| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Laboratoire Plasticité et Physio-Pathologie de la Motricité, Centre National de la Recherche Scientifique (CNRS) and Aix-Marseille Université, CNRS, Marseille, France
Submitted 29 March 2006; accepted in final form 26 June 2006
 |
ABSTRACT |
|---|
|
|
|
INTRODUCTION |
|---|
|
; Takahashi 1984; Wu et al. 1992; Ziskind-Conhaim 1998), because of a high intracellular Cl– concentration ([Cl–]i), which favors Cl– efflux through GABAA- or glycine-operated Cl– channels. GABA- and glycine-mediated depolarizations lead to activation of voltage-dependent Ca2+ channels and Ca2+ oscillations play a key role in neuronal maturation and synaptogenesis (Ben Ari 2002). With development, [Cl–]i decreases, leading to a shift of the chloride equilibrium potential toward further negative values and thereby to a change in glycine and GABA-evoked potentials from depolarization to hyperpolarization (Gao and Ziskind-Conhaim 1995; Takahashi 1984). Up-regulation of a transporter that regulates [Cl–]i, the neuron specific K-Cl cotransporter, KCC2, underlies this shift (Delpire and Mount 2002; Payne et al. 2003; Rivera et al. 1999, 2004a; Stein et al. 2004). Cortical neurons lacking KCC2 expression fail to show a developmental decrease in [Cl–]i (Zhu et al. 2005). KCC2 knockout mice exhibit severe motor deficits and an inability to breathe, which results in perinatal death (Hübner et al. 2001). The upstream mechanisms responsible for the developmental up-regulation of KCC2 are unknown. There is a need to understand these mechanisms because inhibitory amino acids become excitatory again, and KCC2 is down-regulated in some pathological conditions (i.e., neuronal damage, peripheral nerve-induced chronic pain, human temporal lobe epilepsy; Cohen et al. 2002; Coull et al. 2003; Nabekura et al. 2002; Payne et al. 2003; Toyoda et al. 2003). This down-regulation of KCC2 may be a general early response involved in various kinds of neuronal trauma (Rivera et al. 2004b) and seems to reflect a recapitulation of early developmental mechanisms (Rivera et al. 2004a). Counteracting or preventing the down-regulation of KCC2 may be useful in the treatment of these pathological states.
Maturation of inhibitory synaptic transmission in the rat spinal cord occurs during the perinatal period (Gao and Ziskind-Conhaim 1995; Wu et al. 1992), in a time window during which the projections from the brain stem reach the lumbar enlargement (Brocard et al. 1999; Lakke 1997; Vinay et al. 2002). This raises the question of the influence of the brain on the maturation of spinal cord inhibition. In this study, we investigated the effects of a complete spinal cord transection, made at birth (Norreel et al. 2003), on IPSPs recorded in vitro from lumbar motoneurons at the end of the first postnatal week (4th to 7th days). We report that the typical developmental shift from depolarizing to hyperpolarizing IPSPs does not occur in the absence of descending modulatory inputs. Preliminary results have been presented in abstract form (Jean-Xavier et al. 2004).
|
|
METHODS |
|---|
|
Wistar rats aged from postnatal day 0 (P0, defined as the 1st 24 h after birth) to P7 were distributed into three groups: an experimental group (n = 50 at the following ages: P4, n = 8; P5, n = 8; P6, n = 15; P7, n = 19) whose spinal cord was transected at P0; a sham group (n = 5: P5, n = 1; P6, n = 3; P7, n = 1), operated, handled, and treated in the same way as spinal animals except for the spinal cord transection; and a control group (n = 35 : P0, n = 4; P1, n = 1; P2, n = 1; P4, n = 6; P5, n = 11, P6, n = 10; P7, n = 2) of non-operated animals. All surgical and experimental procedures were made to minimize animal suffering and conformed to the guidelines from the French Ministry for Agriculture and Fisheries, Division of Animal Rights.
Neonatal spinal cord transection and preparation of in vitro experiments were performed similar to those described previously (Norreel et al. 2003). Briefly, rats were deeply anesthetized by hypothermia. The spinal cord was transected at the thoracic (T) T8–T10 level, and one to two segments of the cord were removed. The completeness of spinal cord transection was verified postmortem by visual inspection of lack of continuity between the spinal stumps. For in vitro experiments, the spinal cord and ventral roots were removed from sacral segments up to T8–T10. The preparation was pinned down, ventral side up, in the recording chamber. All dissection and recording procedures were performed under continuous perfusion with saline solution (in mM: 130 NaCl, 4 KCl, 3.75 CaCl2, 1.3 MgSO4, 0.58 NaH2PO4, 25 NaHCO3, and 10 glucose; oxygenated with 95% O2-5% CO2; pH = 7.4, temperature of 24–27°C). In CO2/HCO3–-free solution (oxygenated with 100% O2), NaHCO3 was replaced by HEPES (25 mM). The external concentration of K+ ([K+]o) was decreased from 4 to 2 mM in some experiments; the NaCl concentration was raised to 132 mM to keep the Cl– concentration unchanged. All compounds were purchased from Sigma (St. Louis, MO).
Ventral roots were usually dissected into two rootlets. Monopolar stainless steel electrodes were placed in contact with the roots and insulated with petroleum jelly for stimulation. Glass suction electrodes were used to stimulate the ventral funiculus at the L2–L3 level, on the recording side. After the pia had been removed, lumbar motoneurons were recorded intracellularly using glass microelectrodes filled with 2 M K-acetate (90–150 M
resistance). Intracellular potentials were recorded in the discontinuous current-clamp (DCC) mode (Axoclamp 2B amplifier; Digidata 1200 interface, pClamp8 software, Axon Instruments). Only neurons exhibiting a stable (>15 min) resting membrane potential were considered for analysis. Motoneurons [n = 142 in control (n = 69), sham-operated (n = 11), and cord-transected animals (n = 65)] in the L4–L5 segments were identified by the antidromic response to stimulation of one of the ventral rootlets. Stimulation of either the other rootlets or the ventral funiculus usually induced IPSPs in the presence of 2-amino-5-phosphonovaleric acid (AP5, 30–100 µM) and 6-cyano-7-nitroquin-oxaline-2,3-dione (CNQX, 3–10 µM). IPSPs were recorded at various holding potentials (500 ms-long current pulses). The input resistance of motoneurons was measured by injecting moderate (0.2–0.5 nA) hyperpolarizing current pulses. Amplitudes of IPSPs were measured and plotted against holding potentials. At least 22 values were collected for each motoneuron. The EIPSP was given by the intercept of the regression line with the x-axis (Graphpad Prism 4 Software). Stimulation of the ventral funiculus was used to determine EIPSP in most of the cells (76%). When the two inputs were tested in individual neurons, EIPSP was similar (P > 0.05, paired t-test) for the two inputs and the average was considered for subsequent statistical analysis. Because EIPSP, VREST, and input resistance were very similar in control and sham-operated animals at P4–P7 (Table 1), we pooled the data (cord-intact animals) for statistical comparison with cord-transected animals of the same age and controls at P0–P2.
|
KCC2 immunohistochemistry
Three animal groups were used: cord-intact (n = 6) and cord-transected (n = 5) animals at P7 and neonates (P0, n = 3). After fixation in 4% paraformaldehyde, the lumbar spinal cords were embedded in 3% agarose. Each experiment was conducted on one cord-intact and one cord-transected animals from the same litter. Cords were cut, transversally, at 30 µm on a vibratome (Leica VT1000S). Sections were preincubated with normal goat serum (1% in PBS +3% BSA; Sigma) and Triton-X (0.2%). They were incubated overnight with an affinity-purified rabbit anti-KCC2 polyclonal antibody (diluted 1:200; Upstate Biotech, Lake Placid, NY; Fiumelli et al. 2005; Grob and Mouginot 2005; Lohrke et al. 2005; Vale et al. 2003, 2005) followed by goat anti-rabbit IgG coupled to Alexa-546 (diluted 1:400; Molecular Probes). Sections from different animal groups (cord-intact vs. cord-transected animals at P7 and cord-intact P7 vs. neonates) were processed simultaneously and mounted on the same slides. Sections were observed on a confocal microscope (Olympus). To quantify the immunohistochemical pattern, intensity measures were acquired (FluoView Sofware). Two regions were delineated in the pools of motoneurons (Fig. 2A, mn) in the ventral horn and white matter (Fig. 2A, wm) in the L4 segment for analysis. The same areas were considered in successive sections (n = 145). The mean areas of the mn and wm regions were 35,682 ± 523 and 81,899 ± 1,546 µm2, respectively. The ratio of mean pixel intensities in the gray and white matter (mn/wm) was calculated for each section.
|
|
|
|
RESULTS |
|---|
|
IPSPs were elicited by electrical stimulation of either ventral roots (Renshaw cell-mediated recurrent inhibition; Mentis et al. 2005; Nishimaru et al. 2005) or the ventral funiculus of the spinal cord, in the presence of the glutamate receptors antagonists CNQX and AP5. These IPSPs are mediated by glycine and GABAA receptors (Marchetti et al. 2002; Pinco and Lev-Tov 1994; Schneider and Fyffe 1992). Varying membrane potential by injection of depolarizing or hyperpolarizing current steps altered the amplitude of IPSPs (Fig. 1, A and B). The reversal potential of IPSPs (EIPSP) was significantly more depolarized (+7 mV on average) in cord-transected than in cord-intact animals (Table 1; Fig. 1, A–C). Synaptic responses were abolished by picrotoxin (10–20 µM) and strychnine (2–4 µM), showing that they were still mediated by GABAA and glycine receptors after spinal cord transection (n = 3; data not shown). Resting membrane potential (VREST) and input resistance were not significantly affected by spinal cord transection (Table 1). As a result, EIPSP was, on average, below and above VREST in animals with intact and transected spinal cords, respectively (Fig. 1, A, B, and D). In cord-intact animals, motoneurons exhibiting hyperpolarizing IPSPs at VREST were more numerous than those with depolarizing responses (Fig. 1E, left, column). These proportions were reversed in cord-transected animals (Fig. 1E, right, column). When IPSPs were hyperpolarizing at VREST, EIPSP was significantly more negative in cord-intact than in cord-transected animals (Fig. 1F, lower part of columns). Conversely, EIPSP, when above VREST, was more depolarized in cord-transected than in cord-intact animals (Fig. 1F, upper part of columns).
To determine the effect of the transection on the developmental switch from depolarizing to hyperpolarizing IPSPs, we measured EIPSP early after birth (P0–P2, control animals). EIPSP at P0–P2 was above VREST (Table 1; Fig. 1D). It was close to EIPSP measured at P4–P7 in cord-transected animals and significantly less negative than in cord-intact animals at P4–P7 (Fig. 1C). Altogether, these results suggest that the spinal cord transection at birth stopped the developmental shift of EIPSP toward further hyperpolarizing values.
Outward Cl– transport system is less efficient in cord-transected animals
Efflux of HCO3– through the anion pore may contribute to the depolarizing IPSPs (Kulik et al. 2000). To study this issue, we recorded IPSPs in cord-transected animals after CO2/HCO3– had been removed from the superfusate. EIPSP was similar in HCO3–-free (HEPES) and HCO3–-containing salines (Table 1; P > 0.05, t-test), indicating that HCO3– does not contribute to the depolarizing IPSPs after the transection. These experiments suggest that chloride homeostasis was affected in cord-transected animals.
Because the developmental negative shift in EIPSP is attributable to an increased efficacy of Cl– extrusion and the up-regulation of KCC2 (Hübner et al. 2001; Lu et al. 1999), we studied the presence and functionality of this transporter in cord-transected and cord-intact animals at P7. Anti-KCC2 antibody was used to localize the KCC2 cotransporter in the lumbar spinal cord. Specificity of the antibody was confirmed by lack of signal when the secondary antibody was applied in the absence of primary antibody (data not shown). KCC2 staining in cord-intact animals was abundant in the ventral horn (vh). A dense matrix of processes was heavily stained around large dark cell bodies, presumably motoneurons (mn, Fig. 2, A and C ). Some labeled neurites stretched out of the gray matter in all directions into the lateral and ventral funiculi. These labeled neurites in both the gray and white matters are likely dendrites because KCC2 is not expressed in axons (Hübner et al. 2001). In addition, dendrites are known to temporarily extend into the white matter in neonates (Curfs et al. 1993). At higher magnification, KCC2 immunolabeling clustered around large cell bodies, suggesting a preferential plasma membrane localization (Fig. 2C, inset), although this issue cannot be clarified with certainty at this resolution level. In cord-transected animals, these clusters of stained spots were not present around large cells (Fig. 2D, inset), and diffuse cytoplasmic staining was commonly observed in somas around unlabeled nucleoli and in processes (Fig. 2D, inset, arrowheads and asterisk, respectively). A similar KCC2 immunolabeling was present at P0 in cord-intact animals (Fig. 2E).
At low magnification, a lower level of KCC2 immunoreactivity was observed in the ventral horn at P7 after spinal cord transection compared with cord-intact animals of the same age (cf. Fig. 2, A and B). The immunohistochemical data were quantified in two areas of the ventral horn and white matter in the L4 segment (Fig. 2A). The ratio of mean pixel intensities in the gray and white matter (mn/wm) was significantly larger at P7 in cord-intact animals (Fig. 2F) than in both cord-transected animals of the same age and neonates, indicating that the relative KCC2 targeting to the somatic and dendritic compartments changed with age and that this development was affected by neonatal spinal cord transection. Interestingly, P7 cord-transected and P0 animals, which exhibit similar EIPSP values (Fig. 1C) have similar ratios. The larger ratio in cord-intact animals at P7 was caused, at least partly, by the clusters of KCC2 labeling surrounding motoneurons (Fig. 2C). These results suggest that the developmental upregulation of KCC2 staining in the ventral horn was affected by neonatal spinal cord transection.
We tested the functionality of KCC2 cotransporters at P4–P7 by either applying the loop diuretic bumetanide or decreasing [K+]o. Bumetanide blocks both KCC2 and the Na-K-2Cl cotransporter, NKCC1, which mediates Cl– uptake (Payne et al. 2003; Yamada et al. 2004). Because of the lack of data on the effect of bumetanide on the whole spinal cord in vitro, we tested the effect of bath applications (10–20 min) of bumetanide at different concentrations (100, 250, and 500 µM) on motoneurons recorded from control animals at P4–P7 (Fig. 3A). Applications of bumetanide always induced a depolarizing shift of EIPSP (+0.84 ± 0.35 mV, n = 5 at 100 µM; +4.16 ± 2.33 mV, n = 6 at 250 µM; +11.78 ± 2.50 mV, n = 9 at 500 µM); this effect was significant only for the highest concentration tested. Effects were reversible (Fig. 3A). Bumetanide was then tested on cord-transected animals at this concentration of 500 µM, shown to be efficient to block the Cl– transport system in cord-intact animals. Bumetanide depolarized EIPSP significantly (Fig. 3B, left histogram); the shift was, however, smaller than in cord-intact animals (Fig. 3B, right histogram).
|
) and is usually used at concentrations <100 µM to be specific for NKCC1. Bumetanide, at 50 µM, had no significant effect on EIPSP in cord-transected animals (Fig. 3C). If the regulation of neuronal [Cl–]i in cord-transected animals is dominated by the NKCC1 cotransporters, application of bumetanide should cause a hyperpolarizing shift of EIPSP. However, this loop diuretic always depolarized EIPSP, whatever the concentration used in this study. Therefore, the inward Cl– transport system, NKCC1, does not appear to play a significant role in setting EIPSP at more depolarized values after spinal cord transection.
KCC2 is sensitive to [K+]o; the direction and the driving force of the K-Cl cotransport result from the difference in the transmembrane ionic gradient of both K+ and Cl– (Alvarez-Leefmans 2001; Payne 1997; Thompson and Gahwiler 1989; Woodin et al. 2003). Thus [Cl–]i is affected by changing [K+]o (Woodin et al. 2003; Zhang et al. 1991). In cord-intact animals, decreasing [K+]o from 4 to 2 mM caused a rapid negative shift of the EIPSP in motoneurons (Fig. 3D); effects were reversible. The negative shift of EIPSP was significant for motoneurons recorded from cord-intact animals but not for those recorded after spinal cord transection (Fig. 3D). Note that neither VREST nor the slope of IPSPs was significantly affected by reducing [K+]o (P > 0.05; Table 2). The Cl– extrusion mechanisms therefore seem to be less efficient in cord-transected than in cord-intact animals.
|
|
|
DISCUSSION |
|---|
|
; Kakazu et al. 1999; Owens et al. 1996; Rivera et al. 1999).
The down-regulation of the inward-directed Cl– pumps or vice versa, the increased efficacy of the Cl– extruding system can, in theory, contribute to the age-related reversal of transmembrane Cl– gradients. NKCC1 has been proposed to be important in active accumulation of intracellular Cl– in immature neurons (Dzhala et al. 2005; Ikeda et al. 2003; Plotkin et al. 1997; Sun and Murali 1999; Vardi et al. 2000). However, the time window of NKCC1 expression does not match that of depolarizing effects in all neuronal systems (Balakrishnan et al. 2003; Clayton et al. 1998; Yan et al. 2001). This is the case of the spinal cord, in which NKCC1 transcripts are detected at E12 but have almost disappeared at E18.5 (Hübner et al. 2001) and are absent at P0 (Balakrishnan et al. 2003) at a time when the Cl– reversal potential is above VREST, as shown in our experiments. Therefore NKCC1 may not be the major transporter leading to GABA- and glycine-evoked depolarizations in neonatal motoneurons, and its down-regulation does not provide the molecular basis of the ontogenetic switch from depolarizing to hyperpolarizing IPSPs in motoneurons (Hübner et al. 2001). This is supported by our experiments using bumetanide, which blocks both NKCC1 and KCC2 cotransporters: the relatively high concentration of bumetanide required to affect EIPSP and the net depolarizing effect of this loop diuretic on EIPSP, even at the lowest concentrations used, suggest that KCC2 are responsible for the setting of neuronal [Cl–]i in both cord-intact and cord-transected animals at the end of the first postnatal week. However, our experiments do not rule out the possibility that an inward-directed Cl– system is functional after neonatal spinal cord transection. This issue will require further study when the nature of this system in the spinal cord becomes clear.
In contrast to NKCC1, changes—increases in this case—in KCC2 levels have been associated with the developmental transition from GABA/glycine-induced depolarization to hyperpolarization in several regions of the CNS including the spinal cord so that it is widely accepted that KCC2 is an "indicator of neuronal maturation" (Li et al. 2002; Lu et al. 1999; Mikawa et al. 2002; Rivera et al. 1999, 2004a; Shibata et al. 2004; Stein et al. 2004; Wang et al. 2002, 2005). KCC2 mRNA expression can be detected in the ventral part of the rostral spinal cord as early as E12.5 in rodent embryos (Hübner et al. 2001; Li et al. 2002). KCC2 protein levels increase until P3–P7 (Stein et al. 2004). In addition to the up-regulation of KCC2 gene expression or protein synthesis, posttranslational modifications occur that affect the functionality of the transporter (Kelsch et al. 2001; Stein et al. 2004). An important event in the development of the chloride-extruding system is the translocation of the KCC2 protein from intracellular compartments to the plasma membrane at a position of an active cotransporter (Balakrishnan et al. 2003). There are, in addition, posttranslational modifications that activate an initially inactive form of the protein (Kelsch et al. 2001; Vale et al. 2003 2005). In our experiments, the overall KCC2 staining in the ventral horn—where motoneuron cell bodies are located—was fainter after cord transection compared with cord-intact animals of the same age. The comparison with P0 animals suggests that the developmental upregulation of KCC2 in the somatic—relative to dendritic—compartment that occurs during the first postnatal week has been prevented by neonatal spinal cord transection (Fig. 2F). Some cytoplasmic staining was clearly observed in cell bodies from cord-transected animals, whereas the labeling in cord-intact rats seemed to be associated with the plasma membrane. Both the qualitative and quantitative differences in KCC2 immunolabeling between the three animal groups strongly support the observation that EIPSP was more depolarized in neonates and at the end of the first postnatal week after spinal cord transection.
The motoneurons recorded from cord-transected animals were less sensitive to the two experimental manipulations aimed at testing the functionality of the KCC2 system. Although bumetanide significantly depolarized EIPSP, the shift was less pronounced than in cord-intact animals. In addition, a reduction of [K+]o affected EIPSP significantly only in controls (Fig. 3). These results suggest a lower efficacy of the cotransport mechanisms in cord-transected animals, caused likely by fewer KCC2 transporters being present in the plasma membrane, as indicated by immunohistochemistry, and possibly an alteration of the functional status. Further experiments will be required to compare the regulation of KCC2 protein by phosphorylation/dephosphorylation events in cord-intact and cord-transected animals (Kelsch et al. 2001; Vale et al. 2003, 2005).
Afferent activity is important for the ontogenetic switch from depolarizing to hyperpolarizing glycine responses in the auditory brain stem (Shibata et al. 2004). The possibility that inputs from the brain stem and the neurotransmitters they contain (serotonin, noradrenaline,...) may play a similar critical role in the overall maturation of spinal cord inhibitory synaptic transmission, for instance, by up-regulating KCC2 and enabling it to become functional, is a hypothesis that deserves to be tested further. The first supraspinal projections reach the lumbar enlargement before birth (E17; Vinay et al. 2000, 2002), when EIPSP is around –50/–55 mV (Wu et al. 1992). The number of descending axons reaching the lumbar cord increases gradually until the end of the second postnatal week, when inhibitory synaptic transmission is fully mature. Whether the effect of descending pathways is direct, on intracellular events leading to the upregulaton of KCC2, or indirect, through a modulation of lumbar network activity, is unknown.
What are the functional consequences of an 
10-mV positive shift of EIPSP in cord-transected animals? Several studies performed on various regions of the CNS (Chen et al. 1996; Gao and van den Pol 2001; Gulledge and Stuart 2003; Stein and Nicoll 2003), including the spinal cord (Jean-Xavier et al. 2005) have shown that small depolarizing IPSPs (EIPSP 5–10 mV above VREST) can, depending on timing and location, promote action potential firing in response to subthreshold excitatory inputs. In addition, a shift of EIPSP toward more depolarized values causes a reduction of the inhibitory synaptic strength (Vale et al. 2003). The basic rhythmic activity underlying locomotion is generated by spinal cord interneuronal networks, termed central pattern generators (CPGs; Grillner and Wallén 1985). The alternation of muscle activities between the two hindlimbs relies on mutual glycinergic inhibition of the networks on the two sides of the cord (Butt et al. 2002; Hinckley et al. 2005; Kjaerulff and Kiehn 1997; Kremer and Lev-Tov 1997; Pflieger et al. 2002). We examined recently, in animals cord-transected at birth, the capacity of the CPGs to produce locomotor-like activity (Norreel et al. 2003). The excitability of CPGs was increased after the transection and the left/right alternating locomotor pattern was lost at P6–P7. How EIPSP develops in interneurons during the perinatal period and whether EIPSP is more depolarized also in interneurons after neonatal spinal cord transection are important questions that require further exploration. However, the more depolarized EIPSP observed in motoneurons and possibly in interneurons in cord-transected animals may be partly responsible for both of these observations.
The nonsignificant trend for EIPSP to be more positive in cord-transected animals at P4–P7 than at P0–P2 suggests that some dedifferentiation processes may also occur and raises the important question as to whether inhibition becomes excitatory again after a spinal cord injury in adults (Vinay et al. 2006), as shown in other pathological conditions such as peripheral nerve injury–induced neuropathic pain (Coull et al. 2003), axonal injury (Nabekura et al. 2002; Toyoda et al. 2003), and human temporal lobe epilepsy (Cohen et al. 2002).
In conclusion, we show that pathways descending from the brain stem are critical for inhibition to become and possibly to remain hyperpolarizing in lumbar motoneurons. These results shed light on a new upstream mechanism responsible for the developmental regulation of KCC2, a protein that plays a key role in both ontogeny and neurological diseases.
|
|
GRANTS |
|---|
|
|
|
ACKNOWLEDGMENTS |
|---|
|
|
|
FOOTNOTES |
|---|
Address for reprint requests and other correspondence: L. Vinay, CNRS, P3M, 31 Chemin Joseph Aiguier, F-13402 Marseille cx 20, France (E-mail: vinay{at}dpm.cnrs-mrs.fr)
|
|
REFERENCES |
|---|
|
Balakrishnan V, Becker M, Lohrke S, Nothwang HG, Guresir E, and Friauf E. Expression and function of chloride transporters during development of inhibitory neurotransmission in the auditory brainstem. J Neurosci 23: 4134–4145, 2003.
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]
Brocard F, Vinay L, and Clarac F. Gradual development of the ventral funiculus input to lumbar motoneurons in the neonatal rat. Neuroscience 90: 1543–1554, 1999.[CrossRef][Web of Science][Medline]
Butt SJ, Lebret JM, and Kiehn O. Organization of left-right coordination in the mammalian locomotor network. Brain Res Brain Res Rev 40: 107–117, 2002.[CrossRef][Medline]
Chen G, Trombley PQ, and van den Pol AN. Excitatory actions of GABA in developing rat hypothalamic neurones. J Physiol 494: 451–464, 1996.
Clayton GH, Owens GC, Wolff JS, and Smith RL. Ontogeny of cation-Cl- cotransporter expression in rat neocortex. Brain Res Dev Brain Res 109: 281–292, 1998.[Medline]
Cohen I, Navarro V, Clemenceau S, Baulac M, and Miles R. On the origin of interictal activity in human temporal lobe epilepsy in vitro. Science 298: 1418–1421, 2002.
Coull JA, Boudreau D, Bachand K, Prescott SA, Nault F, Sik A, De Koninck P, and De Koninck Y. Trans-synaptic shift in anion gradient in spinal lamina I neurons as a mechanism of neuropathic pain. Nature 424: 938–942, 2003.[CrossRef][Medline]
Curfs MH, Gribnau AA, and Dederen PJ. Postnatal maturation of the dendritic fields of motoneuron pools supplying flexor and extensor muscles of the distal forelimb in the rat. Development 117: 535–541, 1993.[Abstract]
Delpire E and Mount DB. Human and murine phenotypes associated with defects in cation-chloride cotransport. Annu Rev Physiol 64: 803–843, 2002.[CrossRef][Web of Science][Medline]
Dzhala VI, Talos DM, Sdrulla DA, Brumback AC, Mathews GC, Benke TA, Delpire E, Jensen FE, and Staley KJ. NKCC1 transporter facilitates seizures in the developing brain. Nat Med 11: 1205–1213, 2005.[CrossRef][Web of Science][Medline]
Ehrlich I, Lohrke S, and Friauf E. Shift from depolarizing to hyperpolarizing glycine action in rat auditory neurones is due to age-dependent Cl- regulation. J Physiol 520: 121–137, 1999.
Fiumelli H, Cancedda L, and Poo MM. Modulation of GABAergic transmission by activity via postsynaptic Ca2+-dependent regulation of KCC2 function. Neuron 48: 773–786, 2005.[CrossRef][Web of Science][Medline]
Gao B-X and Ziskind-Conhaim L. Development of glycine- and GABA-gated currents in rat spinal motoneurons. J Neurophysiol 74: 113–121, 1995.
Gao XB and van den Pol AN. GABA, not glutamate, a primary transmitter driving action potentials in developing hypothalamic neurons. J Neurophysiol 85: 425–434, 2001.
Grillner S and Wallén P. Central pattern generators for locomotion, with special reference to vertebrates. Annu Rev Neurosci 8: 233–261, 1985.[CrossRef][Web of Science][Medline]
Grob M and Mouginot D. Heterogeneous chloride homeostasis and GABA responses in the median preoptic nucleus of the rat. J Physiol 569: 885–901, 2005.
Gulledge AT and Stuart GJ. Excitatory actions of GABA in the cortex. Neuron 37: 299–309, 2003.[CrossRef][Web of Science][Medline]
Hinckley C, Seebach B, and Ziskind-Conhaim L. Distinct roles of glycinergic and GABAergic inhibition in coordinating locomotor-like rhythms in the neonatal mouse spinal cord. Neuroscience 131: 745–758, 2005.[CrossRef][Web of Science][Medline]
Hübner CA, Stein V, Hermans-Borgmeyer I, Meyer T, Ballanyi K, and Jentsch TJ. Disruption of KCC2 reveals an essential role of K-Cl cotransport already in early synaptic inhibition. Neuron 30: 515–524, 2001.[CrossRef][Web of Science][Medline]
Ikeda M, Toyoda H, Yamada J, Okabe A, Sato K, Hotta Y, and Fukuda A. Differential development of cation-chloride cotransporters and Cl- homeostasis contributes to differential GABAergic actions between developing rat visual cortex and dorsal lateral geniculate nucleus. Brain Res 984: 149–159, 2003.[CrossRef][Web of Science][Medline]
Jean-Xavier C, Cattaert D, and Vinay L. Excitatory actions of depolarizing inhibitory postsynaptic potentials in neonatal rat lumbar motoneurons. Soc Neurosci Abstr 516.13, 2005.
Jean-Xavier C, Pflieger J-F, and Vinay L. Inhibitory post-synaptic potentials in lumbar motoneurons remain depolarizing after neonatal spinal cord transection in the rat. Soc Neurosci Abstr 883.12, 2004.
Kakazu Y, Akaike N, Komiyama S, and Nabekura J. Regulation of intracellular chloride by cotransporters in developing lateral superior olive neurons. J Neurosci 19: 2843–2851, 1999.
Kelsch W, Hormuzdi S, Straube E, Lewen A, Monyer H, and Misgeld U. Insulin-like growth factor 1 and a cytosolic tyrosine kinase activate chloride outward transport during maturation of hippocampal neurons. J Neurosci 21: 8339–8347, 2001.
Kjaerulff O and Kiehn O. Crossed rhythmic synaptic input to motoneurons during selective activation of the contralateral spinal locomotor network. J Neurosci 17: 9433–9447, 1997.
Kremer E and Lev-Tov A. Localization of the spinal network associated with generation of hindlimb locomotion in the neonatal rat and organization of its transverse coupling system. J Neurophysiol 77: 1155–1170, 1997.
Kulik A, Nishimaru H, and Ballanyi K. Role of bicarbonate and chloride in GABA- and glycine-induced depolarization and [Ca2+]i rise in fetal rat motoneurons in situ. J Neurosci 20: 7905–7913, 2000.
Lakke EAJF. The projections to the spinal cord of the rat during development; a time-table of descent. Adv Anat Embryol Cell Biol 135: 1–143, 1997.
Li H, Tornberg J, Kaila K, Airaksinen MS, and Rivera C. Patterns of cation-chloride cotransporter expression during embryonic rodent CNS development. Eur J Neurosci 16: 2358–2370, 2002.[CrossRef][Web of Science][Medline]
Lohrke S, Srinivasan G, Oberhofer M, Doncheva E, and Friauf E. Shift from depolarizing to hyperpolarizing glycine action occurs at different perinatal ages in superior olivary complex nuclei. Eur J Neurosci 22: 2708–2722, 2005.[CrossRef][Web of Science][Medline]
Lu J, Karadsheh M, and Delpire E. Developmental regulation of the neuronal-specific isoform of K-Cl cotransporter KCC2 in postnatal rat brains. J Neurobiol 39: 558–568, 1999.[CrossRef][Web of Science][Medline]
Marchetti C, Pagnotta S, Donato R, and Nistri A. Inhibition of spinal or hypoglossal motoneurons of the newborn rat by glycine or GABA. Eur J Neurosci 15: 975–983, 2002.[CrossRef][Web of Science][Medline]
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. Proc Natl Acad Sci USA 102: 7344–7349, 2005.
Mikawa S, Wang C, Shu F, Wang T, Fukuda A, and Sato K. Developmental changes in KCC1, KCC2 and NKCC1 mRNAs in the rat cerebellum. Brain Res Dev Brain Res 136: 93–100, 2002.[Medline]
Nabekura J, Ueno T, Okabe A, Furuta A, Iwaki T, Shimizu-Okabe C, Fukuda A, and Akaike N. Reduction of KCC2 expression and GABAA receptor-mediated excitation after in vivo axonal injury. J Neurosci 22: 4412–4417, 2002.
Nishimaru H, Restrepo CE, Ryge J, Yanagawa Y, and Kiehn O. Mammalian motor neurons corelease glutamate and acetylcholine at central synapses. Proc Natl Acad Sci USA 102: 5245–5249, 2005.
Norreel J-C, Pflieger J-F, Pearlstein E, Simeoni-Alias J, Clarac F, and Vinay L. Reversible disorganization of the locomotor pattern after neonatal spinal cord transection in the rat. J Neurosci 23: 1924–1932, 2003.
Owens DF, Boyce LH, Davis MB, and Kriegstein AR. Excitatory GABA responses in embryonic and neonatal cortical slices demonstrated by gramicidin perforated-patch recordings and calcium imaging. J Neurosci 16: 6414–6423, 1996.
Payne JA. Functional characterization of the neuronal-specific K-Cl cotransporter: implications for [K+]o regulation. Am J Physiol 273: C1516–C1525, 1997.
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]
Pflieger JF, Clarac F, and Vinay L. Picrotoxin and bicuculline have different effects on lumbar spinal networks and motoneurons in the neonatal rat. Brain Res 935: 81–86, 2002.[CrossRef][Web of Science][Medline]
Pinco M and Lev-Tov A. Synaptic transmission between ventrolateral funiculus axons and lumbar motoneurons in the isolated spinal cord of the neonatal rat. J Neurophysiol 72: 2406–2419, 1994.
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]
Rivera C, Voipio J, and Kaila K. Two developmental switches in GABAergic signalling: the K-Cl cotransporter KCC2, and carbonic anhydrase CAVII. J Physiol 562: 27–36, 2004a.
Rivera C, Voipio J, Payne JA, Ruusuvuori E, Lahtinen H, Lamsa K, Pirvola U, Saarma M, and Kaila K. The K+/Cl- co-transporter KCC2 renders GABA hyperpolarizing during neuronal maturation. Nature 397: 251–255, 1999.[CrossRef][Medline]
Rivera C, Voipio J, Thomas-Crusells J, Li H, Emri Z, Sipila S, Payne JA, Minichiello L, Saarma M, and Kaila K. Mechanism of activity-dependent downregulation of the neuron-specific K-Cl cotransporter KCC2. J Neurosci 24: 4683–4691, 2004b.
Schneider SP and Fyffe RE. Involvement of GABA and glycine in recurrent inhibition of spinal motoneurons. J Neurophysiol 68: 397–406, 1992.
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]
Stein V, Hermans-Borgmeyer I, Jentsch TJ, and Hubner CA. Expression of the KCl cotransporter KCC2 parallels neuronal maturation and the emergence of low intracellular chloride. J Comp Neurol 468: 57–64, 2004.[CrossRef][Web of Science][Medline]
Stein V and Nicoll RA. GABA generates excitement. Neuron 37: 375–378, 2003.[CrossRef][Web of Science][Medline]
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.
Takahashi T. Inhibitory miniature synaptic potentials in rat motoneurons. Proc R Soc Lond B Biol Sci 221: 103–109, 1984.[Medline]
Thompson SM and Gahwiler BH. Activity-dependent disinhibition. II. Effects of extracellular potassium, furosemide, and membrane potential on ECl- in hippocampal CA3 neurons. J Neurophysiol 61: 512–523, 1989.
Toyoda H, Ohno K, Yamada J, Ikeda M, Okabe A, Sato K, Hashimoto K, and Fukuda A. Induction of NMDA and GABAA receptor-mediated Ca2+ oscillations with KCC2 mRNA downregulation in injured facial motoneurons. J Neurophysiol 89: 1353–1362, 2003.
Vale C, Caminos E, Martinez-Galan JR, and Juiz JM. Expression and developmental regulation of the K+-Cl- cotransporter KCC2 in the cochlear nucleus. Hear Res 206: 107–115, 2005.[CrossRef][Web of Science][Medline]
Vale C, Schoorlemmer J, and Sanes DH. Deafness disrupts chloride transporter function and inhibitory synaptic transmission. J Neurosci 23: 7516–7524, 2003.
Vardi N, Zhang LL, Payne JA, and Sterling P. Evidence that different cation chloride cotransporters in retinal neurons allow opposite responses to GABA. J Neurosci 20: 7657–7663, 2000.
Vinay L, Brocard F, Clarac F, Norreel JC, Pearlstein E, and Pflieger JF. Development of posture and locomotion: an interplay of endogenously generated activities and neurotrophic actions by descending pathways. Brain Res Rev 40: 118–129, 2002.[CrossRef][Medline]
Vinay L, Brocard F, Pflieger J, Simeoni-Alias J, and Clarac F. Perinatal development of lumbar motoneurons and their inputs in the rat. Brain Res Bull 53: 635–647, 2000.[CrossRef][Web of Science][Medline]
Vinay L, Jean-Xavier C, Darbon P, and Liabeuf S. Alteration of chloride homeostasis in lumbar motoneurons after spinal cord transection in the rat. Soc Neurosci Abstr 2006.
Wang C, Ohno K, Furukawa T, Ueki T, Ikeda M, Fukuda A, and Sato K. Differential expression of KCC2 accounts for the differential GABA responses between relay and intrinsic neurons in the early postnatal rat olfactory bulb. Eur J Neurosci 21: 1449–1455, 2005.[CrossRef][Web of Science][Medline]
Wang C, Shimizu-Okabe C, Watanabe K, Okabe A, Matsuzaki H, Ogawa T, Mori N, Fukuda A, and Sato K. Developmental changes in KCC1, KCC2, and NKCC1 mRNA expressions in the rat brain. Brain Res Dev Brain Res 139: 59–66, 2002.[Medline]
Woodin MA, Ganguly K, and Poo MM. Coincident pre- and postsynaptic activity modifies GABAergic synapses by postsynaptic changes in Cl- transporter activity. Neuron 39: 807–820, 2003.[CrossRef][Web of Science][Medline]
Wu W-L, Ziskind-Conhaim L, and Sweet MA. Early development of glycine- and GABA-mediated synapses in rat spinal cord. J Neurosci 12: 3935–3945, 1992.[Abstract]
Yamada J, Okabe A, Toyoda H, Kilb W, Luhmann HJ, and Fukuda A. Cl- uptake promoting depolarizing GABA actions in immature rat neocortical neurones is mediated by NKCC1. J Physiol 557: 829–841, 2004.
Yan Y, Dempsey RJ, and Sun D. Expression of Na(+)-K(+)-Cl(-) cotransporter in rat brain during development and its localization in mature astrocytes. Brain Res 911: 43–55, 2001.[CrossRef][Web of Science][Medline]
Zhang L, Spigelman I, and Carlen PL. Development of GABA-mediated, chloride-dependent inhibition in CA1 pyramidal neurones of immature rat hippocampal slices. J Physiol 444: 25–49, 1991.
Zhu L, Lovinger D, and Delpire E. Cortical neurons lacking KCC2 expression show impaired regulation of intracellular chloride. J Neurophysiol 93: 1557–1568, 2005.
Ziskind-Conhaim L. Physiological functions of GABA-induced depolarizations in the developing rat spinal cord. Perspect Dev Neurobiol 5: 279–287, 1998.[Web of Science][Medline]
This article has been cited by other articles:
|
|
 |
|
  A. Sakurai and P. S. Katz Functional Recovery after Lesion of a Central Pattern Generator J. Neurosci., October 21, 2009; 29(42): 13115 - 13125. [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]
|
|
|
|
 |
|
 Y. Lu, J. Zheng, L. Xiong, M. Zimmermann, and J. Yang Spinal cord injury-induced attenuation of GABAergic inhibition in spinal dorsal horn circuits is associated with down-regulation of the chloride transporter KCC2 in rat J. Physiol., December 1, 2008; 586(23): 5701 - 5715. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Delpy, A.-E. Allain, P. Meyrand, and P. Branchereau NKCC1 cotransporter inactivation underlies embryonic development of chloride-mediated inhibition in mouse spinal motoneuron J. Physiol., February 15, 2008; 586(4): 1059 - 1075. [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]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
TOP
ABSTRACT
INTRODUCTION
