INDEX
Key words
Chloride, Development, Na+, K+-2Cl- cotransporter, K+-Cl- cotransporter, GABA
Title
Immature Stage of Intracellular Cl- Homeostasis Regulated by Cation-Cl- cotransporters in Rat Neocortical Neurons
Authors
Yasumasa Yamada, Atsuo Fukuda, Masaki Tanaka, Yasunobu Shimano, Hitoo Nishino , Kanji Muramatsu, Hajime Togari and Yoshiro Wada
Institution
Address of Correspondence:Dr. Yasumasa Yamada, Department of Pediatrics Nagoya City University Medical School Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan Phone: +81-52-853-8246 Fax: +81-52-842-3449 e-mail y-yamada@med.nagoya-cu.ac.jp
Source
Nagoya Medical Journal 43:179-190 (2000)

SUMMARY
In brain slices from rat somatosensory cortex, neuronal intracellular Cl- concentration ([Cl-]i) recordings were made by an optical technique measuring 6-methoxy-N-ethlquinolinium iodide (MEQ) fluorescence. When the resting [Cl-]i was estimated by calibration curve according to Stern-Volmer relationship, the [Cl-]i from postnatal day (P) 10-14 slices was significantly higher than that from P17-19 slices. Then, P10-14 slices were pretreated with inhibitors of cation-Cl- cotransporters to clarify the type that is responsible for the high [Cl-]i status. Furosemide did not alter this higher level of [Cl-]i, whereas bumetanide significantly decreased it. Application of -aminobutyric acid (GABA) induced Cl- efflux in most cortical neurons during P10-14. These results suggest that inwardly directed cation-Cl- cotransporter, possibly bumetanide-sensitive Na+, K+-2Cl- cotransporter, contributes to this intracellular Cl- accumulation. Such an immature stage of Cl- homeostasis would result in the reversal of the action of GABA in immature neurons as compared with mature ones.
INTRODUCTION
GABA is one of the principal inhibitory neurotransmitters in the brain. However, GABA also appears to be excitatory in immature neurons (1, 2, 3, 4), in which the reversal potential for GABA response may be positive with respect to the resting membrane potential (5, 6). It has been speculated that the negative shift in the equilibrium potential for chloride (ECl) during development, as estimated from the reversal potential for GABA response, is a consequence of alterations in the function of the outwardly and/or inwardly directed cation-Cl- cotransporters and/or Cl- pump (7). However, it is still in controversial about factors regulating the [Cl-]i homeostasis that may determine the action of GABA in immature neurons. Rivera et al. (8) reported that, in immature hippocampal neurons, the ECl was more positive than the resting potential because of an insufficient expression of the outwardly directed K+-Cl- cotransporter, KCC2, thus indicating that GABA may cause Cl- efflux. On the other hand, Plotkin et al. (9) reported that the temporal pattern of expression of the Na+, K+-2Cl- cotransporter in the postnatal rat brain supports the hypothesis that the higher expression of the inwardly directed cotransporter is responsible for intracellular Cl- accumulation in immature neurons. It has been suggested that the cerebral cortex of P12-13 rat is comparable to that of the full-term newborn human infant in regard to the degree of maturation (10). Therefore, when the rat is used for studying certain processes assumed to occur in the human cerebral cortex around full-term birth, it should be necessary to clarify the [Cl-]i homeostasis of cortical neurons in this developmental age for the reasons above. We have established a simultaneous recording method of changes in [Cl-]i in individual neurons of neocortical slices, by means of optical fluorescence measurements using MEQ (11). In this report, using optical imaging of [Cl-]i, we have studied [Cl-]i homeostasis in the rat age, which is considered to be similar to the human perinatal period, by comparing the resting [Cl-]i in normal neocortical neurons with those pretreated with inhibitors of cation-Cl- cotransporters (furosemide and bumetanide). Our findings suggest a possibility that intracellular Cl- accumulation, resulting in the depolarizing action of GABA in this developmental age, is partly mediated by Na+, K+-2Cl- cotransporter.
MATERIAL AND METHOD
The techniques used for preparing and maintaining neocortical slices in vitro and for obtaining optical recordings were similar to those described previously (11). Wistar rats (SLC Japan) aged 1-3 weeks were used for this study. Rats were anesthetized deeply with pentobarbital (50 mg/kg ip) and then decapitated. The protocols used conformed with guidelines on the conduct of animal experiments issued by Nagoya City University; all efforts were made to minimize animal suffering and to reduce the number of animals used. A block of the brain including the neocortex was quickly removed and placed in cold (4), 95% O2-5% CO2-saturated modified artificial cerebrospinal fluid (ACSF) containing the following: 230 mM sucrose, 2.5 mM KCl, 1.25 mM NaH2PO4, 10.0 mM MgSO4, 0.5 mM CaCl2, 26 mM NaHCO3, and 30 mM glucose. Coronal slices of somatosensory cortex with a thickness of 200m were cut in the modified ACSF using a vibratome (DTK-1500; Dosaka). Slices were allowed to recover for 60 min in standard ACSF (126 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 2.0 mM MgSO4, 2.0 mM CaCl2, 26 mM NaHCO3, and 20 mM glucose) within a tightly sealed box filled with 95% O2-5% CO2 at a pressure of 50 kPa at room temperature. The techniques used for the production of diH-MEQ, and for the loading and optical measurement of MEQ were also similar to those described previously (11). Briefly, prior to bath loading of the slices, MEQ (5 mg/100l) was reduced to a cell-permeable form, diH-MEQ (12), by addition of 15l of 12% NaBH4 solution while bubbling with N2 for 30 min. DiH-MEQ was extracted from the reaction mixture as a yellow organic layer, a portion of which was added to ACSF to yield a final concentration of 740M. Neurons were loaded with MEQ by incubating slices with diH-MEQ in oxygenated standard ACSF for 90 min in the box described above. Slices were laid on the glass bottom of a submerged-type chamber which was placed on a microscope stage and continuously perfused at a rate of 3-4 ml/min with standard ACSF gassed with 95% O2-5% CO2. The bathing solution, which was maintained at a temperature of 30 (DTC-200B; Dia Medical), had a pH of 7.4 when saturated with 95% O2-5% CO2. MEQ fluorescence was excited at 330 (30) nm and emitted at 450 (33) nm by means of a xenon arc lamp (75W) and a band pass filter set (XF 03; Omega). Fluorescence images were obtained every 30 sec via an intensified charge coupled device (ICCD) camera (C2400; Hamamatsu Photonics) fitted to an upright microscope (Axioskop FS; Zeiss) equipped with a water immersion lens (Achroplan WPH1; Zeiss). Images were digitized on-line, 128 frames being averaged to improve the signal to noise ratio. The MEQ fluorescence was monitored in the center of the soma of pyramidal neurons and pooled for analysis. Data were analysed off-line using an IBM-PC compatible computer with an image processor and data analysis software (Argus-50 CA; Hamamatsu Photonics). Estimation of [Cl-]i in individual neurons in slices was made possible by calibrating intracellular MEQ fluorescence signals at 0 mM [Cl-]o (extracellular Cl- was substituted by equimolar gluconate) in the presence of tributyltin, a Cl--OH- antiporter, and nigericin, a K+-H+ antiporter, and at 150 mM KSCN. In other word, the resting [Cl-]i was calculated by fitting the ratio of fluorescence in the absence of Cl- (F0:FCl=0-FSCN) divided by that at the resting [Cl-]i (F:FCl=rest-FSCN) to the calibration curve we reported previously (11). The following drugs were used: MEQ from Molecular Probe; GABA, furosemide and bumetanide from Sigma; picrotoxin from Wako Chemical. Stock solutions of bumetanide and picrotoxin were made up in dimethylsulfoxide, furosemide in dimethylformamide, and GABA in distilled water. All drugs were added to the ACSF to yield the required final concentration by 1000X dilution. GABA and picrotoxin were applied by bath perfusion. Because we had confirmed some absorption of UV light by furosemide and bumetanide using spectrophotometer (DU-640, Beckman) (11), slices were pretreated with these drugs for 30 min immediately before initiating optical recordings.
RESULT
The fluorescence of MEQ is quenched collisionally by Cl-, so that decrease in intracellular Cl- level are reflected by corresponding increase in fluorescence (FIG. 1). Estimation of [Cl-]i by clamping [Cl-] across the plasma membrane was performed using the method as previously reported (11). To estimate resting [Cl-]i, the perfusion medium was changed from standard ACSF to calibration solution containing 0 mM Cl- and 128.5 mM K+, together with tributyltin (20M) and nigericin (14M) (FIG. 1A1, 1B). In the presence of these reagents for > 20 min, [Cl-] can be assumed to equilibrate across the plasma membrane of neurons in the slice (FIG. 1A2, 1B). At the end of the procedure, total quenchable intracellular MEQ fluorescence was measured by the addition of 150 mM KSCN, as quenching of the MEQ signal is much more efficient with SCN- than with Cl- (FIG. 1A3, 1B). Since some absorption of light (250-500 nm) by solution containing tributyltin, nigericin and gluconate was noted, the initial drop in fluorescence intensity at the beginning of perfusion by the tributyltin/nigericin solution might be due to absorption of both excitation and emission light of MEQ (FIG. 1B). For this reason, we compensated the reduced fluorescence value when carrying out the calibration, by regarding [Cl-]i at the very beginning of perfusion with 0 mM Cl- solution as resting [Cl-]i (see A1 in FIG. 1B). PLACE FIG. 1 ABOUT HERE Because an insufficient Cl- gradient has been suggested as a mechanism that may underlie the characteristic action of GABA (1, 4, 5) in immature neurons, we attempted to compare the resting [Cl-]i of cortical neurons in the developmental age attributable to human perinatal period with that in more mature age. The resting [Cl-]i in cortical neurons in P10-14 was 24.90.7 mM (n=200, 14 slices), which was significantly higher than that in more mature (P17-19) neurons (6.74.2 mM, n=60, 8 slices, Mann-Whitney U test, p<0.0001, FIG. 2). PLACE FIG. 2 ABOUT HERE To gain some insight into the possible mechanism underlying this relatively high [Cl-]i in P10-14, the [Cl-]i in neurons pretreated with a cation-Cl- cotransporter inhibitor, furosemide (1.5 mM), was compared with that in non-treated neurons. The resting [Cl-]i in slices pretreated with furosemide (27.92.3 mM, n=90, 3 slices) was not significantly different from that in non-treated slices in this developmental period (FIG. 3, Mann-Whitney U test). PLACE FIG. 3ABOUT HERE Next, we tested the effect of another inhibitor of cation-Cl- cotransporter, bumetanide (100M), on the resting [Cl-]i. The resting [Cl-]i in slices pretreated with bumetanide was 15.70.8 mM (n=98, 5 slices), which was significantly lower than the control resting [Cl-]i (FIG. 4, Mann-Whitney U test, p<0.0001). Thus, bumetanide-sensitive cation-Cl- cotransporter might be accumulating Cl- in this age. PLACE FIG. 4 ABOUT HERE Application of GABA (200M) elicited a decrease in [Cl-]i in most neurons (42/46, 4 slices) in slices from P10-14 rats (FIG. 5). The results suggest that GABA induces a Cl- efflux by the opening of GABAA channels in most immature neurons in contrast to an influx occurring usually in adult neurons. Interestingly, application of a GABAA receptor antagonist, picrotoxin, induced [Cl-]i increase (FIG. 5), indicating that perpetual extracellular GABA might contribute to balancing the [Cl-]i homeostasis in this age (3, 6). PLACE FIG. 5 ABOUT HERE
DISCUSSION
The results of present study indicate that GABA induces Cl- efflux in cortical neurons in P10-14 rat brain, the age which would attributable to human perinatal period (10), and that the efflux is derived from an intracellular Cl- accumulation mediated by a bumetanide-sensitive cation-Cl- cotransporter. The Na+,K+-2Cl- cotransporter was inhibited by both bumetanide and furosemide, whereas the K+-Cl- cotransporter was preferentially inhibited by furosemide under the same experimental condition as this study (13, also see reviews 7, 14). Thus, it seems possible that a bumetanide-sensitive cation-Cl- cotransporter in question is most likely the Na+,K+-2Cl- cotransporter. The expression and/or function of inwardly directed Na+,K+-2Cl- cotransporter in this developmental stage might surpass that in adult, resulting in intracellular Cl- accumulation. Alternatively, outwardly directed Cl- transporter such as K+-Cl- cotransporter and Cl- pump both of which are insensitive to bumetanide might be insufficient yet at this age. In any case, the resting [Cl-]i in slices pretreated with bumetanide was still higher than that in P17-19 slices, suggesting that factors other than Na+,K+-2Cl- cotransporter are also involved in the mechanism for high [Cl-]i in this developmental stage. If, as Rivera reported (8), the insufficiency of the neuronal Cl--extruding K+-Cl- cotransporter (KCC2) during immaturity is the main reason for the intracellular Cl- accumulation, furosemide which could inhibit Na+,K+-2Cl- cotransporter as well as K+-Cl- cotransporter should have induced a decrease in resting [Cl-]i like bumetanide. However, in our results, furosemide did not decrease the resting [Cl-]i. These results suggest that the expression and function of K+-Cl- cotransporter is not quite insufficient as compared with that of Na+,K+-2Cl- cotransporter in the age we studied here. Thus, in the case of furosemide pretreatment, the [Cl-]i decrease induced by the inhibition of inwardly directed Na+,K+-2Cl- cotransporter might have been compensated by the inhibition of outwardly directed K+-Cl- cotransporter occurring simultaneously. Accumulating evidence suggests that expression of NKCC1 (or BSC2), an isoform of the Na+,K+-2Cl- cotransporter, is developmentally regulated in the postnatal rat brain. Sun and Murali (15), using primary culture of cortical neurons from cerebral cortex of rat fetus, reported that inwardly directed Na+,K+-2Cl- cotransporter is essential in regulation of intracellular Cl- activity in immature neurons. Furthermore, Plotkin et al. (9) reported that NKCC1 mRNA and protein expression in the cerebral cortex was highest in the first week of postnatal life and then diminished from P14 to adult. Thus, in the material used in this study, NKCC1 expression could be highest. On the other hand, Clayton et al. (16) reported that the NKCC1 expression level in the neocortex peaks by the third postnatal week and is maintained into adulthood. Any case, the reported NKCC1 expression in the developmental stage studied here (P10-14) is compatible to the present results. Our preliminary results using reverse transcriptase-polymerase chain reaction has also shown high expression of NKCC1 during P10-14 (13). As expected, application of GABA to P10-14 neurons with high resting [Cl-]i induced a decrease in [Cl-]i. Using optical imaging, Fukuda et al. (11) and Inglefield and Schwartz-Bloom (17) demonstrated that GABA induced an increase in [Cl-]i in more mature neurons (P25 and P12-21, respectively). In the present study, [Cl-]i decreased to about a quarter of P10-14 level at P17-19, indicating that the status of [Cl-]i homeostasis changes drastically after the second postnatal week. Generally, it has been suggested that massive Cl- entry may play a role in neurotoxic injury such as that caused by excitotoxicity and ischemia, and a reduction in Cl- entry may be able to produce a protective effect (18, 19, 20). Jiang et al. (21) reported that the anoxia-induced increase in [Cl-]i was significantly less in neonatal (P2-10) than in adult hypoglossal neurons, thus suggesting the anoxic tolerance in the neonatal brain. However, it is unknown how the immature type high-[Cl]i- homeostasis accounts for it. Alternatively, high-[Cl]i- itself may worsen hypoxic injury . In this concern, it is interesting to investigate if the immature stage of [Cl-]i homeostasis described in this report accounts for characteristics of the hypoxic-ischemic insult in the immature brain. Because acute hypoxic-ischemic cerebral injury in the perinatal infant is a major cause of long-term neurological and neuropsycholoical handicaps in childhood. In conclusion, we have demonstrated that the resting [Cl-]i of somatosensory cortical neurons from P10-14 rats was significantly higher than that from more mature rats. The Na+, K+-2Cl- cotransporter is likely to contribute to this intracellular Cl- accumulation, resulting in the Cl- efflux and probably depolarization by GABA in this age. Since the developmental stage of the material used in this study is similar to the human perinatal period in terms of cortical development (10), the immature stage of [Cl-]i homeostasis should play specific roles in pathogenesis of neural diseases occurring in perinatal infant.
ACKNOWLEDGEMENT
This work was supported by Grants-in-Aid for Scientific Research #11170220 (A.F.) and #09558104 (H.N.) from the Ministry of Education, Science, Sports and Culture, and a grant from the Ministry of Health and Welfare (A.F.), NOVARTIS Foundation (Japan) for the Promotion of Science (A.F.), and CREST of JST (H.N.).
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FIGURE LEGENDS

Nagoya City University
Medical School