, cell volume increases (Hoppensteadt and Peskin 1992). Therefore it is established, experimentally and theoretically, that a hypotonic stress achieved by reduced [NaCl]o causes cell swelling, whereas a hypertonic stress achieved by excess [NaCl]o causes cell shrinking.

It is known that, under the normal osmotic condition, the ECS occupies ~20% of the total volume of brain tissue (Nicholson 1993; Nicholson and Phillips 1981; Nicholson and Syková 1998). The ECS harbors a matrix consisting of long-chain glycoproteins and glycosaminoglycans, including hyaluronate (Bignami et al. 1993; Margolis and Margolis 1993; Ruoslahti 1996), and there is renewed interest in a particular form of the matrix called the perineuronal net (Celio and Blumcke 1994). The matrix may be sufficiently dense to form an obstructive polymer, which would restrict the diffusion of molecules (Ogston and Sherman 1961). In the present experiments, the ECS is the space within which the dextran diffuses because the cell membranes are impermeable to the dextran over the periods of typical measurements. When cells swell, the ECS will become narrower and therefore may hinder the passage of molecules. More importantly, the swelling cells could compress the matrix, making it denser and more obstructive to diffusing molecules, resulting in a lower ADC. This is true especially for larger molecules the dimensions of which may approach the average path width within the matrix. The converse of these arguments would apply in hypertonic medium, as the cells shrank. Therefore the change in the ADC of macromolecules in some way reflects the volume change of cells in tissue. Obviously the quantitative relation between the changes in ADC and cell volume is complicated, and it may depend heavily on the type of tissue and the type of macromolecule (Nicholson et al. 1998).

The asymmetric behavior of the ADC under hypotonic and hypertonic conditions is worth noting. Although the ADC could decrease dramatically with osmolarity, it can only increase a very limited amount. Based on the argument that the change in ADC reflects the change in cell volume, this result may lead to the hypothesis that the decrease in cell volume caused by hypertonic stress is very limited. This interesting and important point has not been predicted by Tosteson and Hoffman's theoretical model. Therefore an extension to their model is needed. The extension might involve another regulation mechanism, which is activated when the cell volume decreases below a certain critical level. The regulation mechanism would prevent any further decrease in cell volume so that cell functions could be maintained. A possible candidate for the regulation mechanism is the uptake channels through which KCl can be taken up rapidly (Cserr et al. 1987a,b; Gullans and Verbalis 1993; Law 1994). As KCl accumulates, the intracellular osmolarity increases and the water efflux is stopped, preventing further decreases in cell volume. Besides the possible regulation mechanism involving ion redistribution across the membrane, physical constraints imposed by the cytoskeleton and the extracellular matrix also may limit the shrinkage at a certain point.

It has been known that cultured brain cells respond to hypotonic stress by initial swelling, which is followed by the regulatory volume decrease (RVD) within minutes. Similarly, their initial shrinkage responding to hypertonic stress is followed, within minutes, by the regulatory volume increase (RVI) (Ballanyi and Grafe 1988; Hoffmann 1987). However, the question of whether RVD or RVI also occurs in brain slices is still not entirely resolved. Although one recent study showed some data supporting RVI in rat hippocampal slices (Chebabo et al. 1995b), another more detailed study provided evidence against the regulatory volume changes in rat hippocampal slices (Andrew et al. 1997). Because of the nature of the IOI technique, it is not possible to obtain a continuous time course of the change in ADC. However, as mentioned before, for some slices the ADC measurement was repeated during a period of 90 min. It has been found that all the decreases and increases of ADC occurred within the first 10 min after a stress had been applied and the changes were monotonic. If the decreases and increases of ADC indeed reflects the changes in cell volume, the monotonic decreases and increases of ADC means the RVD or RVI has not been seen in this study.

Although the present experiment only demonstrated that the ADC is a function of ECS osmolarity, from the above explanation, it is very likely that ADC also depends on several physiological conditions that affect cell volume. This explanation leads to the possibility that measuring the ADC of macromolecules could be a useful method to study the changes in volume of cells in tissue. This is pertinent to clinical issues that arise when the ECS is reduced in size, during ischemia, anoxia, or edema for example (Syková 1997). From the preceding explanation, it is also apparent that under hypotonic conditions, large molecules, especially those that have the dimensions comparable to the average path width of the extracellular matrix, are going to be more hindered than small ones. This may have consequences for therapeutic intervention, not only in stroke but also, for example, in the delivery of nerve growth factor and similar molecules to treat Alzheimer's disease (Krewson et al. 1995).


    ACKNOWLEDGMENTS

I am grateful to Dr. C. Nicholson for very helpful discussion and Dr. S. Hrabetova for assistance with the field potential recording.

This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-28642 to C. Nicholson.


    FOOTNOTES

Address for reprint requests: Dept. of Physiology and Neuroscience, New York University Medical Center, 550 First Ave., New York, NY 10016.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 13 August 1998; accepted in final form 20 January 1999.


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DISCUSSION
REFERENCES

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