| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1Neuroscience Center and 2Department of Biological and Environmental Sciences, University of Helsinki, Helsinki, Finland; and 3Institute of Neurology, University College London, Queen Square, London, United Kingdom
Submitted 29 May 2007; accepted in final form 17 April 2008
 |
ABSTRACT |
|---|
|
20 mV more positive in KCC2hy/null mice than in wild-type (WT) animals. The basic glutamatergic transmission appeared unaltered in the KCC2hy/null mice, yet they displayed lowered threshold for stimulation-induced synchronous afterdischarges in the CA1 area. Also fatigue of field excitatory postsynaptic potentials/excitatory postsynaptic currents in response to repetitious stimulation was smaller in KCC2hy/null mice, indicating altered synaptic dynamics. Interestingly, this effect was present also under blockade of GABA-ARs and was dependent on the extracellular K+ concentration. Moreover, there were no differences in the levels of either long-term potentiation or long-term depression between the genotypes. The local hippocampal CA1 network can in several aspects maintain its functional viability even in the absence of hyperpolarizing inhibition in pyramidal cells. Our results underscore the central role of shunting type of inhibition in controlling the neuronal excitation/inhibition balance. Moreover, our data demonstrate a novel, unexpected role for the KCC2, namely the modulation of properties of glutamatergic transmission during repetitious afferent activity. |
|
INTRODUCTION |
|---|
|
; Staley and Mody 1992).
The specific physiological significance of these different types of inhibition has remained relatively poorly characterized, partially because of the lack of feasible experimental approaches. Due to the effective Cl– extrusion mechanisms in neurons, manipulations of transmembrane Cl– gradients (and consequently, of EGABA-A) are difficult to perform. Pharmacological dissection of hyperpolarizing and shunting inhibition is a complicated task because direct manipulations of the GABA-AR-linked conductance will alter both modes of action. Further, e.g., inhibitors of co-transporters that can be used to produce positive shift in chloride equilibrium potential can have serious unspecific effects. Both transient and long-lasting depolarizing shifts in EGABA-A have been associated with several experimental models, in particular those addressing the mechanisms of epileptogenesis. The depolarizing shift in EGABA-A produced, e.g., by tetanic stimulation results from a massive GABA release and is accompanied by a large decrease in input resistance and changes in field effects (Bracci et al. 1999) Therefore the significance of the sole liability of the GABA-AR-mediated responses on the hippocampal network behavior is difficult to deduce from experiments of this kind. The controversy on the role of depolarizing GABA-AR-mediated responses in various physiological and pathophysiological phenomena may at least partially rise from the complications linked to the interpretation of the results obtained using the tetanic model.
Thus to assess the role of hyperpolarizing inhibition under more physiological conditions, alternative approaches should be taken. Here we have taken an advantage of the mice deficient of the major neuronal chloride exchanger, the chloride-extruding potassium–chloride cotransporter KCC2 (Mercado et al. 2004; Payne 1997; Rivera et al. 1999) to study the hippocampal function under conditions in which the phasic GABA-AR-mediated responses are depolarizing. As shown previously, these KCC2-deficient mice (KCC2hy/null) mice retain 15–20% of normal KCC2 protein levels in the CNS (Tornberg et al. 2005). We have recently shown that these animals show various behavioral deficits (Tornberg et al. 2005, 2006) but unaltered tonic GABA-AR-mediated conductance in hippocampal pyramidal cells. Using conventional and gramicidin-perforated whole cell patch-clamp techniques as well as field potential recordings, we have now characterized the properties of glutamatergic synaptic transmission and plasticity in the KCC2hy/null mice. We focus on the synaptic behaviors known to be sensitive to changes in the efficacy of phasic GABAergic inhibition; synaptic dynamics, long-term plasticity of glutamatergic transmission [long-term depression (LTD) and long-term potentiation (LTP)] as well as the synchronous activity of the CA1 hippocampal network.
|
|
METHODS |
|---|
|
Eighteen- to 30-day-old wild-type (WT) and KCC2hy/null mice were decapitated under deep pentobarbital (30–40 mg/kg) anesthesia. Transverse slices (400 µm) were cut from the hippocampi using a vibratome. Whole cell experiments were carried out under infrared visual guidance in a submerged chamber at 32°C [recordings of miniature excitatory and inhibitory postsynaptic currents (mEPSCs and mIPSCs)] or room temperature (evoked EPSCs). Standard extracellular solution contained (in mM): 124 NaCl, 3.0 KCl, 2.0 CaCl2, 25 NaHCO3, 1.1 NaH2PO4, 1.0 MgSO4, and 10 glucose (pH 7.4, equilibrated with 95% O2-5% CO2). For the field potential recordings, an interface-type of chamber was used, and all recordings were at 32°C.
Drugs
6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 20 µM; Tocris Cookson), D-2-amino-5-phophonopentanoic acid (AP-5, 40 µM; Tocris Cookson), CGP55845 (1 µM; Tocris Cookson), and picrotoxin (PiTX, 100 µM: Sigma) were bath applied.
Animals
Generation and genotyping of KCC2hy/null mice has been described previously (Tornberg et al. 2005). The KCC2hy/null mice are compound-heterozygous for KCC2 null and hypomorphic alleles and retain 15–20% of normal KCC2 protein levels in the brain. In the KCC2 null allele, a neo-cassette in exon 4 of the KCC2 gene disrupts the reading frame, whereas in the KCC2 hypomorphic allele, a neo-cassette lies within intron 3 in the opposite orientation relative to the KCC2 gene. KCC2hy/null and WT littermates in a (C57BL/6 x 129S2)F1 hybrid background were used in all the experiments described in this study. Animal experiments were approved by the ethics committee for animal research at the University of Helsinki.
Electrophysiology
All recordings were made by using Multiclamp 700A or Axoclamp 200B amplifier (Axon Instruments, Foster City, CA). Whole cell recordings were made from CA1 pyramidal cells with patch electrodes (2–5 M
) filled with a solution containing (in mM) 130 CsMeSO4, 10 HEPES, 0.5 EGTA, 4 Mg-ATP, 0.3 Na-GTP, 5 QX-314, and 8 NaCl, 285 mosM, pH 7.2. For the recordings of mIPSCs, the NaCl concentration was increased to 20 mM. Perforated patch-clamp recordings were made with high-resistance electrodes (12–14 M) to prevent spontaneous rupture of the seal. The tip of the electrodes were filled with a solution containing (in mM) 135 K-gluconate, 10 HEPES, 5 EGTA, 2 KCl, and 2 Ca(OH)2, 290 mosM, pH 7.2, and then backfilled with the solution containing gramicidin (100 µg/ml, Sigma). Recordings were started when an access resistance <150 M was reached. Electrodes filled with 150 mM NaCl were used for field-potential recordings. LTP230D (Anderson and Collingridge 2001; www.ltp-program.com), Axoscope and Clampfit 9.2 programs (Axon Instruments, Foster City, CA). and Mini Analysis Program 5.6.6. (Synaptosoft) were used for data acquisition and analysis.
EGABA was determined from the reversal potential of evoked IPSCs obtained at voltages ranging from –90 to –20 mV. Only cells with resting membrane potential more negative than –58 mV were accepted.
Field excitatory postsynaptic potentials (fEPSPs) or population spikes reflecting synchronous discharging of pyramidal cells were recorded in the CA1 stratum radiatum or s. pyramidale, respectively, and were evoked by stimulation of the Schaffer collateral afferents. Baseline stimulation frequency was 0.05 Hz, pulse duration was 0.1 ms. Stimulus intensity was adjusted to evoke half-maximal fEPSP amplitude. Input-output data obtained at different stimulus strengths was collected. Paired-pulse stimulation at 20-, 40-, 60-, 80-, 100-, 150-, and 200-ms interpulse intervals was used to examine paired pulse facilitation (PPF). After 
20 min of stable baseline, LTP was induced by 100-Hz tetanic stimulation for 1 s. One-hertz/900-pulse stimulation was used to induce LTD. The slope of fEPSP was used as an indicator of synaptic efficacy and was calculated between 20 and 80% of the maximal amplitude. In experiments addressing the network excitability, the amplitude of the population spike as well as the coastline bursting index (CBI) (Korn et al. 1987) were measured. The level of LTP/LTD was measured as a percentage change of the fEPSP slope.
EPSCs and IPSCs were evoked by stimulation of Schaffer collateral-commissural fibers and recorded from CA1 pyramidal neurons. The amplitude of the evoked synaptic responses was measured as the peak relative to the average baseline level 2–8 ms before the stimulation. For spontaneous mEPSCs and mIPSCs, the amplitude detection threshold was set between 4 and 6 pA (2 times the baseline root mean square noise level), and the detected events were verified visually. Cumulative plots were constructed from 10 min of stable recording/cell. All the pooled data are given as means ± SE for the number of cells indicated. For statistical analysis, Student's two-tailed t-test and ANOVA were used. P < 0.05 was considered as statistically significant.
|
|
RESULTS |
|---|
|
Using the perforated patch-clamp technique, we first determined the EGABA-A in WT and KCC2hy/null mice. IPSCs were evoked by afferent stimulation in the presence of CNQX and AP5. In line with the established role of KCC2 in maintaining the transmembrane Cl– concentration gradient, we found that the EGABA-A in KCC2hy/null mice was –48 ± 5 mV, whereas in WT mice, it was –69 ± 4 mV (n = 5 cells/5 animals and 6/4, respectively; P < 0.001; Fig 1). Thus with respect to the resting membrane potential which in WT and KCC2hy/null mice was –66 ± 0.5 mV (n = 6) and –65 ± 0.3 mV (n = 5), respectively, the GABA-AR-mediated responses in the KCC2hy/null mice were clearly depolarizing.
|
To start to characterize the properties of basal glutamatergic synaptic transmission in the area CA1 in WT and KCC2hy/null mice, we first measured the slope of the field EPSP obtained at different stimulus strengths. The input-output curve showing the ratio of the stimulus intensity and the slope of the fEPSP in the slices from KCC2hy/null mice was indistinguishable from that obtained from the WT animals [FIG 2A). We next studied PPF, a form of short-term plasticity using an interpulse interval (IPI) range of 20–200 ms. PPF reflects an enhanced neurotransmitter release due to an accumulation of Ca2+ in the presynaptic nerve terminal following previous stimulus; yet pre- and postsynaptic inhibition may also contribute (Manabe et al. 1993). Whereas there were no differences in PPF between the genotypes with IPIs <100 ms, the PPF appeared to be more accentuated in KCC2hy/null mice at 150- and 200-ms intervals (Fig. 2B).
|
We next used repetitive stimulation using 100 pulses given at 1, 10, and 100 Hz to assess the dynamic properties of the Schaffer collateral-CA1 synapses in WT and hypomorphic mice. Both genotypes displayed the characteristic 150–200% short-term facilitation during the first 5–10 pulses at all frequencies studied (Fig. 3, A–C), the initial facilitation being bigger with higher frequencies. However, whereas in WT mice the fEPSPs rapidly decayed back to the baseline level or even below during the stimulation, in KCC2hy/null mice, the responses showed much less fatigue. This difference was most striking when 1-Hz stimulation was used. Here, the KCC2hy/null fEPSPs showed little or no fatigue following initial facilitation (fEPSP slope values at the end of the stimulation 130 ± 15 and 108 ± 10% in KCC2hy/null mice and WTs, respectively; P < 0.05, Fig. 3A). With 10- and 100-Hz stimulation also, the KCC2hy/null mice showed fatigue of fEPSPs, yet the difference in fEPSP slope in the end of the stimulation persisted (Fig. 3B). Moreover, with 100-Hz stimulation, there was a more prominent slow negative shift in field potential in KCC2hy/null mice (Fig. 3C, right), reflecting the tetanus-induced GABA-AR-mediated depolarization (Kaila et al. 1997; Taira et al. 1997).
|
More prominent phasic depolarizing or excitatory GABA-AR-mediated responses in KCC2hy/null mice could at least partially account for the accentuated short-term facilitation of fEPSPs seen in the mice. To explore this possibility, we next performed a series of experiments under blockade of GABA-ARs to see whether the difference in short-term plasticity was indeed dependent on different efficacy of GABAergic inhibition. Because the blockade of the GABA-ARs in hippocampal slices easily results in epileptiform activity thus hampering the measurements of fEPSPs, especially when repetitious stimuli are applied, the experiments were done using the whole cell patch-clamp technique in a voltage-clamp mode in the continuous presence of AP-5 (50 µM) and PiTX (100 µM).
Paired-pulse experiments using IPIs of 50 and 150 ms revealed that the difference in PPF between the genotypes was also seen in the absence of GABA-AR-mediated inhibition. The PPF of excitatory postsynaptic currents (EPSCs) in WT and KCC2hy/null mice at 50 and 150 ms IPIs was 163 ± 12 and 201 ± 13% and 145 ± 10 and 177 ± 11%, respectively (n = 7/3 and 10/4; P < 0.05; Fig. 4A).
|
Difference in synaptic dynamics between the WT and KCC2hy/null mice is dependent on the extracellular K+ concentration
Lowered activity of KCC2 can result in decreased buffering of extracellular K+ (Ko+) and thus during repetitious activity lead to accentuated accumulation of Ko+ (Payne 1997). This, in turn, can result in prolonged depolarization of synaptic terminals and increased transmitter release (Poolos et al. 1987). To test the effect of changes in Ko+ on EPSC dynamics in WT and KCC2hy/null mice, we applied trains of stimuli (10 Hz/20 pulses) in the presence of low (1 mM) and high (6 mM) Ko+. Whole cell recordings from CA1 pyramidal neurons revealed that lowering the Ko+ to 1 mM abolished the difference in short-term plasticity between the genotypes (Fig. 5A). However, in the presence of high (6 mM) Ko+, the difference in EPSC facilitation was still seen (EPSC amplitudes in the end of the stimulus train were 36 ± 7 and 97 ± 2% of the pretetanus values in WT and KCC2hy/null mice, respectively (n = 7/3 and 7/3; P < 0.05) (Fig. 5C).
|
Change in synaptic dynamics might reflect altered release probability, which can be assessed by measuring mEPSCs (see Lauri et al. 2006). Thus we recorded miniature AMPAR-mediated EPSCs in WT and KCC2hy/null mice in the presence of 100 µM PiTX, 50 µM D-AP-5, and 1 µM TTX. Whole cell recordings from CA1 pyramidal neurons showed that in terms of the frequency (WT: 10.3 ± 2; KCC2hy/null: 10 ± 2 event/min, n = 8/3 and 12/4, respectively; Fig. 6A) or the amplitude (WT: 25.4 ± 1 pA; KCC2hy/null: 24.4 ± 1 pA; Fig. 6B) of the mEPSCs, the genotypes were indistinguishable. This finding is consistent with the data showing that the basic transmission in the KCC2hy/null mice was unaltered (cf. Fig. 2).
|
To assess whether the compromised hyperpolarizing GABA-AR-mediated inhibition was accompanied by homeostatic changes in GABAergic synaptic transmission (see Mody 2005), we recorded mIPSCs from CA1 pyramidal cells in WT and KCC2hy/null mice. We found no difference in the mIPSC amplitude between the genotypes (WT: 12.9 ± 2.5 pA; KCC2hy/null: 10.2 ± 0.6 pA, n = 6/5 and 7/4, respectively; Fig. 7A), the result thus suggesting that the postsynaptic GABA-A conductance was unaltered in the mutant mice. In contrast, the frequency of the mIPSCs was lower in the KCC2hy/null mice than in WT mice (6.3 ± 0.6 and 14.5 ± 1.9 Hz, respectively, P < 0.01), suggesting a reduction in the GABA release or in the density of GABAergic synapses in these animals (Fig. 7B).
|
Attenuated GABAergic inhibition is known to easily result in large-scale synchronization and epileptiform activity in the hippocampal neuronal network. We therefore next examined whether the threshold for the induction of synchronous hippocampal bursting activity was altered in KCC2hy/null mice. Field recordings were done in the CA1 somatic layer (s. pyramidale) to get a better estimate of the synchronous action potential firing: the population spike. In line with the results obtained in recordings from s. radiatum, there were no differences in the input/output curves between the genotypes with respect to the initial slope of the fEPSP (Fig. 8B). However, the threshold for evoking population spike for a given stimulus strength was found to be lower in the KCC2hy/null mice than in the WT mice (Fig. 8A). Moreover, the occurrence of synchronous network bursting 20–80 ms after single stimulation pulse at high-intensity appeared to be much higher in the KCC2hy/null mice as indicated by CBI (Fig. 8C).
|
|
GABA-AR-mediated inhibition is one of the critical factors in setting the threshold for the N-methyl-D-aspartate receptor (NMDAR)-dependent forms of long-term synaptic plasticity, namely LTP and LTD (Wikström and Gustafsson 1986). Because activation of the NMDAR-linked cation channel is accentuated at positive membrane voltages, it is to be expected that nonhyperpolarizing or depolarizing GABA-AR-mediated responses would promote activation of the NMDARs, thus enhancing the induction of changes in synaptic efficacy (see Pavlov et al. 2004). Thus in the last series of experiments, we investigated whether the deficiency in the KCC2 expression would also predispose the CA1 synapses to undergo activity-dependent forms of plasticity.
The protocols to induce synaptic plasticity included 100 Hz/100 pulses or 10 Hz/100 pulses (LTP) and 1 Hz/900 pulses (LTD). We found that there were no significant differences in stimulus-induced long-term changes in synaptic efficacy between the genotypes. One hundred and ten-hertz tetanic stimulation resulted in similar potentiation of the fEPSP slope both in the WT and KCC2hy/null mice. The normalized values of fEPSP slopes at 60 min after 100-Hz stimulation were 149.7 ± 10% (10/8) and 149.6 ± 8% (8/8; Fig. 10, A and D) and after 10-Hz stimulation 129 ± 11% (8/8) and 133.4 ± 7% (6/6; Fig. 10, B and D) for WT and KCC2hy/null mice, respectively. Further, 1-Hz stimulation induced no changes in the synaptic efficacy in either mouse strain. Normalized fEPSP slopes for WT and KCC2hy/null mice after 60 min of the 1-Hz stimulation LTD were 101.5 ± 7% (6/5) and 106.7 ± 13% (8/8), respectively (Fig. 10, C and D).
|
|
|
DISCUSSION |
|---|
|
). Further, we have recently shown that the tonic GABA-AR-mediated conductance in KCC2hy/null mice is not different from that seen in the WT mice (Tornberg et al. 2006). Thus unlike the previously reported KCC2 deficient mice, which had serious developmental defects and died either at birth or before the third postnatal week (Hubner et al. 2001; Woo et al. 2002), these mice allowed us to study the consequences of the absence of hyperpolarizing inhibition on hippocampal function in viable, adult animals. Furthermore, because no KCC2-specific pharmacological tools are currently available, the present results provide interesting insight into the physiological role of the transporter in the adult brain.
The prominent positive shift in EGABA-A in CA1 pyramidal cells in KCC2hy/null mice found in the present study corroborates the role of KCC2 as a major neuronal Cl– transport mechanism in CA1 (Hubner et al. 2001; Rivera et al. 1999; Zhu et al. 2005). In line with the previous reports on KCC2-deficient neurons displaying different levels of KCC2 expression, there were no differences in the resting membrane potential between the WT and KCC2hy/null mice (see Hubner et al. 2001; Zhu et al. 2005). As to the basic glutamatergic synaptic transmission, we found no differences in single pulse-evoked dendritic fEPSPs in the area CA1 between the WT and KCC2hy/null mice. However, action potential firing and consequently, the population spike are easily accentuated on erosion of somatic hyperpolarizing GABAA responses (Kaila et al. 1997; Perreault and Avoli 1992; Staff and Spruston 2003; Taira et al. 1997); this is consistent with our present data.
Neuronal communication normally involves brief bursts of activity with variable frequencies rather than single action potential firing (Dobrunz and Stevens 1999; Lisman 1997). Further, it is well known that prolonged activation of GABA-ARs easily results in accumulation of intracellular Cl– thus shifting the EGABA-A toward positive values, sometimes resulting in postsynaptic excitation (Kaila et al. 1997; Lamsa and Taira 2003; Taira et al. 1997). The effect is expectedly even more prominent in neurons having deficient Cl– extrusion mechanism (see Zhu et al. 2005). We found here that the short-term facilitation of fEPSPs during repetitious stimuli was much more persistent in the KCC2hy/null mice than in the WTs regardless of the stimulation frequency used. Although the initial facilitation of the fEPSPs which was typically seen during the first 2–10 stimulation pulses was of similar magnitude in WT and KCC2hy/null mice, there was much less fatigue of the responses toward the end of the stimulus train in the latter. The accentuated slow negative shift in the extracellular potential in KCC2hy/null mice reflects the tetanus-induced GABA-AR-mediated slow depolarization in CA1 (Kaila et al. 1997; Staley et al. 1995; Taira et al. 1997), indicative of the decreased ability of neurons to recover from activity-induced transmembrane anion shifts (see Zhu et al. 2005).
GABAergic inhibition has a central role in gating the flow of information in synaptic circuitries. During afferent activity both feedforward and -back inhibitory circuits can be activated, thus resulting in an attenuation of subsequent postsynaptic excitatory responses, e.g., on paired-pulse stimulation. On the other hand, on repetitive stimulation at 0.5- to 50-Hz autoinhibition of GABA-release results in relieved postsynaptic inhibition, thus ensuing accentuated postsynaptic excitation and long-term plastic changes (Davies and Collingridge 1991; Davies et al. 1993). Curiously, the differences in the short-term facilitation between the WT and KCC2hy/null mice we observed in fEPSP recordings were retained in the absence of GABA-AR-mediated inhibition as shown in whole cell recordings. Moreover we found no differences either in the mEPSC amplitudes or frequencies between the genotypes, thus supporting our view that the basic excitatory synaptic function was intact in the KCC2hy/null mice. It is interesting to note that cultured cortical KCC2-deficient neurons (completely lacking KCC2) have a delayed spine development and a decreased number of functional glutamatergic synapses as indicated by reduction in mEPSC frequency; an effect independent of the transporter's Cl- extrusion function (Li et al. 2007). Thus our result demonstrates that already 15–20% expression level of KCC2 may be sufficient to rescue its effects on glutamatergic function in vivo.
Thus the observed difference in synaptic dynamics between the WT and KCC2hy/null mice was not dependent on the changes in basic synaptic transmission properties or the compromised hyperpolarizing GABA-AR-mediated inhibition in the KCC2hy/null mice. One plausible explanation for the enhanced EPSPs/EPSCs on repetitious stimulation in the hypomorphic mice is decreased buffering of extracellular K+ accumulation leading to a prolonged depolarization of synaptic terminals and a consequent increase in the transmitter release (Payne 1997; Thompson and Gahwiler 1989; Voipio and Kaila 2000; Zhu et al. 2005). This mechanism would also explain the use dependence of the changes in EPSPs/EPSCs as well as the change in the EPSP/EPSC PPF with long IPIs. The fact that in the presence of 1 mM Ko+, there were no differences in the short term facilitation between the genotypes suggests that the KCC2 expression level in our KCC2hy/null mice (15–20% of WT level) was sufficient to maintain the normal transmembrane K+/Cl– gradient under the lowered K+ load. The attenuation of EPSC amplitudes seen both in WT and KCC2hy/null mice in 6 mM Ko+ is most likely due to high K+-induced sustained terminal depolarization and ensuing inactivation of voltage-dependent Na+ channels, thus leading to decreased transmitter release (Poolos et al. 1987). The preceding findings thus reveal a novel physiological role for KCC2, namely the modulation of synaptic dynamics during repetitious synaptic activation.
At the level of CA1 hippocampal network, the KCC2 deficiency and the lack of hyperpolarizing GABA-AR-mediated inhibition was reflected as a lowered threshold for stimulation-induced synchronous neuronal activity. This finding is in line with the data reported by Woo et al. (2002). However, we were surprised to note that the difference between the WT and KCC2hy/null mice became evident only when high stimulation intensities were used. Thus shunting type of inhibition was evidently powerful enough to curtail neuronal excitation and hypersynchronous activity unless high stimulation intensities of the afferents were employed. Our finding that the mIPSC amplitudes were unaltered in the KCC2hy/null mice supports the idea that the postsynaptic GABA-A conductance and thus the efficacy of shunting was still effective in these animals. It was somewhat curious to find that there was a reduction in the mIPSC frequency in the mutant animals. In the light of the compensatory homeostatic mechanisms, one could have expected to see the opposite. However, the finding is consistent with the previous study by Chudotvorova et al. (2005), who showed that KCC2 overexpresson increases the number of functional GABAergic synapses in cultured neurons. Also if one considers that the depolarizing shift in the GABA-AR-mediated responses in KCC2hy/null mice may easily result in GABAergic excitation (e.g., Taira et al. 1997), it is plausible to assume that the decreased mIPSC frequency in fact is a homeostatic mechanism reducing the overall network excitability.
Application of the GABA-AR blocker PiTX evened out the difference in the CBI. Thus the accentuation of network bursting in the KCC2hy/null mice was evidently linked to the lack of hyperpolarizing inhibition in these animals. Moreover, the relative change in the CBI after PiTX application was bigger in WT mice when compared with KCC2hy/null mice, thus indicating that under control conditions the efficacy of GABA-AR-mediated inhibition is weaker in KCC2hy/null mice.
Downregulation of KCC2 has previously been reported after LTP induction (Wang et al. 2006; see also Rivera et al. 2004). However, whether the transporter plays a role in the LTP induction has not been known. Surprisingly, there were no differences in the level of LTP or LTD in CA1 between the WT and KCC2hy/null mice, although hyperpolarizing inhibition has been thought to be one of the key factors controlling the efficacy of the induction of NMDAR-dependent LTP and LTD in the area CA1 (Wikström and Gustafsson 1986). Thus our data again emphasize that shunting is the physiologically relevant mode of inhibition, particularly in the dendrites (e.g., Staley and Mody 1992).
What might then explain the lack of difference in the levels of LTP and LTD between the genotypes? During 10- to 100-Hz stimulation, the LTP induction ensues rapidly already during the first pulses, and thus the EPSP/EPSC facilitation as well as the slow depolarizing shift seen during the 100 Hz tetanic stimulation may no longer affect the induction mechanisms. This idea is in line with the finding that attenuation of GABA-AR-mediated depolarization has no effect on the LTP induction by tetanic stimulation (Debray et al. 1997). Finally, it is plausible that the GABAergic excitation between interneurons is accentuated in the KCC2hy/null mice (see Lamsa and Taira 2003). Thus during elevated neuronal activity (e.g., on high-frequency stimulation), there may be stronger inhibitory shunt to pyramidal neurons in the KCC2hy/null mice, thus compensating for the possible effect of depolarizing GABA-AR-mediated responses on the LTP induction.
As a conclusion, our study provides new information on the significance of the hyperpolarizing inhibition in the hippocampal CA1 circuitry. Surprisingly, it appears that despite the lack of hyperpolarizing GABA-AR-mediated inhibition the local neuronal circuitry in the KCC2hy/null mice can retain its function showing only relatively minor changes when compared with the WT mice. The results underscore the central role of shunting type of inhibition in controlling the neuronal excitation/inhibition balance. Moreover, our data demonstrate a novel, unexpected role for the KCC2, namely the modulation of synaptic dynamics during physiological-type, repetitious afferent activity.
|
|
GRANTS |
|---|
|
|
|
ACKNOWLEDGMENTS |
|---|
|
|
|
FOOTNOTES |
|---|
Address for reprint requests and other correspondence: T. Taira, Neuroscience Center and Department of Biosciences, P.O. Box 65 (Viikinkaari 1), FIN 00014 University of Helsinki, Helsinki, Finland (E-mail: tomi.taira{at}helsinki.fi)
|
|
REFERENCES |
|---|
|
Bracci E, Vreugdenhil M, Hack SP, Jefferys JG. On the synchronizing mechanisms of tetanically induced hippocampal oscillations. J Neurosci 19: 8104–8113, 1999.
Chudotvorova I, Ivanov A, Rama S, Hubner C, Pellegrino C, Ben-Ari Y, Medina I. Early expression of KCC2 in rat hippocampal cultures augments expression of functional GABA synapses. J Physiol 566: 671–679, 2005.
Davies CH, Collingridge GL. The physiological regulation of synaptic inhibition by GABAB autoreceptors in rat hippocampus. J Physiol 472: 245–265, 1993.
Davies CH, Starkey SJ, Pozza MF, Collingridge GL. GABA autoreceptors regulate the induction of LTP. Nature 349: 609–611, 1991.[CrossRef][Medline]
Debray C, Diabira D, Gaiarsa JL, Ben-Ari Y, Gozlan H. Contributions of AMPA and GABA(A) receptors to the induction of NMDAR-dependent LTP in CA1. Neurosci Lett 238: 119–122, 1997.[CrossRef][Web of Science][Medline]
Dobrunz LE, Stevens CF. Response of hippocampal synapses to natural stimulation patterns. Neuron 22: 157–166, 1999.[CrossRef][Web of Science][Medline]
Hubner CA, Stein V, Hermans-Borgmeyer I, Meyer T, Ballanyi K, 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]
Kaila K, Lamsa K, Smirnov S, Taira T, Voipio J. Long-lasting GABA-mediated depolarization evoked by high-frequency stimulation in pyramidal neurons of rat hippocampal slice is attributable to a network-driven, bicarbonate-dependent K+ transient. J Neurosci 17: 7662–7672, 1997.
Korn SJ, Giacchino JL, Chamberlin N, Dingledine R. Epileptoform burst activity induced by potassium in the hippocampus and its regulation by GABA-mediated inhibition. J Neurophysiol 57: 325–340, 1987.
Lamsa K, Palva J-M, Ruusuvuori E, Kaila K, Taira T. Synaptic GABAA activation inhibits AMPA/kainate receptor-mediated bursting in the newborn (P0–P2) rat hippocampus. J Neurophysiol 83: 359–366, 2000.
Lamsa K, Taira T. Use-dependent shift from inhibitory to excitatory GABA-A receptor action in SP-O interneurons in the rat hippocampal CA3 area. J Neurophysiol 90: 1983–1995, 2003.
Lauri S. E, Lamsa K, Pavlov I, Riekki R, Johnston B, Molnar E, Rauvala H, Taira T. Activity-blockade induces formation of functional synapses in the newborn rat hippocampus. Mol Cell Neurosci 22: 107–117, 2003.[CrossRef][Web of Science][Medline]
Li H, Khirug S, Cai C, Ludwig A, Blaesse P, Kolikova J, Afzalov R, Coleman S, Lauri S, Airaksinen M, Keinänen K, Khiroug L, Saarma M, Kaila K, Rivera C. KCC2 interacts with the dendritic cytoskeleton to promote spine development. Neuron 56: 1019–1033, 2008.[CrossRef][Web of Science]
Lisman J.E. Bursts as a unit of neural information: making unreliable synapses reliable. Trends Neurosci 20: 38–43, 1997.[CrossRef][Web of Science][Medline]
Ludwig A, Li H, Saarma M, Kaila K, Rivera C. Developmental up-regulation of KCC2 in the absence of GABAergic and glutamatergic transmission. Eur J Neurosci 18: 3199–3206, 2003.[CrossRef][Web of Science][Medline]
Mody I. Aspects of the homeostatic plasticity of GABA-A receptor-mediated inhibition. J Physiol 562.1: 37–46, 2005.
Manabe T, Wyllie DJ, Perkel DJ, Nicoll RA. Modulation of synaptic transmission and long-term potentiation: effects on paired pulse facilitation and EPSC variance in the CA1 region of the hippocampus. J Neurophysiol 70: 1451–1459, 1993.
Mercado A, Mount DB, Gamba G. Electroneutral cation–chloride cotransporters in the central nervous system. Neurochem Res 29: 17–25, 2004.[CrossRef][Web of Science][Medline]
Pavlov I, Riekki R, Taira T. Synergistic action of GABA-A and NMDA receptors in the induction of LTD in glutamatergic synapses in the newborn rat hippocampus. Eur J Neurosci 20: 3019–3026, 2004.[CrossRef][Web of Science][Medline]
Pavlov I, Voikar V, Kaksonen M, Lauri S, Hienola A, Taira T, Rauvala H. Role of heparin-binding growth-associated molecule (HB-GAM) in hippocampal LTP and spatial learning revealed by studies on overexpressing and knockout mice. Mol Cell Neurosci 20: 330–342, 2002.[CrossRef][Web of Science][Medline]
Payne J. Functional characterization of the neuronal-specific K-Cl cotransporter: implications for [K+]o regulation. Am J Physiol 273, Cell Physiol 42: C1516–C1525, 1997.
Perreault P, Avoli M. 4-aminopyridine-induced epileptiform activity and a GABA-mediated long-lasting depolarization in the rat hippocampus. J Neurosci 12: 104–115, 1992.[Abstract]
Poolos NP, Mauk MD, Kocsis JD. Activity-evoked increses in extracellular potassium modulate presynaptic excitability in the CA1 region of the hippocampus. J Neurophysiol 58: 404–416, 1987.
Rivera C, Voipio J, Payne J, Ruusuvuori E, Lahtinen H, Lamsa K, Pirvola U, Saarma M, Kaila K. The K+/Cl- co-transporter KKC2 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, Kaila K. Mechanism of activity-dependent downregulation of the neuron-specific K-Cl cotransporter KCC2. J Neurosci 24: 4683–4691, 2004.
Staff NP, Spruston N. Intracellular correlate of EPSP-spike potentiation in CA1 pyramidal neurons is controlled by GABAergic modulation. Hippocampus 13: 801–805, 2003.[CrossRef][Web of Science][Medline]
Staley KJ, Mody I. Shunting of excitatory input to dentate gyrus granule cells by a depolarizing GABAA receptor-mediated postsynaptic conductance. J Neurophysiol 68: 197–212, 1992.
Staley K, Soldo B, Proctor WR. Ionic mechanisms of neuronal excitation by inhibitory GABAA receptors. Science 269: 977–981, 1995.
Taira T, Lamsa K, Kaila K. Posttetanic excitation mediated by GABAA receptors in rat CA1 pyramidal neurons. J Neurophysiol 77: 2213–2218, 1997.
Thompson S, Gähwiler 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.
Tornberg J, Segerstrale M, Kulesskaya N, Voikar V, Taira T, Airaksinen M. KCC2-deficient mice show reduced sensitivity to diazepam, but normal alcohol-induced motor impairment, gaboxadol-induced sedation and neurosteroid-induced hypnosis. Neuropsychopharmacology 32: 911–918, 2006.[CrossRef][Web of Science][Medline]
Tornberg J, Voikar V, Savilahti H, Rauvala H, Airaksinen MS. Behavioral phenotypes of hypomorphic KCC2-deficient mice. Eur J Neurosci 21: 1327–1337, 2005.[CrossRef][Web of Science][Medline]
Voipio J, Kaila K. GABAergic excitation and K+-mediated volume transmission in the hippocampus. Prog Brain Res 125: 323–332, 2000.
Wang W, Wang H, Gong N, Xu T-L. Changes of K+–Cl- cotransporter 2 (KCC2) and circuit activity in propofol-induced impairment of long-term potentiation in rat hippocampal slices. Brain Res Bull 70: 444–449, 2006.[CrossRef][Web of Science][Medline]
Woo NS, Lu JM, England R, McClellan R, Dufour S, Mount DB, Deutch AY, Lovinger DM, Delpire E. Hyperexcitability and epilepsy associated with disruption of the mouse neuronal-specific K–Cl cotransporter gene. Hippocampus 12: 258–268, 2002.[CrossRef][Web of Science][Medline]
Wikström H, Gustafsson B. Postsynaptic control of hippocampal long-term potentiation. J Physiol (Paris) 81: 228–236, 1986.[Medline]
Wikström H, Gustafsson B. Facilitation of hippocampal long-lasting potentiation by GABA antagonists. Acta Physiol Scand 125: 159–172, 1995.
Zhu L, Lovinger D, Delpire E. Cortical neurons lacking KCC2 expression show impaired regulation of intracellular chloride. J Neurophysiol 93: 1557–1568, 2005.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
TOP
ABSTRACT
INTRODUCTION
, 
). Right: I-V plots for the GABA-R-mediated currents. **, P < 0.01.
