Astrocytic GABA transporter activity modulates excitatory neurotransmission

Boddum, Kim; Jensen, Thomas P.; Magloire, Vincent; Kristiansen, Uffe; Rusakov, Dmitri A.; Pavlov, Ivan; Walker, Matthew C.

doi:10.1038/ncomms13572

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Published: 25 November 2016

Astrocytic GABA transporter activity modulates excitatory neurotransmission

Nature Communications volume 7, Article number: 13572 (2016) Cite this article

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Abstract

Astrocytes are ideally placed to detect and respond to network activity. They express ionotropic and metabotropic receptors, and can release gliotransmitters. Astrocytes also express transporters that regulate the extracellular concentration of neurotransmitters. Here we report a previously unrecognized role for the astrocytic GABA transporter, GAT-3. GAT-3 activity results in a rise in astrocytic Na⁺ concentrations and a consequent increase in astrocytic Ca²⁺ through Na⁺/Ca²⁺ exchange. This leads to the release of ATP/adenosine by astrocytes, which then diffusely inhibits neuronal glutamate release via activation of presynaptic adenosine receptors. Through this mechanism, increases in astrocytic GAT-3 activity due to GABA released from interneurons contribute to 'diffuse' heterosynaptic depression. This provides a mechanism for homeostatic regulation of excitatory transmission in the hippocampus.

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Introduction

γ-aminobutyric acid (GABA), the main inhibitory transmitter in the brain, binds to post-synaptic ionotropic and metabotropic GABA receptors, and consequently modifies neuronal responses to excitatory inputs by reducing cell excitability. In the hippocampus, GABA is mainly released by interneurons that are recruited through feed-forward or feed-back circuits. This regulates not only network excitability but also the temporal precision of neuronal integration and input discrimination^1,2,3. The precision of afferent input is further increased by a complimentary form of inhibition, heterosynaptic depression, in which activation of one afferent pathway depresses the target cell’s response to a second pathway. It has long been recognized that heterosynaptic depression accompanies long-term potentiation in the Schaffer collateral pathway⁴. NMDA receptor-dependent release of adenosine contributes to this depression, possibly through the recruitment of interneurons paralleled by Ca²⁺ rises in local astrocytes^5,6. Are these two processes, interneuronal recruitment and astrocyte Ca²⁺ rises, causally related?

Activation of interneurons prompts synaptic GABA release and elevates the extracellular GABA concentration, which is in turn regulated by GABA transporters⁷. Four subtypes of GABA transporters (GATs) have been identified in rat and human: GAT-1, 2, 3 and betaine GABA transporter (corresponding in mice to GAT-1, 3, 4 and GAT-2, respectively)⁸. GAT-1 is primarily responsible for neuronal GABA uptake, and predominantly regulates the GABA concentration detected by pyramidal cells. The role of GAT-3, which is mainly expressed in astrocytes⁹, is less clear but it may play a part in regulating the extracellular GABA detected by interneurons¹⁰ and be necessary for extracellular GABA regulation during excessive GABA release¹¹, such as can occur during periods of elevated network activity.

Here we report a previously unrecognized GABA receptor-independent mechanism through which GABA release from interneurons suppresses glutamatergic signalling in the hippocampus. This novel form of inhibitory GABA action depends on astrocytic GAT-3 activation. Increases in GAT-3 activity result in astrocytic Na⁺ accumulation and a consequent increase in astrocytic Ca²⁺ through Na⁺/Ca²⁺ exchange, leading to the astrocytic release of ATP/adenosine. The resultant rise in extracellular adenosine inhibits glutamate release through presynaptic adenosine receptors. This form of inhibition contributes to the detection and homeostatic regulation of network activity by astrocytes.

Results

Activation of GAT-3 inhibits excitatory transmission

Whole-cell patch-clamp recordings from CA1 pyramidal neurons in acute hippocampal slices were performed in the presence of GABA_A-, GABA_B- and NMDA-receptor antagonists to isolate AMPA receptor-mediated excitatory postsynaptic currents (EPSCs) evoked by electrical stimulation of Schaffer collaterals. Surprisingly, despite the presence of GABA receptor antagonists, application of 30 μM GABA reversibly decreased EPSC amplitudes by 22.5±4.9% (P=0.006; n=6; Fig. 1a,c). Since a rise in the extracellular GABA concentration also increases GABA transporter activity¹², we tested the effects of GABA transporter blockers. The glial GABA transporter, GAT-3 inhibitor SNAP5114 (100 μM) prevented the GABA-induced reduction of EPSC amplitude (Fig. 1b,c, P=0.031). Moreover, SNAP5114 without exogenously applied GABA increased the EPSC amplitude by 34.0±13.3% (n=7; P=0.04; Fig. 1d,h), indicating that constitutive GAT-3 activity suppresses EPSCs. SNAP5114 also significantly decreased the paired-pulse ratio and increased 1/CV² of EPSCs (Fig. 1e–g; P=0.03; n=6) suggesting a presynaptic site of action.

**Figure 1: GABA inhibits glutamatergic signalling in a GABA receptor-independent manner through GAT-3 activation.**

In contrast to the effects of SNAP5114, blocking the primarily neuronal GABA transporter GAT-1 with SKF89976A (30 μM) decreased the EPSC amplitude by 11.2±3.4% of control (n=6; P=0.02; Fig. 1h). There was also a trend for SKF89976A to potentiate the effect of SNAP5114 (Fig. 1h). These effects are consistent with enhanced GAT-3-mediated activity when neuronal GABA uptake is blocked (it is, however, difficult to directly compare the effects of exogenous GABA application and transporter inhibition, as it is unknown to what extent extracellular GABA concentration increases in local micro-domains as a consequence of transporter inhibition).

Since GAT-3 is predominantly expressed in astrocytes, we asked whether the effects we observed were mediated by astrocytes. We, therefore, tested whether inhibiting astrocytic metabolism with fluoroacetate (FAC, 5 mM) prevented SNAP5114-induced enhancement of EPSCs. In the presence of FAC, SNAP5114 had no effect on EPSC amplitude (Fig. 1i, P=0.65; paired t-test, n=6), confirming an astrocyte-specific locus of SNAP5114’s action.

To confirm that GAT-3 inhibition resulted in a presynaptic enhancement of excitatory transmission, we used optical quantal analysis of postsynaptic Ca²⁺ responses in CA1 pyramidal cells¹³. By documenting successes and failures of Ca²⁺ rises in individual dendritic spines in response to electrical stimulation of presynaptic fibers, we directly measured the impact of SNAP5114 (and, therefore, tonic GAT-3 activation) on release probability (P_r) and the post-synaptic Ca²⁺ signal (Fig. 2). SNAP5114 increased release probability from 0.32±0.03 to 0.44±0.04 (n=10, P=0.027), but had no effect on the magnitude of rises in post-synaptic Ca²⁺-dependent fluorescence (n=10, P=0.72, Fig. 2b,c), indicating a predominant presynaptic effect.

**Figure 2: GAT-3 inhibition increases release probability at individual CA3-CA1 synapses.**

To further test whether endogenously released GABA from interneurons affects glutamatergic neurotransmission through this mechanism, we optogenetically activated interneurons in hippocampal slices from mice expressing channelrhodopsin-2 (ChR2) in parvalbumin-positive cells. Trains of 470 nm light pulses in the presence of GABA receptor antagonists significantly decreased EPSC amplitudes (by 16.4±2.1%; n=14; P=0.0024; Fig. 3a–c). This effect on EPSC amplitude reversed once photostimulation was stopped (Fig. 3c) and was occluded by blocking GAT-3 with SNAP5114 (Fig. 3d; P=0.017; n=6).

**Figure 3: Interneuronal activity suppresses glutamatergic neurotransmission via a GAT-dependent mechanism.**

GAT-3 activity evokes astrocytic Ca²⁺ signalling

Activation of GABA uptake would be expected to result in accumulation of intracellular Na⁺ due to GABA:Na⁺ co-transport by GAT-3. This could mechanistically alter astrocyte Ca²⁺ dynamics via the Na⁺/Ca²⁺ exchanger and consequently lead to Ca²⁺-dependent Ca²⁺ release from internal stores^14,15. To test this, we first employed a wide-field imaging approach to determine the effect of exogenously applied GABA on Ca²⁺ dynamics in astrocytes identified by Sulforhodamine 101 counterstaining (Fig. 4a). In all, 30 μM GABA led to an increase in the fluorescence intensity of the Ca²⁺-sensitive dye Fura-2 (by 65.6±16.9%; P=0.006; n=8), which was suppressed by SNAP5114 (Fig. 4b,c,e; n=8, P=0.026). This effect is not due to a non-specific inhibition by SNAP5114 of astrocytic Ca²⁺ transients, as activation of the astrocytic metabotropic glutamate receptor with APCD can still elicit Ca²⁺ rises in the presence of SNAP5114 (Supplementary Fig. 1). This also indicates that the increase in the Fura-2 fluorescence intensity by 30 μM GABA is independent from that mediated by APCD.

**Figure 4: GABA induces GAT-3-dependent astrocytic Ca²⁺ signalling.**

Activating GAT-3 with the relatively low affinity substrate, β-alanine (1 mM) (ref. 16), which prevents GABA uptake without abolishing transporter action, also resulted in an astrocytic Ca²⁺ rise (Supplementary Fig. 2a). This, however, was considerably smaller than the Ca²⁺ rise observed in response to 30 μM GABA application (c.f. Fig. 4e) and, similar to the application of a low GABA concentration, did not result in a pronounced depression of EPSC amplitude (Supplementary Fig. 2b). However, a higher concentration of β-alanine (2 mM), as would be predicted for a low affinity substrate, resulted in a significant reduction of EPSC amplitude by 17±2% (n=3, P=0.0004, Supplementary Fig. 2c).

Blocking the Na⁺/Ca²⁺ exchanger using KB-R7943 increased the intracellular Ca²⁺ (n=8, P=0.0004) and occluded the effect of GABA on intracellular Ca²⁺ levels (Fig. 4d,e; P=0.2; n=8). In line with this, KB-R7943 also reduced EPSC amplitude to 84.6±3.7% of baseline (n=4, P=0.002). Furthermore, application of 30 μM GABA in KB-R7943 had no effect on the EPSC amplitude (96.7±2.9% of response in KB-R7943, n=5, P=0.32; GABA wash-out 97.4±4.3%, n=5, P=0.9). Recent studies of astrocyte–synapse interactions suggest that modulation of synaptic neurotransmission could rely on highly compartmentalized Ca²⁺ dynamics in astrocytic processes^17,18. We, therefore, asked whether GABA-induced Ca²⁺ rises occurred in astrocytic processes or were restricted to the astrocytic soma. In order to understand astrocytic Ca²⁺ dynamics at the subcellular level, we used two-photon excitation imaging of astrocytes, individually loaded with Fluo-2 MA in whole-cell mode (Fig. 4f–i). We found that, in addition to overall somatic Ca²⁺ increases in Fura-2 loaded cells (Fig. 4a), GABA application increased local Ca²⁺ transients in fine astrocytic processes (P=0.007, n=5), and that these were also significantly suppressed by KB-R7943 and SNAP5114 (Fig. 4f–i; in these tests, Ca²⁺ increases in the soma, but not in astrocytic processes, were likely to be suppressed due to the proximity of the dialysing pipette tip¹⁹). Furthermore, two-photon excitation Na⁺ imaging with the indicator ANG-2 (ref. 20) confirmed detectable GABA-induced Na⁺ rises in astrocytic processes in response to GABA application (Fig. 5, n=4, P=0.0003).

**Figure 5: GABA induces Na⁺ rise in astrocytic processes.**

To further demonstrate that GAT-3-mediated astrocyte Ca²⁺ rises are indeed causal to the suppression of excitatory transmission, we obtained whole-cell current-clamp recordings from CA1 astrocytes, and were thereby able to combine internal Ca²⁺ concentration clamp with local field excitatory postsynaptic potential (fEPSP) monitoring²¹. By including known Ca²⁺ and Ca²⁺ buffer concentrations within the internal solution, one can clamp [Ca²⁺] within an astrocyte to baseline levels (50–80 nM) while taking advantage of the low membrane resistance of passive astrocytes to measure the fEPSP through the patch pipette (aEPSP). In the absence of any additional Ca²⁺ buffer, GABA significantly suppressed the aEPSP (to 83.1±4.1% of baseline, P=0.01; n=6, Fig. 6a), consistent with Fig. 1a. In contrast, buffering the internal Ca²⁺ concentration to ∼50–80 nM with intracellular Ca²⁺ buffers prevented this decrease in aEPSP amplitude (95.0±3.5% of baseline, P=0.24, n=5, Fig. 6b). Using a repeat measures ANOVA (between-subject factor: presence of buffer; within-subject factors: GABA and interaction of GABA with presence of buffer), there was a significant effect of the interaction of GABA with presence of buffer (F_1,9=5.39, P=0.045). This implies that changes in the internal astrocytic Ca²⁺ concentration modify the effect of GABA on aEPSP amplitude.

**Figure 6: Astrocytic Ca²⁺ clamp prevents suppression of field EPSPs by GABA.**

GAT-3 activity and A₁ receptor activation

Astrocytes display Ca²⁺-mediated adenosine and vesicular ATP release^22,23; extracellular ATP is then rapidly degraded to adenosine by ecto-ATPases²⁴. Therefore, we sought to determine if presynaptic adenosine A₁ receptors mediate the GAT-3-dependent, presynaptic decrease in glutamate release. Blocking A₁ receptors with 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 10 μM) increased the evoked EPSC amplitude (34.1±16.6%; n=6; P=0.001; Fig. 7a,c), similar to that induced by SNAP5114. Moreover, DPCPX occluded the effect of SNAP5114 on EPSC amplitudes (n=6; P=0.8; Fig. 7a,c). Pre-treatment with DPCPX also prevented the effects of GABA on EPSC amplitudes (n=7; P=0.8, Fig. 7b,c).

**Figure 7: GAT-3-mediated EPSC inhibition depends on astrocytic Ca²⁺ signalling and ATP/adenosine release.**

GAT-3 activation and heterosynaptic depression

To understand the role of GAT-3 activation on heterosynaptic depression, we stimulated two distinct Schaffer collateral pathways and recorded fEPSPs in the absence of any neurotransmitter antagonists (Fig. 8a). We intermittently tetanized one pathway (stimulated pathway), and determined the effect of the tetani on the amplitude of the fEPSP in the other pathway (test pathway). The tetani consistently induced depression in the test pathway. This heterosynaptic depression was significantly reduced by inhibiting GAT-3 with SNAP5114 (Fig. 8b,c; n=6; P=0.0002). In a separate set of experiments, we used two-photon excitation three-dimensional (3D) Ca²⁺ imaging to further confirm that GAT-3 activity strongly influences the spatial spread and amplitude of the Ca²⁺ rise that follows such tetanic stimulation (Fig. 8d–g). Tetanic stimulation results in a large rise in Ca²⁺ indicator fluorescence in the processes of the imaged astrocytes that is significantly reduced by SNAP5114 (Supplementary Fig. 3). However, Ca²⁺ signals measured within one focal plane do not fully represent the extent of Ca²⁺ activity or its changes in all three dimensions of the astrocyte arbor. To better understand such activity, we estimated the spatial extent of Ca²⁺ rises across the 3D astrocytic morphology including the overall volume over which the astrocytic Ca²⁺ response peaks (Fig. 8e). SNAP5114 significantly reduced the total volume and spatial extent of Ca²⁺ rise in astrocytes observed during tetanic stimulation by 45.6±7.7% (P=0.004) and 41.8±10.2% (P=0.015), respectively (n=5, Fig. 8f,g).

**Figure 8: Inhibition of glial GABA uptake reduces high-frequency-induced heterosynaptic depression and astrocytic Ca²⁺ response.**

Discussion

Recent work has indicated that astrocytes, which had previously been proposed to have predominantly a supportive role, have crucial functions in determining network excitability, and both respond to and modulate neuronal activity^25,26,27. It has been shown that astrocytes detect network activity through metabotropic and ionotropic receptors, and modulate neuronal excitability by the release of gliotransmitters, in particular glutamate, ATP (adenosine), D-serine and neurotrophins²⁸. Indeed, astrocytes serve a panoply of homeostatic roles including spatial isolation of synapses, buffering of extracellular potassium and regulation of extracellular neurotransmitter concentrations. However, in contrast to the regulation of extracellular glutamate by astrocytic glutamate transporters²⁹, the role of astrocytic GABA transporters in regulating extracellular GABA in the hippocampus remains poorly characterized. Here we show a novel role for astrocyte expressed GABA transporters in the hippocampus to regulate astrocytic release of ATP/adenosine, and so modulate excitatory transmission. Moreover, we show that this process is activated by endogenous GABA release and that it can mediate heterosynaptic depression.

It has previously been suggested that astrocytes express both GABA_A and GABA_B receptors and that activation of these receptors can result in increases in intracellular astrocyte Ca²⁺ concentrations^30,31,32,33. The role of astrocyte expressed GABA_A receptors is not entirely clear; GABA_A receptors activation tends to depolarize astrocytes and can activate voltage-gated Ca²⁺ channels. However, phasic activation of GABA_A receptors by endogenous GABA has yet to be shown. Nevertheless, prolonged activation of astrocytic GABA_A receptors can affect morphological differentiation of astrocytes during development. Endogenous GABA can, however, also activate astrocytic GABA_B receptors resulting in increases in astrocytic Ca²⁺ through release of Ca²⁺ from IP3-sensitive intracellular stores.

In addition to receptor-mediated signalling GABA can affect glial physiology through GABA transporters (for example, ref. 34). GABA transporter expression is cell specific and GAT-3 is predominantly expressed on astrocytes in the hippocampus. It has been previously shown that GAT activation can result in Ca²⁺ rises in astrocytes and that this can contribute to the regulation of vascular tone¹⁴. Here we have demonstrated that GAT-3 activation results in a rise in astrocytic Na⁺ and Ca²⁺ in astrocyte processes. Moreover, inhibiting Na⁺/Ca²⁺ exchange prevents the GAT-3-dependent astrocyte Ca²⁺ rises. This implies that the increase in astrocyte Na⁺ drives the Ca²⁺ rise, similar to that observed for astrocytes in the developing olfactory bulb¹⁴; indeed, this may represent a ubiquitous mechanism in the central nervous system. We have shown that such Ca²⁺ rises occur not only in response to exogenously applied GABA but also during synaptic activity. Astrocytes can, therefore, detect extracellular GABA through three separate mechanisms (ionotropic receptors, metabotropic receptors and transporter activity). These may act over different temporal scales and may be regulated differently at distinct stages of development, but a precise functional division is still unclear.

We observed that tonic activity of astrocytic GAT-3 was sufficient to lead to presynaptic inhibition of excitatory transmission, and that increases in extracellular GABA either through exogenous application or increases in interneuron activity could increase this inhibition. Moreover, by using optical quantal analysis of post-synaptic Ca²⁺ transients, we showed that this is through a direct inhibition of presynaptic release probability.

This inhibition of excitatory transmission could be occluded either by clamping the astrocyte Ca²⁺ concentration to baseline levels (50–80 nM) or by inhibiting presynaptic A₁ adenosine receptors. Altogether, these data imply that the GAT-3-induced Ca²⁺ rises result in increases in extracellular adenosine (through astrocyte ATP and/or adenosine release) that acts on presynaptic A₁ receptors. Thus, this GAT-3-mediated signalling acts in synergy with the proposed mechanism for astrocyte GABA_B receptor mediated inhibition of excitatory transmission⁶. Whether ATP/adenosine is released in this case in a vesicular fashion and involves SNARE machinery³⁵ warrants additional investigation. We also do not know whether the network effects of GABA acting on astrocytic signalling are limited to excitatory neurotransmission or whether GABAergic signalling is also modulated (for example, refs 30, 36, 37). Further research is also needed to establish whether the release of other gliotransmitters such as D-serine, purines and glutamate is affected by GAT-3 activity. It is likely that Ca²⁺ rises that are recorded in astroglia in response to different stimuli have highly distinct sources, underlying molecular cascades and spatiotemporal dynamics that may result in different patterns of gliotransmitter release³⁸.

We have further shown that during high-frequency stimulation, a protocol that is commonly used to induce Schaffer collateral long-term potentiation, there are rises in astrocyte Ca²⁺ and consequent heterosynaptic depression that are GAT-3 dependent. Importantly, these experiments were performed without any exogenous receptor antagonists, and so demonstrate a counterintuitive phenomenon in which inhibition of glial GABA uptake decreases network inhibition. The contribution of GAT-3 activation to heterosynaptic depression is an unexpected observation, and increases the range of mechanisms by which heterosynaptic depression can occur. Heterosynaptic depression through activation of interneurons and consequent ‘release’ of adenosine has long been recognized, but the mechanisms underlying this have only been partially elucidated^5,6. Such heterosynaptic depression permits competitive network activity. This is a computationally powerful process, which enables ‘winner takes all’ during synaptic plasticity and promotes sparse coding, so optimizing information processing and storage in the hippocampus. Moreover, GAT-3 activity is sensitive to low concentrations of GABA¹⁶ making it an ideal detector of neuronal activity, so providing a powerful method for homeostatic network control.

Methods

Hippocampal slice preparation

Animal procedures were subject to local ethical approval and adhered to the United Kingdom Animals (Scientific Procedures) Act of 1986. Male Sprague Dawley rats (P19-P26) were sacrificed using an overdose of isoflurane. Animals were kept in groups (5–8 per cage) under standard housing conditions with 12 h light–dark cycle and free access to food pellets and drinking water. After decapitation brains were rapidly removed, hippocampi dissected out and transverse slices were prepared on a Leica VT1200S vibratome (350 μM thick for electrophysiological and two-photon imaging experiments, or 250 μM thick for Fura-2 imaging experiments). The slicing was performed in ice-cold slicing solution that for electrophysiology contained (in mM): 75 sucrose, 87 NaCl, 22 glucose, 2.5 KCl, 7 MgCl₂, 1.25 NaH₂PO₄, 0.5 CaCl₂ and 25 NaHCO₃; for Fura-2 imaging the solution contained (in mM): 100 NMDG, 20 NaCl, 22 glucose, 2.5 KCl, 1.3 MgSO₄, 1 NaH₂PO₄, 25 NaHCO₃, both 315-330 mOsm. For two-photon imaging slicing solution contained (in mM): 60 NaCl, 105 sucrose, 26 NaHCO₃, 2.5 KCl, 1.25 NaH₂PO₄, 7 MgCl₂, 0.5 CaCl₂, 11 glucose, 1.3 ascorbic acid and 3 sodium pyruvate. All solutions were bubbled with 95% O₂ plus 5% CO₂, pH adjusted to 7.4. Slices were incubated in the oxygenated storage solution at 34 °C for 15 min and then allowed to equilibrate to room temperature for 15 min. Slices used for electrophysiological recordings were then transferred to a continuously oxygenated humid interface chamber and allowed to recover at room temperature for at least 60 min. Recordings were performed in a recording chamber constantly perfused with 32–34 °C oxygenated artificial cerebrospinal fluid (aCSF) solution using a gravity driven perfusion system. aCSF solution contained (in mM): 120 NaCl, 22 glucose, 2.5 KCl, 1.3 MgSO₄, 1 NaH₂PO₄, 25 NaHCO₃, 1.7 MgCl, 2.5 CaCl₂, had osmolality of 296 mOsm and equilibrated with 95% O₂ plus 5% CO₂. Storage solution had Ca²⁺ concentration reduced to 1 mM. A cut was made between the CA1 and CA3 regions to prevent propagation of recurrent excitation through Schaffer collaterals.

Electrophysiological recordings

Whole-cell patch-clamp recordings were performed from CA1 pyramidal cells (input resistance 330±70 MΩ) visualized using an infrared differential contrast imaging system. Standard walled borosilicate glass capillaries were used to fabricate recording electrodes with a resistance of 2.5–3.5 MΩ. Intracellular pipette solution contained (in mM): 120 Cs-methanesulfonate, 10 HEPES, 0.2 EGTA, 8 NaCl, 0.2 MgCl₂, 2 Mg-ATP, 0.3 Na-GTP, 5 QX314-Br, 10 phosphocreatine, pH adjusted to 7.2 and osmolality adjusted to 296 mOsm. Series resistance was monitored throughout experiments using a −5 mV step command. Cells showing a series resistance >20 MΩ, a >20% change in series resistance, or unstable holding current were rejected. EPSCs were evoked by stimulation of Schaffer collaterals with bipolar tungsten electrodes at 0.025 Hz. Paired responses were evoked with 50 ms inter-stimulus intervals.

For astrocyte Ca²⁺ clamp experiments CA1 Stratum radiatum passive astrocytes were patched in whole-cell mode with an internal solution containing (in mM): 135 potassium methanesulfonate, 10 HEPES, 10 di-Tris-Phosphocreatine, 4 MgCl₂, 4 Na₂-ATP, 0.4 Na-GTP (pH adjusted to 7.2 using KOH, osmolarity 290–295), and supplemented with the morphological tracer dye Alexa 594 (50 μM). To maintain intracellular free Ca²⁺ concentration at a steady state level of 50–80 nM, the internal solution was supplemented with 0.2 mM BAPTA, 0.45 mM EGTA and 0.14 mM CaCl₂. Following whole-cell access, astrocytes were identified by criteria previously described³⁹ and left for 25 min for the internal solution and Ca²⁺ buffers to equilibrate across the astrocytic arbor. Excitatory synaptic responses were monitored as field potentials recorded through the astrocyte patch-pipette in current clamp mode (aEPSPs) as previously described²¹ and evoked by stimulation of Schaffer collaterals with bipolar tungsten electrodes 200 μm distant to the astrocyte at a rate of 0.033 Hz. Stimulus intensity was normally set at ∼50% of maximum response amplitude.

FEPSPs were recorded using 1.5–2 MΩ resistance glass electrodes filled with aCSF. Stimulation intensity was set to induce half-maximal responses. Heterosynaptic depression in the CA3-CA1 synapses was induced in the absence of glutamate and GABA receptor antagonists by tetanising one of the two independent Schaffer collateral pathways with three consecutive 1 s 100 Hz trains using bipolar tungsten stimulation electrodes. Pathway independence was established at the start of each experiment using paired-pulse stimulation (20 ms inter-pulse interval). Paired responses of each pathway were compared with those evoked by activating the pathways at 20 ms intervals. Ca²⁺ concentration in the aCSF in these experiments was reduced to 1.5 mM to facilitate the induction of heterosynaptic depression. fEPSP slopes were measured to determine changes in the evoked responses. Since heterosynaptic depression lasts minutes (see Fig. 8b), we represented the amount of depression as area under the curve rather than peak depression.

Recordings were obtained using a MultiClamp 700 B amplifier (Axon Instruments, Foster City, CA, USA), filtered at 4 kHz, digitized and sampled through an AD converter Digidata 1550 (Molecular Devices) or NI PCI-6221M (National Instruments) at 10 kHz and stored on a PC. LabView routines (National instruments, Newbury, UK), pClamp10 software (Molecular Devices) or WINWCP (John Dempster, Strathcylde Elecrophysiology Software) were used for data acquisition and off-line analysis (blinded to group). Average EPSC amplitude values were calculated for 5-min periods immediately before drug applications, and at the end of drug wash-in and wash-out periods.

Optogenetic experiments

PV::cre mice (B6;129P2-Pvalb^tm1(cre)Arbr/J Jackson laboratory stock number: 008069) were crossed with Ai32 mouse line, which has floxed-stop EYFP-tagged excitatory opsin ChR2 (B6;129S-Gt(ROSA)26Sor^{tm32(CAG-COP4*H134R/EYFP)Hze}/J Jackson laboratory stock number: 012569), to produce animals with ChR2 expression in parvalbumin positive (PV⁺) interneurons throughout the brain⁴⁰. Animals were kept under standard housing conditions with 12 h light–dark cycle and free access to food pellets and drinking water. Hippocampal slices were prepared from mice of both sexes aged between postnatal day 25 and 50. Littermates of the same sex were housed in groups of 3–5 animals. Wide-field illumination of the CA1 region of the hippocampus was delivered through 20 × water immersion objective (Olympus). Blue light (wavelength 470 nm) was generated using pE-2 LED illumination system (CoolLED); light intensity at the surface of the slice was in the range of 5–7 mW. PV⁺ interneurons were activated using 5 s trains of 1 ms pulses delivered at 25 Hz. To test the effect of GABA released from PV⁺ interneurons on AMPA receptor-mediated currents in the Schaffer collaterals synapses EPSCs were evoked every 12 s either in the absence of photostimulation, or 1,250 ms after the termination of the light train. Ten EPSCs were averaged at the end of each period: 5 min baseline, 3 min photostimulation and 5 min recovery.

Fura-2 Ca²⁺ imaging

Before Ca²⁺ imaging, slices were incubated with Sulforhodamine 101 (SR101; 5 μM) and the high-affinity Ca²⁺-sensitive dye, Fura-2 AM (5 μM), in aCSF solution for 35 min at 34 °C while bubbled with 95% O₂ plus 5% CO₂. Live cell imaging was performed at room temperature using an Olympus BX51WI (USA) microscope, with a 40 × PlanApo objective (NA 0.8). Fluorescent dyes were excited at 340/380 nm (Fura-2) and 570 nm (SR101) with a xenon light source (Polychrome IV or V, TILL Photonics, Germany). Images were recorded with a SensiCam cooled CCD (charge-coupled device) camera (PCO Imaging, Germany) controlled by TILL-visION imaging software (TILL Photonics, Germany). Fluorescent emission was recorded at 0.5–2 Hz from elliptical regions of interest placed over individual astrocytes. The ratio between the measured values of Fura-2 fluorescence (340/380) was background subtracted, normalized to baseline (F₀) and reported as ΔF/F₀. Average Ca²⁺ response was calculated for 2-min periods 2 min after drug applications, and at the end of drug wash-out periods. Data for GABA+SNAP5114 and KB-R7943+GABA were normalized to baseline fluorescence before GABA or KB-R7943 application correspondingly.

Two-photon excitation imaging

We used a Femtonics Femto3D-RC imaging system (Femtonics Femto 3D-RC, Budapest) optically linked to a femtosecond pulse laser MaiTai (SpectraPhysics-Newport) and integrated with patch-clamp electrophysiology (25 × Olympus objective, NA1.05). For imaging of spontaneous and evoked astrocytic Ca²⁺ dynamics, CA1 Stratum radiatum passive astrocytes were patched with the potassium methanesulfonate-based internal solution described above supplemented with the morphological tracer dye Alexa 594 (50 μM) and either the Ca²⁺ indicator Fluo-2 MA (200 μM) or Na⁺ indicator ANG-2 (200 μM).

Following whole-cell access, astrocytes were identified by criteria previously described³⁹ and left for 25 min for fluorophores to equilibrate across the astrocytic arbor. For time-lapse imaging of astrocytic Ca²⁺ transients and GABA evoked Na⁺ responses, images were collected in frame scan mode for 5 min at a resolution of∼0.4 μm per pixel and image size appropriate for the shape of the astrocyte, frame rate was ∼3 Hz per time point. For imaging of evoked astrocytic Ca²⁺ responses four-dimensional frame scanning was implemented using a fast piezo-objective positioner of the Femto3D-RC setup. In this mode four focal planes separated by five microns were scanned sequentially for 40 s. Two-dimensional image dimensions and scan rate were preserved by reducing the pixel dwell time. Ca²⁺ responses were generated spontaneously in response to GABA application or evoked by 1 s 100 Hz trains of electrical stimulation using the same parameters and method described above for aEPSP recordings.

Analysis of spontaneous and evoked Ca²⁺ transients

For analysis of spontaneous Ca²⁺ transients in astrocytic fine processes, Ca²⁺ responses were defined as regions where an increase in ΔG/R >2.5 × s.d. of the baseline occurred. To obtain these data image stacks were opened in ImageJ (Rasband WS, ImageJ, NIH, Bethesda, Maryland, USA, http://rsb.info.nih.gov/ij/, 1997–2008), pixel binned 2 × 2 (x,y), and processed to make a G-G_min/R image stack where G_min is a minimum intensity projection of the stack in the time domain. This image stack was then median filtered along the time domain to remove shot noise, and 20 frames in which no visually identifiable Ca²⁺ signals occurred were chosen to produce a baseline image stack. Both baseline image stack and complete stack were then imported into MATLAB 2015a and an image corresponding to the mean+2.5 × s.d. of the baseline stack was used as a threshold to segment each frame into active and inactive pixels. Ca²⁺ signalling activity was then expressed as the percentage of the imaged area active (>2.5 × s.d. baseline ΔG/R) per frame. For comparisons between experimental conditions, the mean area active for 1.5 min before drug application was compared with the mean area active for 1.5 min, 1.5 min following drug wash on.

For 3D analysis of the spread of high-frequency stimulus-evoked astrocytic Ca²⁺ transients amplitude thresholding produced qualitatively inaccurate representations of the spread of the tetanic stimulation-evoked Ca²⁺ signal across the volume imaged. As a result a local cross-correlation approach previously described in ref. 41 (code available from http://labrigger.com/blog/2013/06/13/local-cross-corr-images/) was used to produce an activity heat map from the time lapse image stacks for each focal plane imaged. The heat map images were then thresholded to the top 5% cross-correlation scores to distinguish the locations of active regions within each focal plane. To identify the field of activity around the astrocytes soma the thresholded (binary) images from each focal plane were summed and the radial reslice function in ImageJ (Rasband WS, ImageJ, NIH, Bethesda, Maryland, USA, http://rsb.info.nih.gov/ij/, 1997–2008) was used to identify the total radial coverage around the astrocytes soma where active regions were identified. Estimates of the volume of responsive regions were by the sum of active voxel volumes within each image.

Postsynaptic Ca²⁺ imaging in CA1 pyramidal neurones

Postsynaptic imaging of Ca²⁺ responses (optical quantal analysis) in CA1 pyramidal cell dendrites and spines was carried out as previously described^13,42. Local, minimal stimulation was provided by bipolar stimulation using a pair of glass stimulating electrodes filled with aCSF. The electrodes were placed parallel to the targeted dendritic branch, at a distance of ∼20–30 μm form the branch. To identify active synapses fast (20 Hz), frame scans of the local dendrites were viewed while three 100-μs square pulses of 2–10 V were delivered with a 25-ms interstimulus interval using a constant voltage isolated stimulator (model DS2A-mkII; Digitimer). This protocol was repeated until a Ca²⁺ response confined to a spine head was observed. In all, 600-ms line scans of the active spine were then recorded while a dual stimulus (50-ms interstimulus interval) was delivered. Scans were repeated once every 30 s; a minimum of 15 trials in each condition were used to assess the release probability at the imaged synapse.

Pharmacological manipulations

All recordings (except for the field potential recordings of heterosynaptic depression) were done in the presence of DL-2-amino-5-phosphonopentanoic (DL-APV; 100 μM), picrotoxin (100 μM) and (2S)-3-[[(1S)-1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl](phenylmethyl) phosphinic acid (CGP55845; 5 μM) to block NMDA-, GABA_A- and GABA_B-receptors respectively. (1-[2-[tris(4-methoxyphenyl)methoxy]ethyl]-(S)-3- piperidinecarboxylic acid ((s)-SNAP5114; 100 μM), 1-(4,4-diphenyl-3-butenyl)-3-piperi dinecarboxylic acid (SKF89976A; 30 μM), DPCPX (10 μM), 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea mesylate (KB-R7943; 50 μM), (±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (t-ACPD; 50 μM) were added to the perfusion solution. All above drugs were from Tocris Bioscience (Bristol, UK). β-alanine (1 or 2 mM), FAC (5 mM; minimum 30 min of pre-incubation) and γ-Aminobutyric acid (GABA; 5 or 30 μM) were purchased from Sigma-Aldrich (Dorset, UK).

Statistical analysis

Data were analysed using GraphPad Prism software (GraphPad software, San Diego CA, USA) or SPSS (SPSS Inc., USA) and are presented as mean±s.e.m. Imaging and electrophysiological data are available upon request. Statistical analysis was performed using two-tailed paired or unpaired Student’s t-test and ANOVA as detailed. Normality was tested using Shapiro-Wilk test. Differences were considered as significant at P<0.05, and this was corrected for multiple comparisons using the Holm-Bonferroni correction.

Data availability

The data that support the findings of this study are available from the corresponding authors upon request.

Additional information

How to cite this article: Boddum, K. et al. Astrocytic GABA transporter activity modulates excitatory neurotransmission. Nat. Commun. 7, 13572 doi: 10.1038/ncomms13572 (2016).

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Acknowledgements

We thank K. Volynski and D.M. Kullmann for comments. This work was supported by Epilepsy Research U.K. (I.P.), The Worshipful Company of Pewterers (I.P.). A Wellcome Trust Principal Fellowship, all U.K. European Research Council Advanced Grant, FP7 ITN EXTRABRAIN (European Commission), Russian Science Foundation Grant 15-14-30000 (D.A.R.).

Author information

Kim Boddum and Thomas P. Jensen: These authors contributed equally to this work
Ivan Pavlov and Matthew C. Walker: These authors jointly supervised this work
i.pavlov@ucl.ac.uk
m.walker@ucl.ac.uk

Authors and Affiliations

Department of Clinical and Experimental Epilepsy, UCL Institute of Neurology, University College London, Queen Square, London, WC1N 3BG, UK
Kim Boddum, Thomas P. Jensen, Vincent Magloire, Dmitri A. Rusakov, Ivan Pavlov & Matthew C. Walker
Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, Copenhagen, 2100, Denmark
Kim Boddum & Uffe Kristiansen

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Kim Boddum
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Thomas P. Jensen
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Contributions

K.B., T.P.J. and I.P. performed electrophysiological recordings, V.M. performed optogenetic experiments, K.B. carried out Fura-2 Ca²⁺ imaging experiments, T.P.J. carried out patch-clamp experiments combined with 2-photon Ca²⁺ and Na⁺ imaging and respective data analyses. D.A.R. designed imaging experiments, contributed to data analyses and manuscript writing. M.C.W. and I.P. conceived and designed the study, and wrote the manuscript, which was revised by all co-authors.

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Correspondence to Dmitri A. Rusakov, Ivan Pavlov or Matthew C. Walker.

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Boddum, K., Jensen, T., Magloire, V. et al. Astrocytic GABA transporter activity modulates excitatory neurotransmission. Nat Commun 7, 13572 (2016). https://doi.org/10.1038/ncomms13572

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Received: 02 September 2016
Accepted: 14 October 2016
Published: 25 November 2016
DOI: https://doi.org/10.1038/ncomms13572

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