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1
ek21Institute of Biology II, Faculty of Biosciences, Pharmacy and Psychology, University of Leipzig, Leipzig, Germany; and 2Institute of Experimental Biomedicine, Academy of Sciences, Prague, Czech Republic
Submitted 30 October 2006; accepted in final form 27 June 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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). These large neurons integrate excitatory inputs from few auditory nerve terminals (endbulbs of Held) (Brawer and Morest 1975; Ryugo and Sento 1991) with acoustically driven, GABA- and glycine-mediated inhibition (Kopp-Scheinpflug et al. 2002). The inhibitory inputs are provided by nonprimary innervation from sources including the dorsal cochlear nucleus, the superior olivary complex, and the contralateral cochlear nucleus (Wenthold 1991), and they appear to be glycinergic, glycine and GABAergic, and GABAergic only (Altschuler et al. 1993; Juiz et al. 1996). Depolarizing GABAergic and/or glycinergic responses were described as a transient feature of immature mammalian auditory brain stem (Ehrlich et al. 1999; Kakazu et al. 1999; Kandler and Friauf 1995; Löhrke et al. 2005), hippocampus (Ben-Ari et al. 1989; Cherubini et al. 1990), neocortex (Owens et al. 1996; Yuste and Katz 1991), cerebellum (Eilers et al. 2001), cultured midbrain neurons (Jarolimek et al. 1999; Titz et al. 2003), and retina (Huang and Redburn 1996). The excitatory responses are due to the elevated intracellular Cl– concentration in the immature neurons; this is attributable to the prolonged postnatal development of the chloride homeostasis system (for review, see Ben-Ari 2002).
The K+-Cl–-extruding cotransporter KCC2 is expressed exclusively in neurons (Payne et al. 1996) and its upregulation is believed to underlie the functional switch from excitatory to inhibitory action of GABA (Owens and Kriegstein 2002; Rivera et al. 1999). Studies of the chick nucleus magnocellularis (NM; avian homologue to the CN) suggested that GABAA receptor-mediated synaptic responses remain depolarizing throughout the animal's life span due to the outward-directed electrochemical gradient for Cl– at the resting membrane potential. However, depolarizing GABA-evoked responses prevent NM neurons from producing action potentials in developing as well as in mature auditory brain stem (Hyson et al. 1995; Lu and Trussell 2001; Monsivais and Rubel 2001).
GABAA receptors are primarily permeable to Cl–, yet also to HCO3– (relative permeability ca. 0.2) (Bormann et al. 1987; Kaila 1994), and a detailed characterization of the impact that GABA has on the membrane current flow must consider the actual intracellular anion concentration. In the present developmental study, we used the gramicidin-perforated-patch recordings because the membrane pores formed by gramicidin are exclusively permeable to monovalent cations and small, uncharged molecules, leaving the [Cl–]i undisturbed (Akaike 1996; Kyrozis and Reichling 1995). We determined the reversal potential of the SBC responses to GABA through the early postnatal development, up to the time of hearing onset (P12) (Woolf and Ryan 1984) and studied the pattern of KCC2 expression from neonatal to subadult ages. Our results reveal a rather early (at P1) expression of the KCC2 protein in CN. However, the indications of its activity temporally correlate with hyperpolarizing GABAA-mediated responses observed toward the end of the first postnatal week.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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This study was performed in the Neurobiology Laboratories of the Institute of Biology II at University of Leipzig. The Western blot analyses were done in the Neurophysiology Laboratories of the Paul-Flechsig Institute for Brain Research, University of Leipzig. All experimental procedures were approved by the Saxonian district Government, Leipzig. For experimental purposes, we used pigmented (agouti) Mongolian gerbils (Meriones unguiculatus) aging postnatal day one to 30 (P1-30), obtained from the institutes animal care facilities.
Slice preparation
Acute brain stem slices (200 µm) containing the rostral pole of the anteroventral cochlear nucleus (AVCN) were acquired from P3–P12 gerbils using vibratome (Microm HM 650, Walldorf, Germany) as previously described by Oertel (1983). The cold (3–4°C) preparation solution used for cutting contained (in mM) 125 NaCl, 2.5 KCl, 0.1 CaCl2, 3 MgCl2, 1.25 NaH2PO4, 25 NaHCO3, 25 glucose, 2 sodium pyruvate, 3 myo-inositol, and 0.5 ascorbic acid, continuously equilibrated with 5% CO2-95% O2, pH 7.4. The slices were preincubated for 30 min at 37°C and stored at room temperature (RT) until recording in extracellular solution (ACSF) of the following composition (in mM) 125 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 1.25 NaH2PO4, 25 NaHCO3, 10 glucose, 2 sodium pyruvate, 3 myo-inositol, and 0.5 ascorbic acid, pH 7.4 when equilibrated with 5% CO2-95% O2 mixture. For recordings in which ASCF was exchanged with HEPES extracellular solution, sodium bicarbonate was substituted by 20 mM HEPES, NaCl increased to 135 mM, NaH2PO4 was omitted, and pH was adjusted to 7.4 with NaOH. Slices were transferred to a recording chamber (1 ml volume) mounted on the stage of an upright microscope (Axioskop 2, Zeiss, Germany) and continuously perfused at the rate of 2 ml/min with the extracellular solution. All experiments were performed at RT (21–23°C).
Electrophysiology
Patch pipettes were pulled from filamented borosilicate glass capillaries (Harvard Apparatus, Edenbridge, UK) to have resistances of 3–6 M
for whole cell- and 5–6 M for gramicidin-perforated recordings. To measure EGABA in whole cell recordings, pipette solutions with two different Cl– concentrations were used: high [Cl–]i (in mM) 140 CsCl, 5 EGTA, and 10 HEPES, pH 7.3 with CsOH (280mosM); low [Cl–]i (in mM) 125 CsMeSO3, 15 CsCl, 1 MgCl2, 5 EGTA, and 10 HEPES, pH 7.3 with CsOH (286 mosM). Because it was previously suggested that Cs+ ions could interfere with Cl–-extruding capacity of KCC2 (Kakazu et al. 1999, 2000), K+-based internal solutions were used for perforated-patch recordings. Pipettes were tip-filled with (in mM) 140 KCl and 10 HEPES, pH 7.3 with KOH (280 mOsm) or with solution in which 140 mM KCl was substituted by 145 mM K+-gluconate and 5 mM KCl (283 mOsm). The remainder of the pipette was back-filled with the same K+-based solution including gramicidin (gramicidin A, Sigma, Deisenhofen, Germany). A 10 mg/ml stock solution of gramicidin was freshly prepared in dimethylsulfoxide, sonicated, and diluted to a final concentration of 50 µg/ml just prior to experiment.
Membrane currents and membrane potentials were recorded using a single-electrode amplifier (npi electronic, Tamm, Germany) in bridge, discontinuous current- or voltage-clamp mode. Switching frequency was 20 kHz, and signals were filtered at 1 kHz and digitized at 2–5 kHz using npi electronic hardware and software (Cell Works 5.0). Data analysis was performed with pClamp 9.0 software (Axon Instruments, Union City, CA). In gramicidin-perforated-patch recordings, the access resistance was monitored with a –10 mV pulse, which typically reached a steady level within 30 min after the giga-seal formation (>5 G). Voltages were corrected off-line for junction potentials as follows: –8.8 mV (CsMeSO3) and –12 mV (K-gluconate-based pipette solution). During voltage-clamp recordings, extracellular solution was supplemented with 0.3 µM TTX and 25 µM CGP 4638 (both Tocris, Bristol, UK) to block Na+-dependent action potentials and to antagonize GABAB receptors, respectively. GABA (100 µM; Sigma) and muscimol (10 µM; Tocris) solutions were prepared in HCO3–-containing extracellular solution and pressure applied (5 psi, 50 ms) over the soma of the recorded neuron through a wider-tip patch pipette mounted on a Picospritzer (General Valve, Fairfield, NJ). Although the application of agonists via a puff pipette slightly dilutes the antagonist when it is presented in the bath, the blocking effects were efficient with low antagonist concentration (Fig. 1). Recorded cells were characterized as SBC according to their firing of a single action potential at the start of depolarizing current step (Wu and Oertel 1984).
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2-min interval between applications because it was previously shown not to cause the shifts in the [Cl–]i in similar experiments (Ehrlich et al. 1999). Step voltages Vhold ranged from –60 to 40 mV for CsCl-filled electrodes, from –89 to 11 mV for Cs-methanesulphonate, from –92 to –32 mV for K-gluconate, and from –90 to –30 mV for KCl-filled pipettes. Fitting of the I-V relations was calculated with linear regressions and EGABA was determined as the voltage at the zero current. Vrest was estimated as the mean potential of the cell at the zero current in gramicidin-perforated-patch configuration. Immunohistochemistry
Gerbils were killed with sodium pentobarbitol (10 mg/kg body wt, ip), and the tissue was fixed through transcardial perfusion with 4% paraformaldehyde in 0.1 M phosphate buffer. The brains (3–4 for each age investigated) were removed and kept in a fixative for 4 h. For cryoprotection, the brains were kept in 30% sucrose in 0.1 M phosphate buffer until they sank. The brains were shock-frozen to –70°C, and coronal brain stem sections (16 µm) were obtained by means of cryocut. Brain sections were washed thoroughly with PBS and PBS/0.3% Triton/1% DMSO solutions. After blocking of nonspecific binding sites with 5% normal goat serum (NGS) in PBS/0.3% Triton/1% DMSO (30 min at 37°C), the specimens containing AVCN were incubated overnight with polyclonal anti-KCC2 antibody (raised against residues 932-1,043 of the rat KCC2; catalog No. 07–432; Upstate, Lake Placid, NY; 1:200 in blocking solution at 4°C). The antibody corresponds to the antibody published previously by Williams et al. (1999). After washing the specimens with PBS/0.3% Triton/1% DMSO, the secondary goat anti-rabbit Cy2-tagged antibody (20 µg/ml in blocking solution; Jackson Immunoresearch Lab. Dianova, Hamburg, Germany) was incubated for 2.5 h at RT. In animals of younger stages, additional counterlabeling of all cell nuclei was performed with Hoechst 33258 (1:1,000 in PBS, 30 min at RT; Molecular Probes, Leiden, Holland). After rinsing with PBS and dH2O, the sections were dehydrated in xylol, dried, and coverslipped with entellan. Additional staining of large presynaptic calyceal inputs (endbulbs of Held), which terminate at somatas of SBC, was performed with goat anti-calretinin antibody (1:1,000 in blocking solution; Swant, Bellinzona, Switzerland) as previously published (Bazwinsky and Rübsamen 2000; Härtig et al. 2001). This step was conducted to visualize the postsynaptic localization of KCC2 in SBC, the signal of which underlied the calretinin labeling. For this purpose, Cy3-conjugated donkey anti-goat secondary antibody (20 µg/ml in blocking solution) was used. Each cytochemical procedure was controlled by the omission of primary antibodies and the subsequent identical processing of few sections causing the lack of marked structures. Additionally, the fluorophores related to the relevant markers were switched; for example, anti-KCC2 was also revealed by donkey anti-rabbit Cy3 and calretinin by donkey anti-goat Cy2 antibodies. The images were acquired using a confocal laserscanning microscope (LSM 510, Zeiss, Germany).
Western blots
Two experimental approaches were performed for Western blot analysis. First, we took advantage of a clear demarcation of the cochlear nucleus in the dorsolateral brain stem by the posterior cerebellar peduncle; this enabled us to exclusively dissect this nucleus in P1, P5, P9, P14, and P30 gerbils. Whole cell lysates were prepared from this tissue by using the Mammalian Cell Lysis-1 Kit containing protease inhibitors I and II (Sigma, Deisenhofen, Germany). After the homogenization with a Teflon homogenizer (on ice), the lysate was centrifuged at 10,000 g for 10 min, and the protein concentration of the supernatant was determined. Second, membrane-enriched fractions were isolated from the cerebellum, cortex, brain stem, and dissected cochlear nuclei as previously described (Williams et al. 1999). Briefly, dissected tissue was transferred into the homogenization buffer [250 mM sucrose, 10 mM Tris, 10 mM HEPES, 1 mM EDTA, protease inhibitor cocktail (Roche Products); pH adjusted to 7.2] and homogenized in a glass-Teflon homogenizer with 10 strokes. The homogenate was then centrifuged at 10,000 rpm for 10 min at 4°C (Sorval Ultraspeed) and the resulting supernatant was centrifuged again at 20,000 rpm for 30 min at 4°C. The final pellet was resuspended in 
100–500 µl of homogenization buffer, and the protein concentration of the obtained membrane-enriched solution determined. Equal amounts of proteins for each developmental stage and structure (20 µg), previously determined using the Bio-Rad protein assay (Bio Rad, München, Germany) and adjusted to 2 µg/µl, were electrophoretically separated by SDS-polyacrylamide gel electrophoresis (8% resolving gel; Mini Protean 3, Bio Rad) in a Rotiphorese SDS tank buffer (Roth, Karlsruhe, Germany). After transferring the proteins to the nitrocellulose membrane (Mini Trans-Blot, Bio Rad) and blocking unspecific binding sites (2 h in 10% Roti-Block solution, Roth), blots were incubated with rabbit anti-KCC2 antibody (1:1000 in TBS/0.2% Tween-20/10% Roti-Block, overnight at 4°C). The blots were washed 4 x 5min with TBS/0.2% Tween-20 and incubated with alkaline phosphatase-conjugated goat anti-rabbit secondary antibody (1:5,000 in TBS/0.2% Tween-20/10% Roti-Block, 2 h at RT; Sigma). The immunoreactivity was visualized using Sigma Fastblue alkaline phosphatase substrate kit. It revealed a single immunoreactive band between 105 and 160 kDa protein standards, which is consistent with the KCC2 protein (Lu et al. 1999; Williams et al. 1999).
KCC2 expression was previously investigated in membrane-fraction enriched preparations of cortex, hippocampus, cerebellum, brain stem, and retina (Balakrishnan et al. 2003; Stein et al. 2004; Vu et al. 2000; Williams et al. 1999). For the sake of comparison, we performed the analysis of membranes obtained from cortex, cerebellum, brain stem, and cochlear nucleus to the whole cell lysates obtained from cochlear nucleus. This procedure revealed no qualitative difference in the KCC2 staining and was performed because of the low amount of proteins gained after the isolation of the membrane fraction from the cochlear nucleus.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Whole cell patch-clamp recordings were performed to investigate the characteristics of the GABA-induced responses. Focally applied GABA or muscimol evoked inward currents at Vhold of –60 mV and [Cl–]i biased to 140 mM by the patch pipette. The GABA- or muscimol-elicited currents were blocked by 96 ± 1% (n = 8) or by 94 ± 2% (n = 5), respectively, by a bath application of SR95531, a GABAA receptor antagonist (Fig. 1). Furthermore, GABA and muscimol-evoked large ionic currents reversed close to the equilibrium potential predicted for Cl– (Fig. 2). Reversal potentials for IGABA (Fig. 2, A and B) and Imuscimol (C and D) were consistent with activation of Cl– channels (EGABA = 4.8 ± 1.7 mV, n = 9; Emuscimol = 5.3 ± 1.6 mV, n = 7; Nernst potential for Cl– = 1.2 mV) and were independent of age (data not shown). Finally, dependence of the GABAA receptor-mediated currents on [Cl–]i was further exemplified with a low Cl– (17 mM) pipette solution, which yielded a shift in EGABA to –49.2 ± 0.8 mV (n = 7), which is in accordance with the Nernst prediction of –52.6 mV (Fig. 2, E and F).
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; Jones et al. 1998). EGABA in developing SBC
To examine the influence of brief GABA applications on the excitability of SBC with intact [Cl–]i, gramicidin-perforated-patch recordings were performed on brain stem slices containing the rostral pole of the AVCN and isolated from P3 to P12 gerbils. This period was previously shown to be critical for a shift from glycinergic depolarization to hyperpolarization in the superior olivary complex nuclei (SOC) in mammalian brain stem (Kandler and Friauf 1995; Löhrke et al. 2005). Taking advantage of the cation-specific pore-forming agent gramicidin (Akaike 1996; Kyrozis and Reichling 1995), we were able to observe a developmental change in responses of SBC to GABA. As shown in Fig. 3 A, pressure ejection of GABA to the somatic region produced depolarizing responses from rest in P3 and P4 SBC. However, already at P4, GABA exerted hyperpolarization in three of six SBC recorded. GABA-induced hyperpolarization observed between P5 and P8 were followed by a slower depolarization tail. These biphasic responses disappeared by P9, and thereafter the repolarizing phase of the GABA-evoked hyperpolarizations became faster with maturity [
= 0.9 ± 0.5 s for P3-5 (n = 8); = 0.5 ± 0.1 s for P10-12 (n = 12)]. These data indicate that the developmental shift in the action of GABA on SBC temporally correlates to the depolarizing-hyperpolarizing change in the glycine action on the rat lateral superior olive (LSO) and medial superior olive (MSO) neurons (Ehrlich et al. 1999; Löhrke et al. 2005). Biphasic responses as seen in Fig. 3 (recordings were done in bicarbonate buffer) can be explained by a depolarizing gradient for HCO3-, which shapes the late phase of the GABA response when the driving force for Cl– collapses (Cordero-Erausquin et al. 2005). In those cells, in which biphasic responses to GABA were recorded under the voltage clamp, we addressed the possibility of the initial outward component representing the Cl– flux followed by an inward component dominated by HCO3- efflux. Comparable observations were reported in neurons of the hippocampus (Kaila et al. 1997; Staley et al. 1995), in spinal dorsal horn neurons (Coredero-Erausquin et al. 2005), and in the LSO neurons (Ehrlich et al. 1999). As shown in Fig. 3Ba, the slow inward tail of the biphasic response, recorded at a Vhold slightly depolarized with respect to ECl-, was abolished in HEPES-buffered extracellular solution and the remaining outward current represents the Cl– flux. Moreover, the biphasic response observed in bicarbonate buffer was reverted to a monophasic course by shortening the application time of GABA (Fig. 3Bb), most likely due to changes in concentrations of Cl– and HCO3– ions permeating through GABAA channel during the response (Cordero-Erausquin et al. 2005; Staley and Proctor 1999).
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; Kaila et al. 1997; Staley et al. 1995). Comparison of Vrest, estimated in current-clamp mode and of EGABA in the respective neurons (acquired as the interpolated zero current value in I-V relations) revealed that in 56% SBC at age P3-5 (n = 18), EGABA was depolarized with respect to the Vrest (Fig. 4B). On the other hand, only in 1 from 12 (8%) P7 SBC EGABA was found to be depolarized. Hyperpolarizing action of GABA was observed in all SBC from P10 (n = 4), P11 (n = 4), and P12 groups (n = 4; Fig. 4B). Figure 5 clearly demonstrates the significant shift in mean values for EGABA between developmental stages [P3-5: EGABA = –41.7 ± 2.2 mV (n = 18); P7-9: EGABA = –64.9 ± 2.0 mV (n = 30); P10-12: EGABA = –79.0 ± 3.3 mV (n = 12)]. Furthermore, from 14 SBC investigated at P8 in gramicidin-perforated-patch configuration, we recorded seven neurons with 140 mM Cl– and seven neurons with 5 mM Cl– in the pipette solution. The respective mean values for EGABA (low [Cl–]pip –67.9 ± 1.2 mV; high [Cl–]pip –71.2 ± 1.9 mV; P > 0.05) did not differ between the two experimental approaches, suggesting that our recordings were independent of the Cl– in the pipette and that gramicidin ionophores are impermeable to Cl–.
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; Fig. 5]. This observation is consistent with the notion of depolarized neuronal membrane potential being a general feature of immaturity (hippocampus: Cherubini et al. 1990; neocortex: Luhmann and Prince 1991; McCormick and Prince 1987; Owens et al. 1996; LSO: Kakazu et al. 1999). Taken together, the data in Fig. 5 demonstrate the change from mainly depolarizing responses to GABA in SBC up to P5 to the hyperpolarizing responses after P7, a change that is likely due to a decrease in [Cl–]i during development.
Furosemide- and DIOA-sensitive cotransporter is involved in Cl– removal from SBC
The preceding results suggest that by the end of the first postnatal week [Cl–]i in most SBC is lower than it would be predicted if chloride was passively distributed across the membrane. The cation-chloride cotransporter KCC2 is well established as the key factor in Cl– extrusion, and its developmentally regulated activity causes a shift from depolarizing to hyperpolarizing action of GABA in diverse neurons (reviewed in Ben-Ari 2002; Rivera et al. 2005). Consistent with this idea, blocking of functionally active KCC2 by the loop diuretic furosemide (Ehrlich et al. 1999; Jarolimek et al. 1999; Kakazu et al. 1999; Martina et al. 2001) or by [(dihydroindenyl)oxy] alkanoic acid (DIOA) (Coull et al. 2003; Garay et al. 1988) was shown to reverse EGABA or EGly toward more depolarized values. To assess the functional role of KCC2 in chloride homeostasis of SBC, we determined the EGABA for each neuron, then challenged the KCC2 activity by a 20 min bath application of 100 µM furosemide or 50 µM DIOA and thereafter determined EGABA again. As illustrated in Fig. 6, A and B, for a representative recording in a P11 SBC, the otherwise outward IGABA was reversed in the presence of furosemide. Because we aimed at investigation of the effects of KCC2 inhibition on the Cl– extrusion capacity, it was necessary to carefully determine ECl- before and after the application of the blockers. Therefore only the cells showing monophasic responses to GABA (17 of 21 neurons recorded) were analyzed and represented in Fig. 6. The peak amplitudes of the currents, recorded at different holding potentials, were measured and plotted in Fig. 6C revealing a furosemide-induced shift in EGABA by 13 mV in a P11 SBC. To avoid the loading of the cells with Cl– by a repetitive activation of Cl– conductance, which could mimic the effect of KCC2 inhibitors in our in vitro system, we reverted to the voltage protocol established by Ehrlich et al. (1999) and later used by Löhrke et al. (2005), for similar experiments performed in SOC. In three recorded neurons at P11, furosemide caused a change of EGABA toward more depolarized values (from –75.9 ± 5.9 to –60.7 ± 3.2 mV; P < 0.05). Besides interrupting the net outward Cl– transport in older neurons, furosemide was also shown to perturb Cl– accumulation mechanisms in immature neurons (Thompson and Gähwiler 1989; Owens et al. 1996; Jarolimek et al. 1999), but given the maturity of SBC, our finding is consistent with the activity of the furosemide-sensitive Cl– extrusion mechanism. This notion is further supported by an effect of DIOA (Fig. 6D), a more specific KCC2 antagonist, which in P11 SBC caused a significant positive shift in EGABA by 15.1 ± 2.1 mV, P < 0.01 (n = 3); in P7-9 SBC, the shift was 12.5 ± 3.0 mV, P < 0.05 (n = 6); but no significant change in EGABA was observed at P3 [control: –33.2 ± 6.0 mV vs. DIOA: –29.0 ± 4.9 mV (n = 4); paired Student's t-test]. These data imply functional alterations regarding the KCC2 activity between P3 and P7-9 SBC.
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To assess the developmental pattern of the KCC2 protein expression in AVCN neurons, we performed immunohistochemical and Western blot studies. Immunohistochemical staining was conducted on three to four brains per age group and revealed abundant KCC2 protein expression in the AVCN already at P1 (Fig. 7 A). The KCC2 immunoreactivity between P1 and P8 was present in areas which are not labeled by the Hoechst dye, which marks the cell nuclei. The KCC2 signal appeared to be restricted to neuropil at P1 (Fig. 7A). Similar observations were reported for the rat SOC (Balakrishnan et al. 2003; Blaesse et al. 2006; Löhrke et al. 2005) and hippocampal neurons at P0 (Gulyas et al. 2001). As illustrated in Fig. 7, B–F, the staining pattern changed from P5 to P9 with the perisomatic and peridendritic immunoreactivity becoming more pronounced. Figure 7, F–H, indicates that the distribution of KCC2 reaches an "adult-like" pattern at P9, characterized by weak cytoplasm labeling, strong perisomatic, proximal and, possibly distal dendrite labeling. Additionally, the postsynaptic KCC2 immunoreactivity was confirmed by labeling of presynaptic endings (enbulbs of Held) with calretinin (Bazwinsky and Rübsamen 2000), revealing thick labeling pattern around the somata of SBC (Fig. 7I).
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and Stein et al. (2004), a strong immunoreactivity was restricted to a 140-kDa band. These authors used the membrane-enriched fraction from tissue suspension, whereas our preparations were made from the whole cell lysates (due to the small size of the cochlear nucleus). Therefore we additionally compared our data to Western blots of membrane fractions isolated from cerebellum, cortex, brain stem, and dissected cochlear nuclei. A closer inspection of the data in Fig. 8B showed no qualitative difference between the bands obtained from the two different preparations. Together, immunohistochemical and Western blot data strongly corroborate the notion that the KCC2 is expressed in SBC before GABA attains its hyperpolarizing action in these neurons.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Inhibitory neurotransmission in cochlear nucleus of mammals and birds
Despite the independent evolution of the neuronal brain stem system for processing of interaural time differences (ITDs) in birds and mammals (reviewed in Grothe 2003), there are striking similarities in the organization of the auditory brain stem nuclei between the two homeothermic vertebrates with respect to monaural and binaural signal processing in second- and third-order neurons (reviewed in Oertel 1999). Still there are differences in how inhibition is linked into this system.
In the avian NM, GABAergic terminals were found on neuron somata, and GABAergic inhibition originates from intrinsic neurons and from neurons in the superior olivary nucleus (SON) (Carr et al. 1989; Lachica et al. 1994; Yang et al. 1999). The SON seems to serve comparable functions as the mammalian periolivary nuclei. The fact is that glycine is hardly present in the auditory brain stem of birds (Code and Rubel 1989), whereas in the mammalian cochlear nucleus most nonprimary synapses contain GABA, glycine, or colocalize both transmitters (Juiz et al. 1996; Mahendrasingam et al. 2004; Oberdorfer et al. 1988; Saint Marie et al. 1989). Correspondingly, both GABAA and glycine receptors were found at the membranes of bushy cell somata (Juiz et al. 1989, 1994; Lim et al. 2000; Wenthold et al. 1988). GABAergic and glycinergic projections arise bilaterally from nuclei of the superior olivary complex and from the medial nucleus of the trapezoid body (Benson and Potashner 1990; Ostapoff et al. 1990, 1997), whereas inhibitory projections originating from the ipsilateral dorsal cochlear nucleus and from contralateral CN are exclusively glycinergic (Wenthold 1987; Wickesberg and Oertel 1990). Glycinergic synaptic activity was demonstrated in several in vitro studies in the rat AVCN (P12-16) (Lim et al. 1999, 2000, 2003). In vivo electrophysiology using acoustic stimulation reported significant effects of both GABAergic and glycinergic signal transmission on the precisely timed electrical signaling of bushy cells (Backoff et al. 1997, 1999; Caspary et al. 1994; Kopp-Scheinpflug et al. 2002). Recordings of spontaneous and stimulus-evoked currents before and after the onset of hearing, suggest functional synaptic inputs on SBC mediated through both GABAA and glycine receptors (unpublished data).
In the avian nucleus laminaris neurons, GABAergic inhibition is achieved by membrane depolarization throughout the life span (Funabiki et al. 1998; Yang et al. 1999), whereas in mammals, both MNTB and SOC neurons show a shift from depolarizing to hyperpolarizing gradients for Cl– before the onset of hearing (Löhrke et al. 2005). To get a better understanding of the development of GABAergic inhibition in the cochlear nucleus, we tracked the postnatal changes in responses of SBC. In the chick, the GABAA-receptor-mediated responses inhibit NM neurons via a depolarizing shunting inhibition (Hyson et al. 1995; Monsivais and Rubel 2001; Lu and Trussell 2001), yet in the gerbil GABA hyperpolarizes SBC by the end of the first postnatal week. This is consistent with the recent data by Price and Trussell (2006), who recorded hyperpolarizing responses to glycine from rat globular bushy cells at P9-11. The present study shows the time course of maturation of inhibitory neurotransmission in mammalian anteroventral cochlear nucleus and provides evidence on the underlying cellular mechanisms causing a shift from depolarization to hyperpolarization before the onset of hearing.
Hyperpolarizing GABA responses correlate with the activity of KCC2
The neuron-specific K+-Cl–-cotransporter KCC2 was proposed as the key molecule to render low [Cl–]i during development in various CNS regions and, as a result, to gradually convert depolarizing and excitatory GABA responses to the well-established hyperpolarizing inhibition seen in the adult (Rivera et al. 2005). In the neocortex, hippocampus, and retinal neurons of rats, KCC2 expression increases postnatally up to the end of the second postnatal week, a period during which EGABA becomes hyperpolarizing with respect to Vm (Gulyas et al. 2001; Lu et al. 1999; Rivera et al. 1999; Shimizu-Okabe et al. 2002; Vu et al. 2000). In the auditory brain stem SOC, the shift from depolarizing to hyperpolarizing action of glycine is staggered in time and occurs over a period of almost 2 weeks (Ehrlich et al. 1999; Kandler and Friauf 1995; Löhrke et al. 2005; Sanes and Friauf 2000). The effect is due to an age-dependent decrease in [Cl–]i in SOC neurons, and KCC2 was proposed to be the key mediator of this change (Ehrlich et al. 1999; Kakazu et al. 1999). Our immunohistochemical and Western blotting data from the CN in gerbils show KCC2 expression already at P1. A similar study focusing on the CN in rats also found an early KCC2 expression at P3 (earliest time point examined) and constant expression levels throughout subsequent auditory development (Vale et al. 2005). According to our data, at P1 the KCC2 immunoreactivity appears to be mainly distributed in the neuropil and then gradually becomes perisomatic and possibly peridendritic between P5 and P9 (Fig. 7, B–F), indicating subcellular changes of the KCC2 expression.
Interestingly, neither our nor previous studies of the auditory brain stem (LSO, Balakrishnan et al. 2003; CN, Vale et al. 2005; SOC, Löhrke et al. 2005) showed any increase in the KCC2 protein level throughout the postnatal development, which has been described as the rate-limiting step for KCC2 functionality in forebrain areas (Khirug et al. 2005; Rivera et al. 1999; Stein et al. 2004). In accordance with our endeavor, also a broader developmental study in mice by Stein et al. (2004) reported adult-like protein levels of KCC2 at birth in the spinal cord as well as in the brain stem.
Considering our data and those of Balakrishnan et al. (2003) and Löhrke et al. (2005), the mechanisms underlying Cl– extrusion in CN and SOC neurons could be very similar: although the expression of KCC2 occurs early in postnatal development, its efficiency in Cl– extrusion still seems to be very low (Fig. 6D). At this early developmental stage, inhibitory neurotransmitters GABA and glycine cause membrane depolarization in CN neurons (M. Witte, unpublished observations). The KCC2 activity in SBC, possibly accomplished through phosphorylation (Kelsch et al. 2001; Vale et al. 2005), is most likely achieved by the end of the first postnatal week. In a recent study of the rat LSO, Blaesse et al. (2006) reported that oligomerization of KCC2 protein correlates with its activation. The present study affirms the notion that, unlike in forebrain areas, KCC2 expression in auditory brain stem neurons does not reflect its functional state per se.
This hypothesis is corroborated by our gramicidin-perforated-patch-clamp experiments. We could demonstrate that in SBC the KCC2 inhibitor DIOA (Coull et al. 2003) or furosemide (Ehrlich et al. 1999; Jarolimek et al. 1999; Kakazu et al. 1999; Martina et al. 2001) reverse EGABA toward depolarized values. The experiments with DIOA also suggest that KCC2 most likely contributes to Cl– extrusion at P7 and P11, whereas it does not seem to be functional at P3 when GABA is still depolarizing.
In another set of earlier studies, it was shown that KCC2 and the Cl–-accumulating cotransporter NKCC1 both display sensitivity to furosemide and also that they are reciprocally regulated during development (Kanaka et al. 2001; Shimizu-Okabe et al. 2002; Yamada et al. 2004). We acquired our furosemide data from P11 SBC, when in all cells the application of GABA had a hyperpolarizing effect, consistent with the active Cl– extrusion by KCC2. Thus we assumed that the observed DIOA- and furosemide-induced depolarizing shifts of EGABA in SBC are due to the inhibition of KCC2 activity.
Technical consideration
We observed a sustained shift in EGABA toward negative values (from approximately –42 to –79 mV) from P3-5 up to the time of hearing onset at P12 (Woolf and Ryan 1984). During this period, the resting membrane potential gradually became less depolarized, and the difference in Vrest was significant between the P3-5 and P10-12 groups (–45 mV compared with –61 mV, respectively). We estimated –61 mV for Vrest at P10-12, which is comparable to the respective values for bushy cells in rats (–62 mV, P9-11) (Price and Trussell 2006) and mice (–64 mV, P16-19) (McGinley and Oertel 2006). More depolarized values of the membrane potential seem to be a general feature of neuronal immaturity (Ben-Ari et al. 1989; Lo Turco et al. 1991; Luhmann et al. 2000; Zhou and Hablitz 1996). Still it should not be concealed that estimation of Vrest by gramicidin-perforated-patch measurements could potentially show a small systematic bias as demonstrated by Tyzio et al. (2003). These authors showed a trend toward more depolarized values of the membrane potential during perforated-patch and whole cell recordings in small neurons with high-input resistances like hippocampal pyramidal cells. The SBC recorded here, however, are significantly larger and have considerably lower input resistances throughout early postnatal development (McGinley and Oertel 2006; Wu and Oertel 1987), Still we cannot rule out the possibility that the recorded values for Vrest at P3-5 are slightly shifted to more depolarized values, but such a bias would not interfere with the description of the developmental change of EGABA shown here. More important, slightly augmented depolarized Vrest values in younger animals would not interfere with our conclusion on the action of GABA: at P2-5 the driving force for Cl– is depolarizing while already at P7-9, when EGABA reaches –65 mV, the driving force for Cl– is hyperpolarized even to the Vrest of P10-12 neurons (–61 mV).
Physiological relevance
Throughout the early developmental period GABA could have a trophic effect and promote stabilization of synapses in the CN as previously shown in various neuronal structures (reviewed in Ben Ari 2002; Lauder 1993; Owens and Kriegstein 2002). In the present study, we show functional GABAA receptors at P3 (the earliest time point investigated), which mediate depolarizing responses. Such depolarizing responses to GABA and also to glycine were shown to induce transient elevation of cytoplasmic Ca2+ by activating voltage-dependent Ca2+ channels (Reichling et al. 1994; Takahashi 1984; Wu et al. 1992). However, GABAergic currents last longer than glycinergic ones and thus are thought to be more effective in triggering intracellular Ca2+ signals (Chery and de Koninck 1999; Yoshimura and Nishi 1995; unpublished observations in CN neurons). The increased [Ca2+]i might be causally related to mechanisms underlying development and stabilization of functional neuronal networks (Kocsis et al. 1993; Spitzer 1994). In rat cultured hippocampal neurons, for example, the shift from depolarizing to hyperpolarizing effects of GABA is mediated by the GABAA receptors through the regulation of the mRNA level for KCC2 (Ganguly et al. 2001; but see also Ludwig et al. 2003 and Titz et al. 2003). Furthermore, a recent report on rat hippocampal cultures suggests that early KCC2 expression enhances the formation of functional GABAergic synapses (Chudotvorova et al. 2005). Thus it is tempting to assume the utilization of similar mechanisms for strengthening the inhibitory neurotransmission in the CN.
In adult CN, the precise temporal information given by bushy cells is determined by the interaction between excitatory (auditory nerve fibers) and nonprimary inhibitory (both GABAergic and glycinergic) inputs, as concluded from in vivo electrophysiological studies (chinchilla, Backoff et al. 1997, 1999; Caspary et al. 1994; gerbil, Kopp-Scheinpflug et al. 2002). Through this inhibition SBC improves the temporal precision of postsynaptical neuronal discharges as seen from the neurons' phase locking accuracy to pure-tone stimuli. Such temporal precision in the tenth of microsecond range is beneficial for the ITD processing in the MSO (reviewed in Grothe 2003).
In conclusion, the present study suggests a change from depolarizing to hyperpolarizing GABA responses in SBC by the end of the first postnatal week and points to the possible involvement of KCC2 in rendering GABA hyperpolarizing. Although KCC2 is already expressed at birth, the onset of its activity coincides with changes of EGABA and, thus it is likely to play a role in the reduction of the [Cl–]i to the "adult-like" level before hearing onset.
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ek was supported by the Wellcome Trust 073966 and by the Grant Agency of the EUSynapse, Czech Republic 309/03/1158 and 309/06/1304.
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Address for reprint requests and other correspondence: R. Rübsamen, Institute of Biology II, University of Leipzig, Talstr. 33, D-04103 Leipzig, Germany (E-mail: rueb{at}uni-leipzig.de)
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