| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Section on Developmental Neurobiology, Laboratory of Neural Control, National Institute of Neurological Diseases and Stroke, National Institutes of Health, Bethesda, Maryland
Submitted 15 February 2005; accepted in final form 24 September 2005
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
|---|
, 2002; O'Donovan 1999) and is believed to play an important role in neural and network development (Casavant et al. 2004; Hanson and Landmesser 2004; Milner and Landmesser 1999). In the spinal cord of E10–E12 chick embryos, spontaneous activity occurs as recurring rhythmic episodes 
1 min in duration that are followed by longer (
10–15 min) inter-episode intervals (Landmesser and O'Donovan 1984). During these rhythmic episodes, flexor and extensor motoneurons alternate their discharges in a manner resembling locomotion in the mature animal (Landmesser and O'Donovan 1984). At E10–E12, chloride-mediated GABAergic transmission is an important component of the rhythmic synaptic drive to ventral horn neurons during spontaneous episodes (Sernagor et al. 1995). In the chick embryo (Chub and O'Donovan 2001) as in other developing vertebrates (Gao and Ziskind-Conhaim 1995; Rohrbough and Spitzer 1996; Serafini et al. 1995), GABA depolarizes spinal cord neurons and is functionally excitatory. This action is the result of high intracellular chloride concentration ([Cl–]in), which creates an outward driving force for Cl– ions during the activation of GABAA channels. In the hippocampus, depolarizing GABAA responses become hyperpolarizing later in development (Ben-Ari et al. 1989; Khazipov et al. 2004), and this process itself appears to require activation of GABAergic transmission (Ganguly et al. 2001). In spontaneously active developing spinal networks, GABAergic depolarizations periodically trigger intracellular Ca2+ elevations (Lev-Tov and O'Donovan 1995; O'Donovan et al. 1994; Wenner and O'Donovan 2001), which are believed to be important for structural development of neurons and the maturation of network connections (Ben-Ari 2002; Demarque et al. 2002; Sernagor et al. 2003). Therefore the short- and long-term regulation of [Cl–]in during development is of considerable interest.
We have shown previously that [Cl–]in is reduced in ventral spinal neurons after an episode and is restored before the next episode (Chub and O'Donovan 2001). Because these measurements were made using whole cell electrodes, the [Cl–]in would have been modified by the intracellular pipette solution. Furthermore, they could only be made infrequently because they required computing the current-voltage relation for locally applied GABAA agonists. Therefore in the present work, we have employed a noninvasive optical method to monitor the [Cl–]in changes in spinal motoneurons during spontaneously occurring episodes of activity. For this purpose, we developed a new technique for loading neurons with the Cl–-sensitive 6-methoxy-N-ethylquinolinium iodide (MEQ) dye. Traditionally, the use of MEQ requires synthesis of the cell-permeable form of the dye—dihydro-MEQ (DiH-MEQ)—which is then bath-applied to label neurons (Biwersi and Verkman 1991). In the present work, we retrogradely loaded MEQ into motoneurons by applying the dye to the cut ends of muscles nerves. This method has the advantages of labeling a specific cell population and not requiring an initial reduction reaction to render the dye cell-permeant.
We have used the technique to resolve [Cl–]in changes in motoneurons in response to bath application of the GABAA receptor agonist isoguvacine, bumetanide a Na+/K–/2Cl– co-transporter (NKCC1) inhibitor, and to investigate the changes of intracellular chloride during spontaneously occurring episodes of activity.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
|---|
; Landmesser and O'Donovan 1984) and therefore will be described only briefly. The embryo was pinned to the silicone-elastomer Sylgard-covered bottom of the dissection chamber. After the embryo was eviscerated, and a ventral laminectomy was performed. A portion of the thoracic and the lumbosacral part of the spinal cord (T4–LS8) with the sartorius (SART) and femorotibialis (FEM) motor nerves was dissected from the vertebral column and hind limb muscles. All dorsal roots were cut to eliminate labeling of afferent terminals. The dura and pia mater were removed close to the lateral edge of the spinal cord to improve visualization of the labeled motoneurons.
In the initial experiments, we used bath-loading of the cell-permeable chloride-sensitive MEQ dye (Biwersi and Verkman 1991). MEQ is a largely cell-impermeant organic compound, which exhibits a maximum emission at 420 nm in response to illumination at 360 nm. This fluorescence is collisionally quenched in a concentration-dependent manner by Cl– without shifting the emission spectra. It is known that other halide anions, such as I–, Br–, and SCN– (Biwersi and Verkman 1991) can also quench the MEQ fluorescence. For bath-application, the dye must be initially reduced to a lipophilic Cl–-insensitive form DiH-MEQ and allowed to enter cells where it can be re-oxidized to the Cl–-sensitive MEQ (Biwersi and Verkman 1991). In our initial experiments, we found that the DiH-MEQ did not penetrate deeply into the cord and was only slowly re-oxidized to Cl–-sensitive MEQ dye once inside cells. For example, in E10–E11 preparations, 3–4 h after bath application of the DiH-MEQ (50 µM for 60 min) some cells still exhibited green fluorescence indicating incomplete intracellular DiH-MEQ re-oxidation.
For these reasons, we developed another labeling technique in which motoneurons were retrogradely loaded with the Cl–-sensitive MEQ by applying the dye to the cut end of motor nerves. This technique is similar to retrogradely labeling neurons with calcium-sensitive dyes (O'Donovan et al. 1993). For this type of labeling, the spinal cord was isolated together with attached muscle nerves. In most experiments, both the SART and FEM nerves were pulled into the tip of a plastic pipette by applying gentle mouth suction to the end of the pipette. Another smaller diameter (insertion) plastic tube, connected to a syringe, was used to deliver the MEQ dye (50–100 mM solution in Dulbecco's phosphate-buffered saline with calcium and magnesium, DPBS, Mediatech) into the pipette holding the motor nerves. After 2–4 h of dye application, the MEQ solution was extracted with the insertion tube and the motor nerves were removed from the suction pipette by applying weak positive pressure. To verify that MEQ specifically labeled motoneurons, in six experiments, we loaded the muscles nerves with MEQ and the retrograde tracer Texas Red Dextran (40–50 mM in DPBS; 10,000 MW; Invitrogen). We used Texas Red Dextran because this dye does not cross gap junctions or synapses and has been widely used to retrogradely label motoneurons (Bonnot et al. 2005; Mentis et al. 2005). During the 8–10 h required for dye loading, the spinal cord was continuously superfused with re-circulating at 21–23°C Tyrode's solution.
The preparation was then transferred to a recording chamber mounted on the stage of an inverted microscope (Nikon Diaphot 300) equipped for epi-fluorescence. For MEQ fluorescence measurements, we used illumination from a 100-W xenon-tungsten lamp, and a fluorescence filter combination UV-2E/C (Nikon) with 20x magnification using a Fluor 0.75 (160/0.17) objective (Nikon). An intensified CCD (XR GEN III+, Stanford Photonics Inc) or a digital (CoolSNAP ES, Photometrics) camera was used for capturing images. The duration of image acquisition was set by an electronic shutter controlled by MetaMorph software (Molecular Devices). This software was also used for capturing images and off-line analysis. We calculated the fractional change in fluorescence (
F/F) following various experimental manipulations by first measuring the mean fluorescence within a region of interest (ROI) over the motoneurons. In all cases, unless otherwise mentioned, mean background fluorescence (measured from an unlabeled area) was subtracted from this measurement and the resulting time series was divided by the initial or the predrug fluorescence. The percentage changes of this normalized fluorescence are reported.
We found that MEQ fluorescence decayed over time in the absence of illumination as reported previously and attributed to a gradual leakage of dye from the cells (Biwersi and Verkman 1991). This rate of decay was measured and subtracted from records shown in Figs. 5–8. Fluorescence decay for long time periods (7 h) was exponentially fitted with SigmaPlot (SPSS), and the resulting time constants are reported.
|
|
In some experiments, we used a "low-Na+" bath solution, which comprised (in mM) 144 Tris-Cl, 25 Tris-OH, 5 KHCO3, 12 NaHCO3, 1 MgCl2, 3 CaCl2, and 12 glucose and was saturated with 95% O2-5% CO2; pH 7.3. This solution has the same [Cl–] as control Tyrode's solution, but higher osmolarity. Therefore for these experiments, 40 mM sucrose was added to the control Tyrode's solution to minimize the osmolarity difference between control and "low-Na+" solutions.
After the recordings, some of the spinal cords were fixed by immersion in 4% paraformaldehyde (in PBS) for 4 h at 4°C. The cords were subsequently embedded in warm 5% agar (in PBS) and 50-µm sections cut with a Vibratome. The sections were collected in wells and processed for either morphological analysis or immunohistochemistry. To further confirm motoneuron identity, we used a polyclonal antibody against choline acetyltransferase (ChAT) raised in goat (1:100, Chemicon). To maximize the binding sites of the antibodies, the immunohistochemistry was performed in free floating sections. Initially, the sections were washed once for 10 min in PBS, followed by three washes in PBS-T (0.1% Triton in PBS). Nonspecific binding of the secondary antibodies was minimized by block with Normal Donkey Serum (1:10 in PBS-T). The primary antibodies (diluted in PBS-T) were incubated for 8–12 h at room temperature. The primary antibodies were subsequently washed six times (10 min each) in PBS-T. Immunoreactive sites were revealed by the binding of secondary antibodies conjugated to a specific fluorochrome (Jackson Immuno Research Laboratories). ChAT was visualized by anti-Goat-Cy5. The secondary antibodies were incubated for 3 h at room temperature and were subsequently washed six times (10 min each) in PBS. Finally, the sections were mounted on glass slides and coverslipped.
Images were obtained with confocal microscopy using a four-channel two-photon (Chameleon, Coherent) 510META (Carl Zeiss, Germany) confocal microscope equipped with three single photon lasers (488, 543, and 633 nm). Images from sections containing three fluorochromes (MEQ, Texas-Red Dextran, and ChAT) were acquired by multi-tracking. The MEQ dye was excited with the two-photon laser-locked mode at 720 nm and images acquired with a 390–465 nm filter. Texas Red Dextran was excited with a 543 nm laser and acquired with a 565–615 nm filter. ChAT immunoreactivity was excited with a 633 nm laser and acquired with a 650–710 nm filter.
Chemicals were purchased from Sigma, unless otherwise mentioned. Drugs were prepared as a 10 mM stock solution in distilled H2O and were applied to the continuously recirculating Tyrode's solution. All data are expressed as means ± SE. Statistical analyses were performed by t-test, and a significant difference was assumed when P < 0.05.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
|---|
|
|
1 h) could be linearly approximated (Fig. 3B, inset). The average time constant of fluorescence decay measured over a 7-h period in four experiments was 0.31 + 0.04 h (n = 6, 4 experiments). The decay rates measured from linear fit for short time intervals (1 h) were 14.5 ± 1.4% h–1 for the first 3 h and 5.1 ± 0.6% h–1 for the next 4 h (n = 6, 4 experiments; P < 0.05). We attribute this fluorescence decline to a gradual leakage of the MEQ dye from the labeled cells (Biwersi and Verkman 1991).
|
). The linear regression fit (Fig. 4B) was used to estimate the decline of the fluorescence intensity under continuous illumination.
|
20 mV higher than the resting membrane potential of ventral horn neurons (Chub and O'Donovan 2001). Therefore activation of GABA-A, Cl-conductances should result in an outwardly directed Cl– flux and a reduction of [Cl–]in. We found that bath-application of 100 µM GABAA agonist isoguvacine for 10 min, in the presence of 0.5 µM tetrodotoxin (TTX) to block presynaptic effects, led to an increase of MEQ fluorescence and a depolarization of the MEQ labeled motoneurons that was recorded from the muscle nerve (Fig. 5). Because MEQ dye is quenched collisionally by neuronal Cl– ions, an increase of the dye fluorescence corresponds to an reduction of [Cl–]in (Jayaraman and Verkman 2000). We also found that the isoguvacine-induced changes of [Cl–]in differed between the soma and dendrites of the labeled motoneurons. The largest percentage changes of fluorescence were recorded over the dendrites 16.7 ± 1.5%, and smaller changes were detected over the somata 7.4 ± 0.5% (n = 32, 6 experiments; P < 0.05). The time course of the fluorescence transient recorded from labeled motoneurons corresponded closely with the depolarization electrotonically recorded from the SART nerve (Fig. 5B, bottom). The slow potential recorded electrotonically from the muscle nerve has been shown to have the same time course as the intracellularly recorded membrane potential of individual motoneurons (O'Donovan 1989). Because Cl– is the dominant ionic current activated by isoguvacine, the time course of the slow potential will represent the time course of the Cl– efflux. The mean amplitude of the depolarization simultaneously recorded from the SART nerve was 0.94 ± 0.12 mV (n = 9, 4 experiments).
We have hypothesized that the elevated [Cl–]in in ventral spinal neurons results from the action of an inwardly directed Cl– co-transporter (Chub and O'Donovan 2001; Marchetti et al. 2005). Consistent with this idea, bumetanide, an antagonist of the inward chloride transporter NKCC1 (Haas 1994; Payne et al. 2003; Plotkin et al. 1997; Sun and Murali 1999) can reduce the frequency of spontaneous episodes in the isolated spinal cord of the chick embryo (Marchetti et al. 2005). To test if such a transporter was active in motoneurons, we bath-applied bumetanide and measured the corresponding changes in MEQ fluorescence. Figure 6 shows the effect of 60-min bath application of 20 µM bumetanide. We found that bumetanide increased the neuronal MEQ fluorescence by 5.3 ± 0.8% (n = 11, 4 experiments).
|
; Payne et al. 2003), only NKCC1-dependent Cl– accumulation requires an inwardly directed Na+ gradient. Therefore low extracellular [Na+] as well as bumetanide can inhibit NKCC1-mediated Cl– in-flux (Russell 2000). Figure 7 shows effect of 40-min bath application of 12 mM Na+ solution on motoneuronal MEQ fluorescence. In these experiments, application of the low Na+ solution increased MEQ fluorescence by 11.4 ± 0.9% (n = 11, 3 experiments), consistent with the participation of NKCC1 in inwardly directed neuronal Cl– transport.
|
For these experiments, percentage changes of the MEQ fluorescence (F/F) were calculated as the mean motoneuronal fluorescence after the episode minus the fluorescence before the episode and divided by the pre-episode fluorescence. The largest post-episode fluorescence increase was observed over the motoneuron dendrites 19.7 ± 1.8% and smaller changes were recorded over motoneuron somata 5.2 ± 0.6% (n = 49, 3 experiments; P < 0.05). No fluorescence changes were detected over motoneuron axons.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
|---|
). In addition, we have demonstrated that the elevated [Cl–]in of developing chick motoneurons described previously (Chub and O'Donovan 2001) is controlled at least in part by the action of a bumetanide-sensitive NKCC1 co-transporter. Finally, we have established that motoneuron dendrites, rather than somata, are the major locus for changes of intracellular chloride during spontaneous episodes of network activity.
The rates of neuronal MEQ fluorescence decay and photobleaching in our experiments were similar to those employing bath-loading of DiH-MEQ in fibroblast cultures (Biwersi and Verkman 1991) and in hippocampal (Sah and Schwartz-Bloom 1999) or brain slice preparations (Fukuda et al. 1998; Inglefield et al. 1999; Shibata et al. 2004). In addition, the fluorescence of MEQ-labeled motoneurons was increased by bath-application of the GABAA agonist isoguvacine consistent with Cl– outflow. These findings indicate that intracellular MEQ behaves similarly whether loaded retrogradely or by bath-application. Of course, the advantage of the retrograde method is that a specific population of neurons can be loaded allowing the cellular origin of the Cl– signals to be defined unambiguously.
We used two methods to demonstrate that MEQ labeling was restricted to motoneurons. First, we identified motoneurons by retrogradely labeling them with Texas Red Dextran. Because of the high molecular weight of this dye (10,000), it does not leak through gap junctions and remains localized within motoneurons. Motoneurons were also identified by their ChAT immunofluorescence. When these procedures were used in conjunction with MEQ labeling, we found that only motoneurons were labeled by the Cl-sensitive dye.
In our previous work, we used whole cell recording to show that the [Cl–]in falls after an episode and then is restored during the interepisode interval (Chub and O'Donovan 2001). We hypothesized that the restoration of [Cl–]in was due to the action of an inwardly directed NKCC1 co-transporter. The results of the present work provide functional evidence for the presence of NKCC1 on chick embryo motoneurons at E10–E12. Specifically, the fluorescence of MEQ-loaded motoneurons was increased by the NKCC1 blocker bumetanide and by a low [Na+] in the extracellular solution. Both of these procedures effectively decreased the [Cl–]in. The presence of this transporter on developing chick motoneurons is in agreement with other studies on immature neurons (Plotkin et al. 1997; Rohrbough and Spitzer 1996; Sun and Murali 1999) where electro-neutral NKCC1 co-transport was demonstrated. Our data do not exclude the possibility that other chloride ion transporters, for example, the Cl–/HCO–3 exchanger (Kaila 1994; Staley and Proctor 1999), may also be active at this stage of development in chick embryo motoneurons.
Perhaps the most significant finding in the present work was the observation that periodic variations in the [Cl–]in accompany the discharge of spontaneously active motoneurons. We found that [Cl–]in falls after an episode and slowly recovers during the interepisode interval to the pre-episode level. These activity-dependent variations of MEQ fluorescence were consistent with our recent data indicating that [Cl–]in decreased 15 mM after an episode (Chub and O'Donovan 2001). These earlier data were obtained with whole cell electrodes and were therefore complicated by dialysis of the [Cl–]in by the pipette solution. The fact that we were able to detect significant post-episode changes using whole cell recording raised the possibility that the most significant [Cl–]in changes were occurring in the dendrites. Consistent with this hypothesis we found that the largest changes of MEQ fluorescence during spontaneous episodes were over motoneuron dendrites. This observation may indicate that GABAergic synapses, which are active during spontaneous episodes, are not uniformly distributed on the motoneuron surface. Alternatively, it may indicate that dendrites are more sensitive to the ion redistribution because of their smaller cytoplasmic volume in comparison with that of the soma. Finally, it is possible that the density of Cl– transporters might be different on the dendrites and on the soma, which could be investigated using immunocytochemistry. Additional progress in understanding the spatial distribution of [Cl–]in requires the use of more sensitive chloride dyes and more sophisticated imaging such as that afforded by two-photon confocal microscopy (Marandi et al. 2002). In comparison with patch-clamp recording, Cl-dependent changes of MEQ fluorescence are relatively small. As a result, some important neural network events like local [Cl–]in changes after quantal GABA release (e.g., spontaneous miniature postsynaptic currents) remain beyond detection of the MEQ dye.
In conclusion, our data show that the neuronal Cl– outflow during an episode exceeds the inwardly directed neuronal Cl– transport capacity. Therefore after spontaneous episodes, [Cl–]in is reduced and then slowly restored during the interepisode interval. Recently we have shown that bumetanide can block spontaneous episodes in the isolated chick spinal cord following antagonism of glutamatergic transmission (Marchetti et al. 2005). In addition, the rate of spontaneous activity is greatly slowed following blockade of GABAA receptor activity (Chub and O'Donovan 1998; Marchetti et al. 2005). These findings, together with the results of the present work, are consistent with the idea that the bumetanide-sensitive NKCC1 co-transporter is responsible, in part, for restoring the [Cl–]in during the inter-episode interval. This activity-dependent variation of [Cl–]in leads to corresponding changes in the chloride equilibrium potential and the driving force for GABAergic synaptic potentials. A recent modeling study (Marchetti et al. 2005) has shown that these variations in excitability can themselves lead to the expression of spontaneous episodes of activity. Thus the action of GABAergic Cl– conductances and the slower inward pumping of Cl– by NKCC1 may be an essential mechanism underlying the genesis of spontaneous activity in the developing spinal cord.
|
GRANTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
|---|
|
ACKNOWLEDGMENTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
|---|
|
FOOTNOTES |
|---|
Address for reprint requests and other correspondence: N. Chub, Lab. of Neural Control, NINDS/NIH, Rm. 3BC911, 35 Convent Dr., Bethesda, MD 20892-3700 (E-mail: chubn{at}ninds.nih.gov)
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
|---|
Ben Ari Y. Excitatory actions of gaba during development: the nature of the nurture. Nat Rev Neurosci 3: 728–739, 2002.[CrossRef][Web of Science][Medline]
Ben Ari Y, Cherubini E, Corradetti R, and Gaiarsa JL. Giant synaptic potentials in immature rat CA3 hippocampal neurones. J Physiol 416: 303–325, 1989.
Biwersi J and Verkman AS. Cell-permeable fluorescent indicator for cytosolic chloride. Biochemistry 30: 7879–7883, 1991.[CrossRef][Medline]
Bonnot A, Mentis GZ, Skoch J, and O'Donovan MJ. Electroporation loading of calcium-sensitive dyes into the CNS. J Neurophysiol 93: 1793–1808, 2005.
Casavant RH, Colbert CM, and Dryer SE. A-current expression is regulated by activity but not by target tissues in developing lumbar motoneurons of the chick embryo. J Neurophysiol 92: 2644–2651, 2004.
Chub N and O'Donovan MJ. Blockade and recovery of spontaneous rhythmic activity after application of neurotransmitter antagonists to spinal networks of the chick embryo. J Neurosci 18: 294–306, 1998.
Chub N and O'Donovan MJ. Post-episode depression of GABAergic transmission in spinal neurons of the chick embryo. J Neurophysiol 85: 2166–2176, 2001.
Demarque M, Represa A, Becq H, Khalilov I, Ben Ari Y, and Aniksztejn L. Paracrine intercellular communication by a Ca2+- and SNARE-independent release of GABA and glutamate prior to synapse formation. Neuron 36: 1051–1061, 2002.[CrossRef][Web of Science][Medline]
Fukuda A, Tanaka M, Yamada Y, Muramatsu K, Shimano Y, and Nishino H. Simultaneous optical imaging of intracellular Cl- in neurons in different layers of rat neocortical slices: advantages and limitations. Neurosci Res 32: 363–371, 1998.[CrossRef][Web of Science][Medline]
Ganguly K, Schinder AF, Wong ST, and Poo M. GABA itself promotes the developmental switch of neuronal GABAergic responses from excitation to inhibition. Cell 105: 521–532, 2001.[CrossRef][Web of Science][Medline]
Gao BX and Ziskind-Conhaim L. Development of glycine- and GABA-gated currents in rat spinal motoneurons. J Neurophysiol 74: 113–121, 1995.
Haas M. The Na-K-Cl cotransporters. Am J Physiol Cell Physiol 267: C869–C885, 1994.
Hanson MG and Landmesser LT. Normal patterns of spontaneous activity are required for correct motor axon guidance and the expression of specific guidance molecules. Neuron 43: 687–701, 2004.[CrossRef][Web of Science][Medline]
Inglefield JR and Schwartz-Bloom RD. Confocal imaging of intracellular chloride in living brain slices: measurement of GABAA receptor activity. J Neurosci Methods 75: 127–135, 1997.[CrossRef][Web of Science][Medline]
Inglefield JR and Schwartz-Bloom RD. Fluorescence imaging of changes in intracellular chloride in living brain slices. Methods 18: 197–203, 1999.[CrossRef][Web of Science][Medline]
Jayaraman S and Verkman AS. Quenching mechanism of quinolinium-type chloride-sensitive fluorescent indicators. Biophys Chem 85: 49–57, 2000.[CrossRef][Web of Science][Medline]
Kaila K. Ionic basis of GABAA receptor channel function in the nervous system. Prog Neurobiol 42: 489–537, 1994.[CrossRef][Web of Science][Medline]
Khazipov R, Khalilov I, Tyzio R, Morozova E, Ben Ari Y, and Holmes GL. Developmental changes in GABAergic actions and seizure susceptibility in the rat hippocampus. Eur J Neurosci 19: 590–600, 2004.[CrossRef][Web of Science][Medline]
Landmesser LT and O'Donovan MJ. Activation patterns of embryonic chick hind limb muscles recorded in ovo and in an isolated spinal cord preparation. J Physiol 347: 189–204, 1984.
Lev-Tov A and O'Donovan MJ. Calcium imaging of motoneuron activity in the en-bloc spinal cord preparation of the neonatal rat. J Neurophysiol 74: 1324–1334, 1995.
Marandi N, Konnerth A, and Garaschuk O. Two-photon chloride imaging in neurons of brain slices. Pfluegers 445: 357–365, 2002.[CrossRef]
Marchetti C, Tabak J, Chub N, O'Donovan MJ, and Rinzel J. Modeling spontaneous activity in the developing spinal cord using activity-dependent variation of intracellular chloride. J Neurosci 25: 3601–3612, 2005.
Mentis GZ, Alvarez FJ, Bonnot A, Richards DS, Gonzalez-Forero D, Zerda R, and O'Donovan MJ. Noncholinergic excitatory actions of motoneurons in the neonatal mammalian spinal cord. Prog Natl Acad Sci USA 102: 7344–7349, 2005.
Milner LD and Landmesser LT. Cholinergic and GABAergic inputs drive patterned spontaneous motoneuron activity before target contact. J Neurosci 19: 3007–3022, 1999.
Mount DB, Delpire E, Gamba G, Hall AE, Poch E, Hoover RS, and Hebert SC. The electroneutral cation-chloride cotransporters. J Exp Biol 201: 2091–2102, 1998.[Abstract]
O'Donovan MJ. Motor activity in the isolated spinal cord of the chick embryo: synaptic drive and firing pattern of single motoneurons. J Neurosci 9: 943–958, 1989.[Abstract]
O'Donovan MJ. The origin of spontaneous activity in developing networks of the vertebrate nervous system. Curr Opin Neurobiol 9: 94–104, 1999.[CrossRef][Web of Science][Medline]
O'Donovan MJ, Ho S, Sholomenko G, and Yee W. Real-time imaging of neurons retrogradely and anterogradely labelled with calcium-sensitive dyes. J Neurosci Methods 46: 91–106, 1993.[CrossRef][Web of Science][Medline]
O'Donovan M, Ho S, and Yee W. Calcium imaging of rhythmic network activity in the developing spinal cord of the chick embryo. J Neurosci 14: 6354–6369, 1994.[Abstract]
Payne JA, Rivera C, Voipio J, and Kaila K. Cation-chloride co-transporters in neuronal communication, development and trauma. Trends Neurosci 26: 199–206, 2003.[CrossRef][Web of Science][Medline]
Plotkin MD, Snyder EY, Hebert SC, and Delpire E. Expression of the Na-K-2Cl cotransporter is developmentally regulated in postnatal rat brains: a possible mechanism underlying GABA's excitatory role in immature brain. J Neurobiol 33: 781–795, 1997.[CrossRef][Web of Science][Medline]
Rohrbough J and Spitzer NC. Regulation of intracellular Cl– levels by Na(+)-dependent Cl– cotransport distinguishes depolarizing from hyperpolarizing GABAA receptor-mediated responses in spinal neurons. J Neurosci 16: 82–91, 1996.
Russell JM. Sodium-potassium-chloride cotransport. Physiol Rev 80: 211–276, 2000.
Sah R and Schwartz-Bloom RD. Optical imaging reveals elevated intracellular chloride in hippocampal pyramidal neurons after oxidative stress. J Neurosci 19: 9209–9217, 1999.
Serafini R, Valeyev AY, Barker JL, and Poulter MO. Depolarizing GABA-activated Cl– channels in embryonic rat spinal and olfactory bulb cells. J Physiol 488(Pt 2): 371–386, 1995.
Sernagor E, Chub N, Ritter A, and O'Donovan MJ. Pharmacological characterization of the rhythmic synaptic drive onto lumbosacral motoneurons in the chick embryo spinal cord. J Neurosci 15: 7452–7464, 1995.[Abstract]
Sernagor E, Young C, and Eglen SJ. Developmental modulation of retinal wave dynamics: shedding light on the GABA saga. J Neurosci 23: 7621–7629, 2003.
Shibata S, Kakazu Y, Okabe A, Fukuda A, and Nabekura J. Experience-dependent changes in intracellular Cl– regulation in developing auditory neurons. Neurosci Res 48: 211–220, 2004.[CrossRef][Web of Science][Medline]
Staley KJ and Proctor WR. Modulation of mammalian dendritic GABA(A) receptor function by the kinetics of Cl– and. J Physiol 519: 693–712, 1999.
Sun D and Murali SG. Na+-K+-2Cl– cotransporter in immature cortical neurons: a role in intracellular Cl– regulation. J Neurophysiol 81: 1939–1948, 1999.
Wenner P and O'Donovan MJ. Mechanisms that initiate spontaneous network activity in the developing chick spinal cord. J Neurophysiol 86: 1481–1498, 2001.
This article has been cited by other articles:
|
|
 |
|
  J. N. Chabwine, K. Talavera, L. Verbert, J. Eggermont, J.-M. Vanderwinden, H. De Smedt, L. Van Den Bosch, W. Robberecht, and G. Callewaert Differential contribution of the Na+-K+-2Cl- cotransporter NKCC1 to chloride handling in rat embryonic dorsal root ganglion neurons and motor neurons FASEB J, April 1, 2009; 23(4): 1168 - 1176. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Gonzalez-Islas, N. Chub, and P. Wenner NKCC1 and AE3 Appear to Accumulate Chloride in Embryonic Motoneurons J Neurophysiol, February 1, 2009; 101(2): 507 - 518. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Mukhtarov, O. Markova, E. Real, Y. Jacob, S. Buldakova, and P. Bregestovski Monitoring of chloride and activity of glycine receptor channels using genetically encoded fluorescent sensors Phil Trans R Soc A, October 13, 2008; 366(1880): 3445 - 3462. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. C. Wilhelm and P. Wenner GABAA transmission is a critical step in the process of triggering homeostatic increases in quantal amplitude PNAS, August 12, 2008; 105(32): 11412 - 11417. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. I. Rocha-Gonzalez, S. Mao, and F. J. Alvarez-Leefmans Na+,K+,2Cl- Cotransport and Intracellular Chloride Regulation in Rat Primary Sensory Neurons: Thermodynamic and Kinetic Aspects J Neurophysiol, July 1, 2008; 100(1): 169 - 184. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Jean-Xavier, G. Z. Mentis, M. J. O'Donovan, D. Cattaert, and L. Vinay Dual personality of GABA/glycine-mediated depolarizations in immature spinal cord PNAS, July 3, 2007; 104(27): 11477 - 11482. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Xu, A. Clement, T. M. Wright, and P. Wenner Developmental Reorganization of the Output of a GABAergic Interneuronal Circuit J Neurophysiol, April 1, 2007; 97(4): 2769 - 2779. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. T. Sipila, S. Schuchmann, J. Voipio, J. Yamada, and K. Kaila The cation-chloride cotransporter NKCC1 promotes sharp waves in the neonatal rat hippocampus J. Physiol., June 15, 2006; 573(3): 765 - 773. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
