Coincident pre- and postsynaptic activity downregulates NKCC1 to hyperpolarize E Cl during...

Coincident pre- and postsynaptic activity downregulatesNKCC1 to hyperpolarize ECl during development

Trevor Balena and Melanie A. WoodinDepartment of Cell & Systems Biology, University of Toronto, Toronto, Ontario, Canada

Keywords: cation-chloride cotransporter, GABA, inhibitory synaptic plasticity, inhibitory synaptic transmission, Sprague–Dawley

Abstract

In the mature CNS, coincident pre- and postsynaptic activity decreases the strength of c-aminobutyric acid (GABA)A-mediatedinhibition through a Ca2+-dependent decrease in the activity of the neuron-specific K+-Cl– cotransporter KCC2. In the present studywe examined whether coincident pre- and postsynaptic activity can also modulate immature GABAergic synapses, where the Na+-K+-2Cl– (NKCC1) cotransporter maintains a relatively high level of intracellular chloride ([Cl–]i). Dual perforated patch-clamprecordings were made from cultured hippocampal neurons prepared from embryonic Sprague–Dawley rats. These recordings wereused to identify GABAergic synapses where the reversal potential for Cl– (ECl) was hyperpolarized with respect to the action potentialthreshold but depolarized with respect to the resting membrane potential. At these synapses, repetitive postsynaptic spiking within± 5 ms of GABAergic synaptic transmission resulted in a hyperpolarizing shift of ECl by 10.03 ± 1.64 mV, increasing the strength ofsynaptic inhibition. Blocking the inward transport of Cl– by NKCC1 with bumetanide (10 lm) hyperpolarized ECl by 16.14 ± 4.8 mV,and prevented this coincident activity-induced shift of ECl. The bumetanide-induced hyperpolarization of ECl occluded furosemide, aK+-Cl– cotransporter antagonist, from producing further shifts in ECl. Together, this indicates that brief coincident pre- andpostsynaptic activity strengthens inhibition through a regulation of NKCC1. This study further demonstrates ionic plasticity as amechanism underlying inhibitory synaptic plasticity.

Introduction

Early in the development of the mammalian nervous system, theconcentration of intracellular chloride ([Cl–]i) is relatively high, dueto the electrically neutral inward transport of Cl– by Na+-K+-2Cl–

cotransporter (NKCC1; Xu et al., 1994; Plotkin et al., 1997; Delpire,2000; Mercado et al., 2004; Dzhala et al., 2005). Underphysiological conditions, this cation-chloride cotransporter (CCC)uses energy from the inward Na+ gradient to transport K+ and Cl–

into the cell. Because c-aminobutyric acid (GABA)A receptors havea high Cl– permeability (Kaila, 1994), the elevated [Cl–]i ofimmature neurons renders the reversal potential for GABA (EGABA)more depolarized than the resting membrane potential (RMP). In thisscenario, GABAA receptor activation leads to membrane depolariza-tion and neuronal excitation (Mueller et al., 1984; Ben-Ari et al.,1989; Luhmann & Prince, 1991; Zhang et al., 1991). Shortly afterbirth in rodents there is a change in the expression level of the CCCsmaintaining EGABA in the hippocampus; NKCC1 is downregulated,while the neuron-specific K+-Cl– cotransporter (KCC2) is upregu-lated (Mercado et al., 2004; Rivera et al., 2005). KCC2 derivesenergy from the K+ gradient to extrude Cl–. The increasedupregulation of KCC2 results in EGABA hyperpolarization, whichrenders GABAergic transmission inhibitory (Rivera et al., 1999;Hubner et al., 2001; Payne et al., 2003).Disrupting the expression or regulation of NKCC1 or KCC2 during

development can change the normal inhibition–excitation balance,

which is critical for proper neuronal circuit development and function(Turrigiano & Nelson, 2004; Dzhala et al., 2005; Fukuda, 2005;Hensch & Fagiolini, 2005; Tao & Poo, 2005; Akerman & Cline, 2006;Kanold & Shatz, 2006). Moreover, alterations in neuronal activity candisrupt normal CCC expression and regulation (Fiumelli & Woodin,2007). For example, chronic blockade of GABAA receptors duringdevelopment can prevent the normal hyperpolarization of EGABA bothin cultured hippocampal neurons (Ganguly et al., 2001) and in vivo inthe turtle retina (Leitch et al., 2005). Neuronal activity can also alterCCC function in the mature CNS (Fiumelli & Woodin, 2007). In vitro,brief coincident pre- and postsynaptic activity at mature GABAergicsynapses weakens inhibition through a KCC2-mediated increase in[Cl–]i, which depolarizes EGABA (Woodin et al., 2003). This raises thequestion, are immature GABAergic synapses also modified by spike-timing-dependent plasticity (STDP)?We examined whether coincident pre- and postsynaptic activity

could also regulate the strength of GABAergic transmission duringdevelopment, when it is in transition from excitation to inhibition,which normally occurs during the second postnatal week ofhippocampal development (Rivera et al., 1999). Using dual perforatedpatch-clamp recordings, we examined whether brief coincident pre-and postsynaptic activity could regulate the strength of shuntinginhibitory synapses [action potential (AP) threshold > EGABA >RMP). Shunting inhibition decreases the input resistance leadingto a short-circuiting of neighboring excitatory currents (Alger &Nicoll, 1979; Andersen et al., 1980; Stuart et al., 1997; Banke &McBain, 2006), and occurs when GABAergic transmission ishyperpolarizing as well as when it is depolarizing during immatureGABAergic transmission. We found that coincident activity led to a

Correspondence: Dr M. A. Woodin, as above.E-mail: [email protected]

Received 10 January 2008, revised 18 February 2008, accepted 5 March 2008

European Journal of Neuroscience, Vol. 27, pp. 2402–2412, 2008 doi:10.1111/j.1460-9568.2008.06194.x

ª The Authors (2008). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd

NKCC1-mediated hyperpolarizing shift of chloride reversal potential(ECl), which effectively increases synaptic inhibition.

Materials and methods

Hippocampal cultures

Low-density cultures of dissociated embryonic rat hippocampalneurons were prepared as previously described (Bi & Poo, 1998;Woodin et al., 2003). In brief, E18–19 pregnant Sprague–Dawley ratswere briefly exposed to carbon dioxide and cervically dislocated inaccordance with guidelines from the Canadian Council on AnimalCare. Hippocampi were then removed from E18–19 embryonic ratsand treated with trypsin for 15 min at 37 �C, followed by gentletrituration. The dissociated cells were plated at a density of50 000 cells ⁄ mL on poly-l-lysine-coated 25-mm glass coverslips in35-mm Petri dishes. Cells were plated in Neurobasal medium(Invitrogen, Carlsbad, California, USA), supplemented with 2%B-27 (Invitrogen). Twenty-four hours after plating, half of the mediumwas replaced with the original plating medium containing 20 mm KCl.Every three days following, one-third of the medium was replacedwith the same KCl-supplemented solution. Both glia and neurons werepresent under these culture conditions. Cells were recorded from after10–16 days in culture.

Electrophysiology

Whole-cell perforated patch recordings using either amphotericin B(150 lg ⁄ mL; Sigma-Aldrich, Oakville, Ontario, Canada) or gramici-din (50 lg ⁄ mL; Sigma-Aldrich) were performed on pairs ofsynaptically connected cultured hippocampal neurons. Specifically,gramicidin was used for all experiments that required the addition ofbumetanide or furosemide (Figs 2 and 5); amphotericin was used in allremaining experiments (Figs 1 and 3). The recording pipettes weremade from glass capillaries (World Precision Instruments, Sarasota,Florida, USA), with a resistance of 4–12 MW. The pipettes were filledwith an internal solution containing (in mm): K-gluconate, 154; NaCl,9; MgCl2, 1; HEPES, 1; EGTA, 0.2; and either amphotericin orgramicidin, pH 7.4, osmolarity ¼ 300 mOsmol. The cultures werecontinuously perfused (approximately 1 mL ⁄ min) with extracellularrecording solution containing (in mm): NaCl, 150; KCl, 3;CaCl2Æ2H2O, 3; MgCl2Æ6H2O, 2; HEPES, 10; glucose, 5; pH 7.4,osmolarity ¼ 307–315 mOsmol. Recordings were performed with aMultiClamp 700B (Molecular Devices, Sunnyvale, California, USA)patch-clamp amplifier. Signals were filtered at 5 kHz using amplifiercircuitry. Data were acquired and analysed using Clampfit 9(Molecular Devices). Recordings started after the series resistancehad dropped below 30 MW, and were only continued if it did notchange by more than 5%. For assaying synaptic connectivity, eachneuron was stimulated at a low frequency (0.05 Hz) by a 1-ms stepdepolarization from )70 to +20 mV in voltage-clamp mode.GABAergic postsynaptic currents (GPSCs) were distinguishable fromexcitatory postsynaptic currents (EPSCs) by longer decay times andsensitivity to 10 lm of the GABAA receptor antagonist gabazine(tested at the end of the experiment). Upon occasion we did detectautaptic GABAergic synapses in our cultures, however, we did notexamine these synapses in the present study. In all recordings, thepostsynaptic neuron was voltage clamped at )70 mV resulting ininward GPSCs. During the STDP induction protocol both neuronswere switched to current-clamp mode and injected with current (2 nA,2 ms) both pre- and postsynaptically to generate an AP in each cell, ata frequency of 5 Hz for 30 s. This protocol resulted in 150 pairs of

pre- and postsynaptic APs. For coincident STDP protocols, there wasa ± 5 ms delay between pre- and postsynaptic APs; for non-coincidentprotocols the interval was increased to ± 100 ms. All recordings wereperformed at room temperature (25 �C).The RMP and AP threshold were determined in current-clamp

mode; the RMP was determined in the absence of current injection orsynaptic activity, while the AP threshold was determined as the pointof inflection in the upward AP waveform. ECl was determined byvarying the holding potential of the postsynaptic cell in 10-mVincrements and measuring the resulting GPSC amplitude; each set ofcurrent–voltage (I–V) measurements was repeated after a 5-mininterval. A linear regression of both sets of GPSC amplitudemeasurements was then used to calculate the voltage dependence ofGPSCs. The intercept of this line with the abscissa was taken as ECl.The slope of the same line was taken as GPSC conductance. Valueshave been corrected for the liquid junction potential of 7 mV. Theliquid junction potential was calculated experimentally by filling arecording pipette and the bath with a solution containing (in mm):K-gluconate, 154; NaCl, 9; MgCl2, 1; HEPES, 1; EGTA, 0.2; pH 7.4.In current-clamp mode (with no commands) the pipette offsetpotentiometer was used to null the voltage. The bath solution wasthen replaced with the extracellular solution used for recordings andthe voltage was noted. The liquid junction potential was taken as theinverse of this voltage reading.GABAA receptors are permeable to both HCO3

– and Cl– (�0.2–0.4ratio; Kaila, 1994). Due to the relatively positive HCO3

– equilibriumpotential (�)10 mV), which is set by mechanisms that controlintracellular pH regulation (Kaila & Voipio, 1987), HCO3

– mediatesan inward, depolarizing current (Kaila & Voipio, 1987; Kaila et al.,1993; Gulledge & Stuart, 2003). However, our experiments wereperformed in bicarbonate-free solution buffered with HEPES, and thusGABAA receptor activation was solely mediating a Cl– current. Forthis reason we report ECl and not EGABA.

Chemicals

When bumetanide (10 lm; Sigma-Aldrich) and furosemide (25 and100 lm; Sigma-Aldrich) were required throughout the entire experi-ment they were added to the perfusion. When they were requiredacutely, the perfusion was terminated and the antagonists were addeddirectly to the bath. Bumetanide and furosemide were prepared bymaking 100 mm stock solutions in anhydrous dimethylsulfoxide,which were then diluted in extracellular solution. Gabazine (10 lm;Tocris, Ellisville, Missouri, USA) was also added directly to the bathin the absence of perfusion.

Statistical analysis

All data are presented as mean ± SEM. The P-value reported forcontrol average GPSC amplitude (Fig. 3D) was obtained bycomparing the current amplitude in three 10-min bins using a one-way anova. The P-values reported for the CCC-inhibitor-inducedchanges in the driving force (DF; Fig. 2D) were obtained using a one-way repeated-measures anova followed by a Tukey test. Unlessotherwise noted all other P-values reported were obtained usingunpaired t-tests. We performed linear regression analyses and obtainedthe correlation coefficients in order to determine the relationshipbetween: (1) the coincident activity-induced hyperpolarization of ECl

and the initial GPSC amplitude; and (2) the coincident activity-induced hyperpolarization of ECl and the amplitude variability. Allstatistical analysis was performed using SigmaStat 2.03.

Activity-induced hyperpolarization of ECl 2403

ª The Authors (2008). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing LtdEuropean Journal of Neuroscience, 27, 2402–2412

Results

Cultured hippocampal GABAergic synapses are shuntingduring development

Dual perforated patch-clamp recordings were made from pairs ofhippocampal neurons cultured at a low density. Hippocampal culturescontain glia, pyramidal neurons and GABAergic interneurons. GPSCsand GABAergic postsynaptic potentials (GPSPs) were characterizedby their relatively long decay time constants (35.50 ± 3.37 ms; n ¼ 6;Fig. 1A), in comparison to EPSCs (5.76 ± 0.64 ms; n ¼ 5; Fig. 1A),and by their sensitivity to the GABAA receptor antagonist gabazine

(10 lm; Fig. 1A). For each synapse we characterized the RMP and APthreshold in current-clamp mode (Fig. 1Ci). We then determined theECl in voltage-clamp mode by constructing an I–V curve (Fig. 1Cii).Based on the relation of ECl to RMP, we characterized GABAergicsynapses as one of two populations: (1) shunting inhibition, defined byan ECl more depolarized than the RMP and more hyperpolarized thanthe AP threshold (Fig. 1B middle and 1C); or (2) hyperpolarizinginhibition, defined when ECl was hyperpolarized with respect to theRMP. Because we were interested in examining whether activityduring development could regulate Cl– homeostasis and thus synapticinhibition, we selected GABAergic synapses that were from the

Fig. 1. Cultured hippocampal GABAergic synapses are shunting during development. (A) Stimulation of presynaptic GABAergic neurons produced GPSCs thatwere inward (top panel) and GPSPs that were depolarizing (middle panel). GPSCs and GPSPs were both blocked by the GABAA receptor antagonist gabazine(10 lm). EPSCs had relatively short decay time constants (bottom panel) compared with GPSCs. (B) A diagram illustrating the normal hyperpolarization of ECl inhippocampal neurons during development. Left: early in development ECl is more depolarized than AP threshold, rendering GABAergic transmission excitatory.Middle: when ECl is more hyperpolarized than AP threshold, but more depolarized than RMP, GABAergic transmission is largely shunting in nature. This is thecategory of GABAergic synapses that was examined in the present study. Right: at mature GABAergic synapses, ECl is often hyperpolarized with respect to the RMP,rendering GABAergic transmission hyperpolarizing inhibitory. (C) Example of how shunting inhibition was characterized at each GABAergic synapse. The RMPand the AP threshold were determined in current-clamp mode (i), while ECl was determined in voltage-clamp mode (ii) by stepping the postsynaptic membranepotential in 10-mV increments from )80 mV to )20 mV while stimulating GABAergic synapses in order to generate an I–V curve. Inset: sample traces of GPSCsrecorded during the construction of this I–V curve. Scale bars: 50 ms and 100 pA. Only synapses where ECl was more depolarized than the RMP, and hyperpolarizedwith respect to the AP threshold, were characterized as having shunting inhibition and examined further.

2404 T. Balena and M. A. Woodin


Fig. 2. Shunting inhibition is maintained by NKCC1. (A) Representative experiment showing that the acute application of 10 lm bumetanide hyperpolarized ECl

(black line: I–V curve obtained in antagonist-free extracellular solution; gray line: I–V curve obtained with extracellular solution containing 10 lm bumetanide).Inset: GPSC recorded in antagonist-free extracellular solution (black trace), and GPSC recorded in extracellular solution containing 10 lm bumetanide (gray trace).Voltage-clamped at )70 mV. Scale bars: 15 ms and 30 pA. (B) Representative experiment showing that the acute application of 25 lm furosemide alsohyperpolarized ECl (black line: I–V curve obtained in antagonist-free extracellular solution; solid gray line: I–V curve obtained with extracellular solution containingfurosemide; dashed gray line: I–V curve obtained following washout of bumetanide with drug-free extracellular solution). Inset: GPSC recorded in antagonist-freeextracellular solution (black trace), and GPSC recorded in extracellular solution containing 25 lm furosemide (gray trace). Voltage-clamped at )70 mV. Scale bars:15 ms and 20 pA. (C) Summary of antagonist-induced changes in ECl. Both bumetanide and furosemide significantly hyperpolarized ECl (bumetanide, n ¼ 5,P ¼ 0.035; 25 lm furosemide, n ¼ 4, P ¼ 0.04; 100 lm furosemide, n ¼ 7, P ¼ 0.006). There was no significant difference in the magnitude of the ECl shiftinduced by bumetanide, 25 lm furosemide, or 100 lm furosemide (one-way anova P ¼ 0.599). (D) Acute application of bumetanide hyperpolarized ECl,regardless of whether ECl was initially shunting or hyperpolarizing (black or gray lines, respectively; upper panel). This bumetanide-induced shift in ECl resulted in asignificant decrease in the DF for shunting inhibition synapses (lower panel), with no significant change in the DF for hyperpolarizing inhibitory synapses (lowerpanel). Subsequent application of furosemide produced no further shifts in ECl for synapses that were initially shunting (top panel), and no further change in the DF(bottom panel). In contrast, subsequent application of furosemide significantly depolarized ECl (top panel), and significantly decreased the DF (bottom panel), forsynapses that were initially hyperpolarizing. Upper panel: black solid lines represent the results of individual GABAergic synapses that were initially shunting innature, gray lines represent individual GABAergic synapses that were initially hyperpolarizing inhibitory. The dashed black line is the mean ± SEM of allGABAergic synapses that were initially shunting in nature, the dashed gray line is the mean ± SEM of all GABAergic synapses that were initially hyperpolarizinginhibitory. *Indicates statistical significance.



shunting inhibition population (Fig. 1B middle panel and 1C). Onaverage, these synapses had an ECl of )48.50 ± 3.08 mV (n ¼ 28),which was more depolarized than the RMP )67.36 ± 1.13 mV(n ¼ 69; P < 0.001), but more hyperpolarized than AP threshold)29.98 ± 1.16 mV (n ¼ 51; P < 0.001). Under these conditions,GPSCs were inward and GPSPs were depolarizing (Fig. 1A), thusthese GABAergic synapses did not exhibit any hyperpolarizinginhibition.

Shunting inhibition is maintained by NKCC1

EGABA is largely determined by the electrochemical gradient for Cl–

(Kaila, 1994), which is dependent upon the balance of NKCC1 andKCC2 (Rivera et al., 1999; Mercado et al., 2004; Rivera et al., 2005).The normal hyperpolarization of EGABA in development is due to acombination of downregulated NKCC1 and upregulated KCC2(Rivera et al., 1999; Mercado et al., 2004; Rivera et al., 2005). Inorder to determine the relative contributions of NKCC1 and KCC2 toCl– homeostasis in our cultured hippocampal neurons, we recordedfrom presynaptic GABAergic neurons and their postsynaptic partners,and examined the shift in ECl induced by acute application of CCCantagonists. Gramicidin was used as the perforating agent in this set ofexperiments because it forms pores that are permeable to monovalentcations and small uncharged molecules but not to Cl–, permittingreliable measurements of ECl (Kyrozis & Reichling, 1995; Owenset al., 1996). Addition of the NKCC1 antagonist bumetanide (10 lm;Payne, 1997; Hannaert et al., 2002; Dzhala et al., 2005) produced ahyperpolarization of ECl by 16.14 ± 4.8 mV (n ¼ 5; P ¼ 0.035;Fig. 2A and C), with no significant change in GPSC conductance(n ¼ 5; P ¼ 0.868). Similarly, application of the non-specific CCCantagonist furosemide also hyperpolarized ECl (25 lm furosemide:13.9 ± 3.86 mV, n ¼ 4, P ¼ 0.04; 100 lm furosemide:11.13 ± 2.46 mV, n ¼ 7, P ¼ 0.006; Fig. 2B and C). Whilefurosemide is routinely used at a concentration of 100 lm (Woodinet al., 2003), we found in the present study that this concentrationproduced a 37.08 ± 25.24% decrease in GABAergic conductance(corresponding to a decrease of 0.52 ± 0.17 pS; n ¼ 7; P ¼ 0.02).Furosemide at a concentration of 100 lm has previously been reportedto decrease GABAA conductance (Wafford et al., 1996). For thisreason we used a lower concentration of furosemide (25 lm), which inour experiments significantly hyperpolarized ECl without changingGPSC conductance (n ¼ 4; P ¼ 0.134). There was no differencebetween the magnitude of ECl hyperpolarization induced bybumetanide, 25 lm furosemide or 100 lm furosemide (one-wayanova P ¼ 0.599; Fig. 2C). These results suggest that shuntinginhibition is being largely maintained by NKCC1. However, becausefurosemide is not a specific antagonist for KCC2 it is difficult todetermine if there is a KCC2-mediated regulation of Cl– in theseneurons.In order to gain further insight into the effects of bumetanide and

furosemide on Cl– homeostasis we asked whether the bumetanide-

induced hyperpolarization of ECl occluded any further furosemide-induced shifts in ECl. In this experiment we examined bothpopulations of neurons: those that exhibited shunting inhibition andthose with hyperpolarizing inhibition. In addition to measuring theinhibitor-induced regulation of ECl, we also quantified the resultingchange in the DF for Cl–, which is the difference between the RMPand ECl (a + value indicates an outward DF for Cl–). For GABAergicsynapses that were shunting in nature (AP threshold > ECl > RMP),acute bumetanide application (10 lm) induced a significant hyperpo-larization of ECl from )46.5 ± 3.6 mV to )65.01 ± 5.15 mV (n ¼ 5;P ¼ 0.025; Fig. 2D top), and effectively eliminated the DF by shiftingit from 20.85 ± 3.6 mV to 2.35 ± 5.15 mV (P ¼ 0.004; Fig. 2Dbottom). Subsequent addition of 25 lm furosemide produced nofurther shift in ECl (n ¼ 3; P ¼ 0.424; Fig. 2D) or DF (P ¼ 0.795).Thus, the bumetanide-induced hyperpolarization of ECl and decreasein DF occluded any additional shifts in ECl or DF induced byfurosemide. Because both bumetanide and furosemide act on NKCC1,these results suggest that this subpopulation of neurons has lowCl– extrusion and high Cl– uptake largely mediated by NKCC1.We then asked whether bumetanide also occluded the effects of

furosemide when we examined synapses from the populationcharacterized by hyperpolarizing inhibition (RMP > ECl). There wasa significant difference in ECl and DF between the shunting andhyperpolarizing inhibition groups under control conditions (ECl:)46.5 ± 3.6 mV vs )77.07 ± 3.38 mV, P < 0.001; DF: 20.85 ± 3.6vs )9.71 ± 3.38, P < 0.001; Fig. 2D). Acute application of bumeta-nide to hyperpolarizing inhibitory synapses induced a hyperpolariza-tion of ECl to )83.53 ± 4.091 mV (n ¼ 5; P ¼ 0.016; Fig. 2D) andan increase in the DF from )9.71 ± 3.38 to )16.170 ± 4.091 mV(n ¼ 5; Fig. 2D). Unlike with shunting inhibition, these bumetanide-induced shifts in ECl and DF did not occlude further shifts byfurosemide. When furosemide was added to hyperpolarizing inhibi-tory synapses already in the presence of bumetanide, ECl depolarizedto )65.89 ± 3.63 mV (n ¼ 5; P ¼ 0.009; Fig. 2D) and the DFdecreased to 1.471 ± 3.636 (n ¼ 5; P < 0.001; Fig. 2D), effectivelyeliminating the DF for Cl–. Thus, the bumetanide-induced hyperpo-larization of ECl and increase in DF does not occlude a furosemide-induced depolarization of ECl at GABAergic synapses that are initiallyhyperpolarizing inhibitory. These results would suggest that thebumetanide-induced block of NKCC1 at hyperpolarizing inhibitorysynapses isolates the contribution of KCC2 to ECl regulation. Thiscontribution by KCC2 was then abolished by the addition offurosemide.

Coincident activity hyperpolarizes ECl in developing neurons

Mature inhibitory GABAergic synapses in both hippocampal culturesand slices are sensitive to coincident pre- and postsynaptic activity(Woodin et al., 2003; Fiumelli & Woodin, 2007), and repetitivepostsynaptic spiking (Fiumelli et al., 2005). STDP occurs when thepostsynaptic neuron repetitively fires APs within 20 ms before or after

Fig. 3. Coincident activity hyperpolarizes ECl during development. (Ai) Coincident pre- and postsynaptic activity (5 Hz for 30 s, +5 ms STDP interval)hyperpolarized ECl (black line, I–V curve obtained during the control period; gray line: I–V curve obtained 10 min after STDP induction). Inset: the current traces ofGPSCs recorded during the construction of this I–V curve. Scale bars: 50 ms and 120 pA. (Aii) STDP induction (+5 ms, given at arrow) decreased GPSC amplitudedue to a decrease in the DF for Cl–. (Bi) Non-coincident activity (5 Hz for 30 s, +100 ms STDP interval) produced a decrease in GPSC conductance with no changein ECl. (black line, I–V curve obtained during the control period; gray line: I–V curve obtained 10 min after non-coincident STDP induction). Inset: the current tracesof GPSCs recorded during the construction of this I–V curve. Scale bars: 50 ms and 350 pA. (Bii) STDP induction (+100 ms, given at arrow) decreased GPSCamplitude due to a decrease in GPSC conductance. (C) Summary of the activity-induced changes in ECl and GPSC conductance induced by coincident and non-coincident STDP protocols. Coincident activity induced a significant hyperpolarization of ECl (P ¼ 0.01). Non-coincident activity significantly decreased GPSCconductance (P ¼ 0.05). (D) Using the perforated patch-clamp configuration GABAergic synapses could be recorded for over 30 min with no significant change inGPSC amplitude (n ¼ 6; P ¼ 0.34).





the activation of a GABAergic synapse (Woodin et al., 2003).GABAergic STDP is due to a Ca2+-dependent decrease in KCC2activity, which increases intracellular Cl–. The resulting depolarizationof ECl effectively reduces the strength of inhibition. In the presentstudy we wanted to know whether similar physiological patterns ofactivity can also regulate the strength of inhibition during develop-ment. STDP was induced by injecting current pulses into thepostsynaptic neuron to fire APs in synchrony with repetitivepresynaptic stimulation at a frequency of 5 Hz for 30 s. The spike-timing interval was termed coincident when pre- and postsynapticspiking was ± 5 ms; when the interval was increased to ± 100 ms theactivity was referred to as non-coincident. Coincident activity resultedin an ECl hyperpolarization of )10.03 ± 1.64 mV (n ¼ 8; P ¼ 0.011;Fig. 3A and C), with no change in synaptic conductance (n ¼ 8;P ¼ 0.796; Fig. 3Ai and C). The hyperpolarization of ECl decreasedthe driving force for Cl– (as ECl approached Vclamp), accounting for the

significant decrease in GPSC amplitude of 36% (n ¼ 8; P ¼ 0.001;Fig. 3Aii). ECl remained hyperpolarized throughout the recordings,which at a maximum were maintained for 40 min following STDPinduction. Whether these activity-induced changes in [Cl–]i aremaintained longer than 40 min was not examined in the presentstudy. Moreover, because these experiments were performed with dualpatch-clamp recordings we can not identify whether the activity-induced plasticity was input specific. Non-coincident activityproduced no significant change in ECl ()3.24 ± 1.08 mV; n ¼ 5;P ¼ 0.479; Fig. 3Bi and C), but did decrease the GPSC conductanceby 34.8 ± 10.45% (n ¼ 5; P ¼ 0.05; Fig. 3B and C). The decrease inconductance can account for a 17% decrease in GPSC amplitude(n ¼ 5; Fig. 3Bii).The use of amphotericin as the perforating agent did not affect the

GPSC amplitude. When synaptic amplitudes were recorded for over30 min in the absence of STDP, there was no significant change in

Fig. 4. STDP-induced ECl hyperpolarization does not depend on initial GPSC amplitude. (A) The relationship between initial GPSC amplitude and the change inECl induced by coincident pre- and postsynaptic activity. The solid line is a linear regression (r ¼ 0.05; P ¼ 0.901). (B) The relationship between the amplitudevariability and the change in ECl induced by coincident pre- and postsynaptic activity. The amplitude variability was taken as the standard deviation (SD) of theGPSC amplitude throughout the duration of the recording. The solid line is a linear regression (r ¼ 0.12; P ¼ 0.776).



GPSC amplitude (n ¼ 6; P ¼ 0.34; Fig. 3D). Moreover, stable controlrecordings of GPSC amplitudes and the ability to induce GABAergicSTDP have both been previously demonstrated using amphotericin asthe perforating agent (Woodin et al., 2003).

The magnitude of the STDP-induced modification of glutamatergicsynapses between cultured hippocampal neurons depends upon theinitial synaptic strength (Bi & Poo, 1998). By performing a linearregression we determined that at GABAergic synapses the magnitudeof the STDP-induced ECl hyperpolarization did not depend on either

the initial GPSC amplitude (r ¼ 0.05; P ¼ 0.901; Fig. 4A) or theamplitude variability during the control recording (r ¼ 0.12;P ¼ 0.776; Fig. 4B).

NKCC1 is required for activity-inducedhyperpolarization of ECl

Based on the predominant action of NKCC1 in maintaining shuntinginhibition in our neurons (Fig. 2), we rationalized that coincident

Fig. 5. NKCC1 is required for activity-induced hyperpolarization of ECl. (Ai) Example of I–V curves obtained in the presence of 10 lm bumetanide; pre-STDP(black), post-STDP (gray). (Aii) The current traces of GPSCs recorded during the construction of the I–V curves in (Ai). Scale bars: 10 ms and 100 pA.(B) When coincident pre- and postsynaptic activity was given in the presence of 10 lm bumetanide, there was no significant change in ECl (P ¼ 0.84; dashed line).Solid black lines represent ECl before and after coincident STDP for individual experiments (n ¼ 5). (C) Following STDP, ECl hyperpolarized by)10.03 ± 1.54 mV; this shift was significantly larger than the change in ECl following STDP performed in the presence of bumetanide (n ¼ 5; *P ¼ 0.013).



activity may be hyperpolarizing ECl through a regulation of thiscotransporter. In order to determine whether the coincident activity-induced hyperpolarizing ECl required NKCC1, we repeated the aboveSTDP experiments in the presence of bumetanide, again usinggramicidin as the perforating agent. Bumetanide prevented thecoincident activity-induced hyperpolarization of ECl (pre-STDP:)90.06 ± 2.71 mV; post-STDP )93.11 ± 3.26 mV; n ¼ 5;P ¼ 0.492; Fig. 5A and B), and left GPSC conductance unchanged(P ¼ 0.841). This non-significant change in ECl following STDPperformed in the presence of bumetanide was significantly differentthan the )10.03 ± 1.54 mV hyperpolarization of ECl that occurred inthe absence of bumetanide (P ¼ 0.013; Fig. 5C). Thus, coincidentactivity hyperpolarizes ECl during development in an NKCC1-dependent manner, effectively increasing the strength of inhibition.

Discussion

At mature inhibitory GABAergic synapses coincident pre- andpostsynaptic spiking alters the activity of KCC2, resulting in adepolarization of EGABA, which decreases the strength of inhibition(Woodin et al., 2003). Our present results demonstrate that immatureGABAergic synapses, which are largely shunting in nature, are alsosensitive to the temporal pattern of pre- and postsynaptic activity. Atthese developing synapses, coincident activity acted via NKCC1 tohyperpolarize ECl, which effectively increased synaptic inhibition.When pre- and postsynaptic activity were non-coincident, synapticinhibition was decreased through a decrease in GPSC conductance,with no change in ECl. These results are particularly important in lightof recent findings that suggest that spike-timing is critical in neuralcircuit information processing and storage (Dan & Poo, 2006).The ability of activity to regulate the strength of synaptic inhibition

through a shift in ECl is supported by results from a number of otherstudies that have also demonstrated activity-induced ionic plasticity(Fiumelli & Woodin, 2007). Spontaneous activity in the isolated spinalcord of the chick embryo induces a depression of GABAergictransmission through a hyperpolarization of EGABA (Chub & O’Dono-van, 2001). At more mature synapses, repetitive postsynaptic spiking(Fiumelli et al., 2005) and coincident pre- and postsynaptic activity(Woodin et al., 2003) resulted in a weakening of GABAA-mediatedtransmission through a decrease in KCC2 activity. Furthermore, whenGABAergic transmission is antagonized during development synapticinhibition fails to develop (Leitch et al., 2005). This present study buildson these previous demonstrations of ionic plasticity by providing thefirst evidence that activity-induced plasticity of synaptic inhibition canoccur at immature synapses via NKCC1 regulation. However, to fullyunderstand how activity regulates GABAergic transmission, we stillneed to examine whether excitatory GABAergic transmission betweenhippocampal neurons is also modified by coincident pre- andpostsynaptic activity.As mentioned previously, repeated postsynaptic spiking (10 Hz,

5 min) produces a KCC2-mediated hyperpolarization of ECl in matureneurons (Fiumelli et al., 2005). We did not examine whether repeatedpostsynaptic spiking regulated ECl in immature neurons with shuntinginhibition because we were more interested in examining theimportance of the temporal order of spikes, which is known to becritical for information processing in the CNS (Froemke & Dan, 2002;Dan & Poo, 2006). Moreover, the mechanisms underlying thecoincident pre- and postsynaptic activity-induced hyperpolarizationof ECl differ from the postsynaptic spiking-induced hyperpolarizationof ECl. In particular, the postsynaptic spiking-induced hyperpolariza-tion of ECl requires the release of Ca2+ from internal stores (Fiumelli

et al., 2005), while the coincident activity-induced regulation of ECl

does not (Woodin et al., 2003).Shunting inhibition is independent of the polarity of GABAergic

transmission; that is to say, it occurs when GPSCs are bothdepolarizing or hyperpolarizing (as long as EGABA is hyperpolarizedwith respect to the AP threshold). When GPSCs are depolarizing (butsubthreshold), the nature of the inhibition is largely shunting (Alger &Nicoll, 1979; Andersen et al., 1980; Stuart et al., 1997). In contrast,when GPSCs are hyperpolarizing the nature of the inhibition ismediated by both hyperpolarization of the postsynaptic membrane andshunting inhibition. While we did not examine shunting inhibition atmature GABAergic synapses in the present study, Woodin et al.(2003) previously demonstrated that coincident pre- and postsynapticactivity at GABAergic synapses with hyperpolarizing and shuntinginhibition produced a KCC2-mediated hyperpolarization of ECl.Moreover, we did not demonstrate in the present study that theimmature GABAergic transmission produces a decrease in inputresistance or a short-circuiting of neighboring excitatory synapses; wesimply defined synapses as having shunting inhibition based on therelationship between ECl, RMP and AP threshold.During development of the hippocampus, shunting inhibition is

observed transiently onto pyramidal cells, as GABAA-mediatedcurrents pass from excitatory to hyperpolarizing inhibitory (Riveraet al., 1999; Ben-Ari, 2002; Rivera et al., 2005). However, shuntinginhibition has recently been identified onto interneurons in the CA3region of the more mature hippocampus (Banke & McBain, 2006;Szabadics et al., 2006). In our cultures we recorded from a mixedpopulation of hippocampal neurons and thus can not distinguishwhether coincident activity-induced ECl hyperpolarization was cell-type specific. In the future it will also be interesting to determinewhether coincident activity can also regulate GABAergic synapsesthat have reverted to an immature state as a result of trauma (van denPol et al., 1996; Nabekura et al., 2002), pain (Coull et al., 2005) orepileptic activity (Rivera et al., 2002; Payne et al., 2003; Rivera et al.,2005; Fiumelli & Woodin, 2007).Through the use of the NKCC1-specific antagonist bumetanide

(Gillen et al., 1996; Holtzman et al., 1998; Race et al., 1999; Hannaertet al., 2002), we demonstrated that depolarizing GABAA-mediatedresponses are largely maintained by Na+-K+-Cl– cotransport. Similarto reports from other studies (Yamada et al., 2004; Dzhala et al., 2005;Nakanishi et al., 2007), bumetanide hyperpolarized ECl. Wethen wanted to know whether there was any additional CCC-mediatedCl– regulation in these neurons. Unfortunately there is no specificantagonist to the neuron-specific KCC2. As an alternative we used thenon-specific CCC antagonist furosemide, which has similar Ki valuesfor NKCC1 and KCC2 (Payne, 1997; Payne et al., 2003). Our rationalwas that if after the bumetanide-induced hyperpolarization of ECl

furosemide produced no further shift in ECl, then we could concludethat there was no significant KCC2 regulation in these immatureneurons. Moreover, in the presence of both antagonists there should beno Cl– regulation and thus no driving force for Cl–. What we foundwas that for neurons from the shunting inhibition population, the useof bumetanide occluded any further furosemide-induced shifts in ECl.Moreover, in the presence of either bumetanide, or bumetanide andfurosemide, there was effectively no driving force for Cl–. Thus, wecan conclude that shunting inhibition at immature GABAergicsynapses was largely maintained by NKCC1.As a control experiment, we repeated the bumetanide–furosemide

occlusion experiments on mature neurons with hyperpolarizinginhibition. We note that furosemide is used routinely to antagonizeKCC2 in more mature neurons when there is a high KCC2 : NKCC1expression ratio (Woodin et al., 2003). We hypothesized that if



furosemide produced a further change in ECl, following thebumetanide shift, this change could be attributable to KCC2. Wefound that bumetanide alone produced an initial hyperpolarizing shiftin ECl. The ability of bumetanide to hyperpolarize ECl in matureneurons suggests that when GABAergic transmission is hyperpolariz-ing inhibitory, these neurons still have some functional NKCC1-mediated Cl– regulation. However, bumetanide did not occludeadditional furosemide-induced shifts in ECl at these mature synapses.Addition of furosemide to mature synapses already in the presence ofbumetanide depolarized ECl. Moreover, in the presence of bumetanideand furosemide there was no effective driving force for Cl–.Collectively these results demonstrate that shunting inhibition inimmature neurons is largely maintained by NKCC1, while hyperpo-larizing inhibition in mature neurons is maintained by both NKCC1and KCC2.

Furthermore, we demonstrated that the presence of bumetanideoccluded the hyperpolarization of ECl induced by coincident activity,suggesting that coincident activity mediates a decrease in NKCC1activity during development. In contrast, at mature inhibitorysynapses, coincident activity depolarizes EGABA through a decreasein KCC2 activity (Woodin et al., 2003). However, in the present studyno such depolarization of ECl was observed in the presence ofbumetanide, suggesting that during development coincident activity isprimarily regulating NKCC1. One similarity between the presentfindings on immature neurons and the findings by Woodin et al.(2003) on mature neurons is that non-coincident activity produced nochange in ECl, but did produce a decrease in GABAergic conductance.The decrease in conductance resulted in a decrease in GPSCamplitude. The mechanism underlying the non-coincident activity-induced change in conductance has not been examined in either study.

Phosphorylation and dephosphorylation are firmly established in theregulation ofNKCC1 transport in non-neuronal cells (Payne&Forbush,1995). For example, in secretory tubules, decreasing [Cl–]i leads tophosphorylation of NKCC1, which activates the transporter (Lytle &Forbush, 1996). Based on the time-course of NKCC1 downregulation inthe present study (< 5 min; Fig. 3A andB),we suggest that activity leadsto the dephosphorylation of NKCC1, which in turn hyperpolarizes ECl.This suggested mechanism does not preclude changes in NKCC1membrane expression, which may be required to maintain the activity-induced hyperpolarization of ECl in the long term.

Our pharmacological evidence suggests that at the shunting inhibitorysynapses in our cultures there was little KCC2-mediated Cl– regulation.However, because KCC2 expression levels do not always correspondwith predicted EGABA values, the present results do not preclude thepossibility that KCC2 was expressed in our cultures. In the lateralsuperior olive, where KCC2 is expressed early in development, there isineffective KCC2-mediated Cl– extrusion (Blaesse et al., 2006). Incultured hippocampal neurons, the developmental expression of KCC2also fails to parallel the functional activity of KCC2, due to a kinase-dependent rate-limiting step that is required for KCC2 transport (Khiruget al., 2005). Moreover, protein kinase C-dependent phosphorylation ofKCC2 has been demonstrated to increase the targeting of KCC2 to theneuronal cell surface (Lee et al., 2007).

Taken together with previous work, a model emerges wherecoincident activity can induce bi-directional plasticity of GABAergictransmission, with the direction of the modification dependent uponthe maturity of the synapse. When GABAergic synapses havematured, the same pattern of activity weakens inhibition through adepolarization of EGABA (Woodin et al., 2003). However, at immatureGABAergic synapses, where a relatively depolarized ECl is maintainedby NKCC1, coincident activity hyperpolarizes ECl, increasing thestrength of inhibition.

Acknowledgements

We acknowledge John Ormond, Hubert Fiumelli and MuMing Poo for theirhelpful comments on the manuscript. This work was supported by a NaturalSciences and Engineering Research Council of Canada (NSERC) Discoverygrant to M.A.W.

Abbreviations

AP, action potential; CCC, cation-chloride cotransporter; DF, driving force; ECl,chloride reversal potential; EGABA, GABA reversal potential; EPSC, excitatorypostsynaptic current; GABA, c-aminobutyric acid; GPSC, GABAergicpostsynaptic current; GPSP, GABAergic postsynaptic potential; KCC2,neuron-specific K+-Cl– cotransporter; NKCC1, Na+-K+-2Cl– cotransporter;RMP, resting membrane potential; STDP, spike-timing-dependent plasticity;[Cl–]i, concentration of intracellular chloride.

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