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Neuroscience

J Physiol 594.13 (2016) pp 3651–3666

Biphasic modulation of parallel fibre synaptic transmission by co-activation of presynaptic GABAA and GABAB receptors in mice Rebecca D. Howell and Jason R. Pugh University of Texas Health Science Center at San Antonio, Department of Physiology, San Antonio, TX 78229, USA

Key points

The Journal of Physiology

r Many excitatory synapses co-express presynaptic GABAA and GABAB receptors, despite their r r r r

opposing actions on synaptic transmission. It is still unclear how co-activation of these receptors modulates synapse function. We measured presynaptic GABA receptor function at parallel fibre synapses onto stellate cells in the cerebellum using whole-cell patch-clamp recording and photolytic uncaging of RuBi-GABA. Activation of presynaptic GABA receptors results in a transient (100 ms) enhancement of synaptic transmission (mediated by GABAA receptors) followed by a long lasting (>500 ms) inhibition of transmission (mediated by GABAB receptors). When activated just prior to high-frequency trains of stimulation, presynaptic GABAA and GABAB receptors work together to reduce short-term facilitation/enhance depression, altering the filtering properties of synaptic transmission. Inhibition of synaptic transmission by GABAB receptors is more sensitive to GABA than enhancement by GABAA receptors, suggesting GABAB receptors may be activated by ambient GABA or release from greater distances.

Abstract GABAA and GABAB receptors are co-expressed at many presynaptic terminals in the central nervous system. Previous studies have shown that GABAA receptors typically enhance vesicle release while GABAB receptors inhibit release. However, it is not clear how the competing actions of these receptors modulate synaptic transmission when co-activated, as is likely in vivo. We investigated this question at parallel fibre synapses in the cerebellum, which co-express presynaptic GABAA and GABAB receptors. In acute slices from C57BL/6 mice, we find that co-activation of presynaptic GABA receptors by photolytic uncaging of RuBi-GABA has a biphasic effect on EPSC amplitudes recorded from stellate cells. Synchronous and asynchronous EPSCs evoked within 100 ms of GABA uncaging were increased, while EPSCs evoked 300–600 ms after GABA uncaging were reduced compared to interleaved control sweeps. We confirmed these effects are presynaptic by measuring the paired-pulse ratio, variance of EPSC amplitudes, and response probability. During trains of high-frequency stimulation GABAA and GABAB receptors work together (rather than oppose one another) to reduce short-term facilitation when GABA is uncaged just prior to the onset of stimulation. We also find that GABAB receptor-mediated inhibition can be elicited by lower GABA concentrations than GABAA receptor-mediated enhancement of EPSCs, suggesting GABAB receptors may be selectively activated by ambient GABA or release from more distance synapses. These data suggest that GABA, acting through both presynaptic GABAA and GABAB receptors, modulate the amplitude and short-term plasticity of excitatory synapses, a result not possible from activation of either receptor type alone.

 C 2016 The Authors. The Journal of Physiology  C 2016 The Physiological Society

DOI: 10.1113/JP272124

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(Received 6 January 2016; accepted after revision 30 March 2016; first published online 9 April 2016) Corresponding author J. R. Pugh, Department of Physiology, University of Texas Health Science Center San Antonio, 7703 Floyd Curl Drive, South Texas Research Facility, MSC 8253, San Antonio, TX 78229-3900, USA. Email: [email protected] Abbreviations ISI, interstimulus interval; MLI, molecular layer interneuron; PPR, paired-pulse ratio; PTX, picrotoxin; RRP, readily releasable pool.

Introduction In the classical view of synaptic transmission, neurotransmitter is released from presynaptic terminals and activates receptors located in the postsynaptic membrane. However, it has long been known that neurotransmitter receptors can also be expressed in presynaptic terminals (Eccles et al. 1963). In fact, nearly every class of receptor expressed in postsynaptic membranes can also be found at presynaptic terminals, and some terminals co-express a remarkable diversity of receptors. For example, parallel fibre boutons in the cerebellum have been shown to express GABAA (Stell et al. 2007), GABAB (Dittman & Regehr, 1996), kainate (Delaney & Jahr, 2002), metabotropic glutamate (Abitbol et al. 2008), cannabinoid type 1 (Takahashi & Linden, 2000), adenosine (Drejer et al. 1987) and possibly NMDA receptors (Casado et al. 2000, but see Shin & Linden, 2005). While the effects of each of these receptors have been studied in isolation, it is largely unknown how the actions of presynaptic receptors may compliment or oppose one another. GABA receptors (both GABAA and GABAB ) are among the earliest known presynaptic receptors (Eccles et al. 1963; D´esarm´enien et al. 1983). Interestingly, these receptors, which respond to the inhibitory neurotransmitter GABA are co-expressed at a wide range of glutamatergic synaptic terminals, including hippocampal mossy fibres (Ruiz et al. 2003; Cabezas et al. 2012), Schaffer collaterals (Wu & Saggau, 1995; Jang et al. 2005), the calyx of Held (Turecek & Trussell, 2002; Sakaba & Neher, 2003), excitatory afferents to the dorsal raphe nucleus (Soiza-Reilly et al. 2013), axons of cortical pyramidal neurons (Christie & Jahr, 2009; Wang et al. 2010) and cerebellar parallel fibres (Dittman & Regehr, 1996; Stell et al. 2007; Pugh & Jahr, 2011). The widespread co-expression of these receptors suggests they may be involved in a general mechanism by which GABA dynamically regulates excitatory synaptic transmission. These receptors are generally activated by spillover of GABA from neighbouring inhibitory synapses (Pugh & Jahr, 2011, 2013), or by ambient GABA in the extracellular space (Ruiz et al. 2003; but see Alle & Geiger, 2007). Presynaptic GABAA receptors generally depolarize presynaptic terminals, due to high chloride concentrations in axons (Price & Trussell, 2006), and enhance vesicle release (Trigo et al. 2008). Presynaptic GABAB receptors

on the other hand reduce vesicle release by inhibiting voltage-gated calcium channels (Mintz & Bean, 1993; Dittman & Regehr, 1996). Despite extensive studies of the function of each of these receptors in isolation, it is not known how co-activation of these receptors, as is likely to happen in vivo, influences synapse function. Do the opposing actions of these receptors simply cancel each other out with little or no net effect on release probability? Does one receptor effect dominate over the other with a net inhibition or enhancement of release? Or, do temporal and spatial differences in receptor activation allow selective activation of receptors? We have addressed these questions by examining the physiological function of presynaptic GABAA and GABAB receptor co-activation at parallel fibre synapses in the cerebellum. We recorded parallel fibre EPSCs in stellate cells of the cerebellar cortex and GABA was applied on alternating sweeps by photolytic uncaging of RuBi-GABA. We find that GABA induces a rapid enhancement of vesicle release mediated by GABAA receptors, followed by a slow inhibition of release mediated by GABAB receptors. We determined that these changes are presynaptic based on the observations that enhancement of EPSCs is associated with a decrease in the paired pulse ratio, an increase in the variance of response amplitudes, and an increase in the response success rate, while the opposite results were observed for inhibition of EPSCs. GABA uncaging just prior to high-frequency trains of activity reduced short-term facilitation/enhanced depression. This was due to both rapid activation of GABAA receptors (resulting in increased vesicle depletion) and slow activation of GABAB receptors (resulting in reduced release in the middle or end of the train). On the other hand, GABA uncaging hundreds of milliseconds before the onset of a train enhanced short-term facilitation, largely due to GABAB receptor-mediated reduction in release probability. Furthermore, presynaptic GABAB receptors respond to lower concentrations of GABA than GABAA receptors, suggesting they are more likely to respond to ambient GABA or GABA spillover from more distant synapses. These data indicate that presynaptic GABA receptors work together to temporally and spatially regulate synaptic transmission in a pattern not possible from activation of either receptor alone.

 C 2016 The Authors. The Journal of Physiology  C 2016 The Physiological Society

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Biphasic action of presynaptic GABAA and GABAB receptors

Methods Ethical approval

All experimental procedures involving animals were approved by the Institutional Animal Care and Use Committee at the University of Texas Health Science Centre San Antonio. Slice preparation

Acute parasagittal brain slices were prepared from the cerebella of male and female postnatal day (P)14–P30 C57BL/6 mice (Charles River, Wilmington, MA, USA). Mice were deeply anaesthetized with isoflurane before rapid dissection of the cerebellum in accordance with the University of Texas Health Science Centre San Antonio protocols and guidelines. The cerebellum was immediately placed in ice-cold oxygenated ACSF containing the following (in mM): 119 NaCl, 26.2 NaHCO3 , 2.5 KCl, 1.0 NaH2 PO4 , 11 glucose, 2 CaCl2 , 1.3 MgCl2 . Slices (300 μm) were cut from the vermis of the cerebellum using a vibratome (Leica Biosystems, Buffalo Grove, IL, USA) and then incubated at 34°C for 30 min after which they were incubated at room temperature. During recording, slices were superfused with ACSF at room temperature at a flow rate of 2 ml min−1 (where indicated, slices were superfused with ACSF at 32–34°C). For GABA uncaging experiments, 10 ml of ACSF containing 60 μM RuBi-GABA (Tocris, Bristol, UK) was recirculated using a fluid pump (Cole-Parmer, Vernon Hills, IL, USA). Where indicated ACSF also contained one or more of the following: 3 μM CGP 55845 ([(2S)-3-[[(1S)-1-(3,4-dichlorophenyl)ethyl]amino]-2hydroxypropyl]-(phenylmethyl)phosphinic acid; Abcam, Cambridge, MA, USA), 200 μM picrotoxin (PTX; Abcam), 50 μM bicuculline (Tocris), or 20 μM EGTA-AM (Life Technologies, Carlsbad, CA, USA). For some experiments, RuBi-GABA (1–5 mM) was applied to the surface of the slice by pressure ejection (PDES-02DX; NPI electronic, Tamm, Germany) from a patch pipette. In this case, the bath solution was not recirculated. Stellate cells were visually identified in the outer third of the molecular layer and patched with borosilicate pipettes (4–6 M) filled with internal solution containing the following (in mM): 137 potassium gluconate, 2 KCl, 4 MgCl2 , 10 Hepes, 5 EGTA, 4 Na-ATP, 0.5 Na-GTP. The pH was adjusted to 7.2–7.4 using KOH and the osmolarity was 280–300 mosmol l−1 . Electrophysiological currents were recorded with a Multiclamp 700B amplifier (Molecular Devices, Sunnyvale, CA, USA), filtered at 5 kHz and digitized at 50 kHz. Data were collected using pCLAMP software (Molecular Devices). EPSCs were evoked by stimulation of parallel fibres with 1–3 pulses (100 μs, 10–60 V) at 50 Hz through a patch pipette filled with

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ACSF. On alternating sweeps, GABA was photolytically uncaged by full-field 5 ms light pulses from a 470 nm light-emitting diode (LED) (CoolLED, Andover, UK); at 20% power prior to parallel fibre stimulation. In most experiments, EPSCs were evoked either 50 ms after GABA uncaging or 350 ms after uncaging. Five millisecond light pulses were used to maximize activation of axonal GABA receptors and to mimic the relatively slow ‘spillover’ GABA transient experienced by parallel fibre GABA receptors (Pugh & Jahr, 2013). The postsynaptic membrane potential was held near the chloride reversal potential to minimize postsynaptic currents following GABA uncaging. Asynchronous EPSCs were identified by eye. Only synaptic events occurring more than 5 ms after the onset of the second stimulus artifact (in a pair of stimuli delivered at 50 Hz) were counted as asynchronous events. Stimulus artifacts have been digitally removed in all figures. Experiments were performed at room temperature, except where noted. For experiments using minimal electrical stimulation, the stimulating electrode was placed in the tissue and the stimulus intensity was slowly increased until synaptic responses were observed. The stimulus intensity was then increased by 10–20% to ensure reliable activation of the presynaptic axon(s). The synaptic success rate was measured by the number of sweeps with a response on the first stimulus as a proportion of the total number of sweeps. Only cells with a control success rate greater than or equal to 0.1 and less than or equal to 0.9 were included for analysis, as changes in release probability can be obscured at the limits of all successes or all failures. In order to determine how much GABA was uncaged by 5 ms light pulses, we made whole cell patch-clamp recordings from cerebellar granule cells using a high chloride internal solution containing (in mM): 135 KCl, 4 MgCl2 , 10 Hepes, 5 EGTA, 4 Na-ATP, 0.5 Na-GTP, to maximize GABAA receptor-mediated responses. We then placed a borosilicate pipette containing 2.5, 5, or 10 μM GABA in ACSF into the tissue 5–10 μm from the patched cell body. On alternating sweeps (30 s inter-sweep interval to allow clearance of GABA) GABA was applied by either pressure ejection from the pipette (PDES-02DX; NPI electronic) for 200 ms, or by photolytic uncaging of RuBi-GABA (60 μM) using a 5 ms blue light flash. This protocol was repeated with two to four different light intensities (5–20% LED power). From these data we extrapolated the LED power required to match the GABA current produced by a known concentration of GABA (applied by pressure ejection) by calculating the ratio of the peak current during pressure ejection and the peak current during uncaging and multiplying this by the uncaging light intensity used. For each cell this value was averaged across all uncaging light intensities used (only one concentration of GABA was applied by pressure ejection per cell).

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Analysis

Data were analysed using IgorPro (Wavemetrics, Lake Oswego, OR, USA). Statistical significance was determined using paired Student’s t tests in Excel (Microsoft, Redmond, WA, USA). In order to determine the effects of GABA on the profile of short-term plasticity during high-frequency trains of EPSCs, we performed repeated measures two-way ANOVAs on the normalized trains (Sigmaplot, San Jose, CA, USA). GABA application and stimulus number were considered as factors. To determine the effects of PTX and CGP 55845 on trains of EPSCs, we performed two-way ANOVAs on the normalized trains using pharmacological treatment and stimulus number as factors. Bonferroni t tests were used for post hoc comparison. The initial release probability during 100 Hz trains was calculated by fitting the last 15 points of the cumulative EPSC trace with a linear function and back-extrapolating the y-intercept from this fit, a common technique for estimating the size of the readily releasable pool of vesicles (Schneggenburger et al. 1999; Chu et al. 2012). The initial release probability was then calculated by dividing the y-intercept value by the amplitude of the first EPSC for each cell. A value of P ≤ 0.05 was considered significant. Data are reported as means ± SEM.

Results Temporal modulation of synaptic transmission by GABA receptors

Presynaptic GABAA and GABAB receptors have opposite effects on vesicle release at parallel fibre synapses. They also have very different kinetics of activation and deactivation, suggesting these receptors may modulate synaptic transmission over different time scales. In order to determine the relative effects and time course of presynaptic GABAA and GABAB receptors, we made whole-cell patch-clamp recordings from cerebellar stellate cells and pairs of EPSCs (20 ms interval) were evoked by parallel fibre stimulation. On alternating sweeps, GABA was applied by photolytic uncaging of RuBi-GABA (60 μM) using a 470 nm LED, 20–550 ms prior to parallel fibre stimulation (Fig. 1A, top). EPSCs evoked less than 100 ms after GABA uncaging were enhanced (50 ms: 156.2 ± 17.7% of control, n = 30, P < 0.001; Fig. 1A–C), while EPSCs evoked more than 300 ms after GABA uncaging were inhibited (350 ms: 63.0 ± 5.4% of control, n = 28, P = 0.001; Fig. 1A–C). Because channel kinetics can be sensitive to temperature, we repeated this experiment at 32–34°C. At elevated temperature we found the same relationship between GABA uncaging and modulation of EPSC amplitudes (20 ms: 120.3 ± 7.5% of control, n = 13, P = 0.027; 350 ms: 66.1 ± 9.4% of control, n = 8, P = 0.008). However, at elevated temperatures inhibition of EPSCs is

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more rapid, with significant inhibition of EPSCs beginning 150 ms after GABA uncaging. In order to determine whether these effects are due to activation of pre- or postsynaptic GABA receptors, we measured the paired-pulse ratio (PPR) with or without GABA uncaging (20 ms interval between stimuli). EPSCs evoked 50 ms after GABA uncaging showed a decreased PPR (2.9 ± 0.3 vs. 2.2 ± 0.2, n = 28, P = 0.006), while EPSCs evoked 350 ms after uncaging showed an increased PPR (2.4 ± 0.2 vs. 3.2 ± 0.3, n = 25, P = 0.01; Fig. 1D), suggesting a presynaptic effect. However, in these conditions, measurement of PPR may be influenced by activation of molecular layer interneurons (MLIs) during the first stimulus and subsequent release of GABA leading to activation of presynaptic GABA receptors prior to the second stimulus. This would probably increase the apparent PPR in control sweeps, but not uncaging sweeps (when GABA is already present and MLIs are hyperpolarized during the first stimulus). We therefore took the further step of measuring changes in the variance of the first EPSC amplitude following GABA uncaging 50 or 350 ms before the first stimulus. The change in EPSC variance directly correlated with the change in EPSC amplitude (r = 0.52; Fig. 1E), consistent with a presynaptic change of release probability. We also found that asynchronous release events were increased by GABA application 50 ms before the first stimulus (179 ± 33% of control, n = 24, P = 0.05; Fig. 1F), and decreased by GABA application 350 ms before the first stimulus (81 ± 10.5% of control, n = 23, P = 0.036), further confirming that GABA acts through presynaptic changes in release probability. These results show that a single application of GABA both enhances and inhibits vesicle release from parallel fibre synapses in a temporal sequence lasting hundreds of milliseconds. Given the previously described effects of GABAA and GABAB receptors at these synapses (Dittman & Regehr, 1996; Stell et al. 2007; Pugh & Jahr, 2011) and the relative activation kinetics of these receptors (ionotropic GABAA receptors activate rapidly, while metabotropic GABAB receptors activate slowly), we reasoned that the enhancement of EPSCs up to 100 ms after GABA uncaging is probably mediated by GABAA receptors and the inhibition of EPSCs at longer intervals is probably due to GABAB receptors. In order to clearly distinguish between GABAA and GABAB receptor-mediated effects, we measured changes in EPSC amplitude 50 ms or 350 ms after GABA uncaging in the presence of the GABAA receptor antagonist picrotoxin (PTX; 100 μM) and/or the presence of the GABAB receptor antagonist CGP 55845 (3 μM). In PTX, the enhancement of EPSCs at 50 ms was abolished (89.1 ± 9.1% of control, n = 7, P = 0.1), while inhibition of EPSCs at 350 ms was unchanged (80.6 ± 7.1% of control, n = 7, P = 0.04; Fig. 2A (top) and B), suggesting the rapid enhancement of EPSCs is mediated by GABAA receptors. The trend towards  C 2016 The Authors. The Journal of Physiology  C 2016 The Physiological Society

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inhibition of EPSCs in the presence of PTX indicates there may also be a small underlying GABAB receptor-mediated inhibition at this time point that is normally overcome by GABAA receptor activation. Bath application of CGP 55845 abolished the inhibition of EPSCs 350 ms after GABA uncaging (126.5 ± 11.7% of control, n = 16, P = 0.02; Fig. 2A (upper middle) and B), suggesting the slow inhibition is GABAB receptor dependent. However, CGP 55845 also abolished the enhancement of EPSCs 50 ms after uncaging (93.9 ± 5.3% of control, n = 15, P = 0.21; Fig. 2A (upper middle) and B). Previous work has shown that GABAB receptor activation can increase GABAA receptor currents (Connelly et al. 2013; Tao et al. 2013), raising the possibility that blocking GABAB receptors in our experiments reduces the GABAA receptor

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current to the point that enhancement of EPSCs is no longer observable. We tested this possibility by applying higher concentrations of RuBi-GABA (1–5 mM) to the slice through a puffer pipette. Under these conditions GABA enhanced EPSCs at 50 ms in the presence of CGP 55845 (179.4 ± 20.3% of control, n = 14, P = 0.0003; Fig. 2A (lower middle) and B), suggesting enhancement is not GABAB receptor mediated. This enhancement was blocked by the addition of bicuculline (50 μM) and PTX to the bath solution (104.6 ± 10.0% of control, n = 12, P = 0.88), suggesting that enhancement of EPSCs is, indeed, mediated by GABAA receptors, but requires higher GABA concentrations when GABAB receptors are blocked. Consistent with results using 60 μM RuBi-GABA and GABAB receptor-mediated inhibition of EPSCs,

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ISI (ms) Figure 1. Bi-phasic effect of GABA on vesicle release A, top: diagram of GABA uncaging (LED) and parallel fibre stimulation (stim) protocol. Representative average synaptic response in control (black) or following GABA uncaging (red/grey). Synaptic responses are enhanced 50 ms after uncaging (middle) and inhibited 350 ms after uncaging (bottom). B, expanded view of traces in A to show changes in EPSC amplitudes. Traces are baselined to period just before the first stimulus. C, average change in EPSC amplitudes following GABA uncaging as a function of the interstimulus interval (ISI) between uncaging and the first synaptic stimulus. D, paired-pulse ratio in control (black) and after GABA uncaging (red/grey) for responses evoked 50 or 350 ms after GABA uncaging. The control paired-pulse ratios with a 50 ms or 350 ms ISI were not significantly different (P = 0.12). E, for each cell, the change in the average EPSC amplitude 50 ms (black) or 350 ms (grey) after GABA uncaging is plotted against the change in variance of EPSC amplitudes. F, example traces of asynchronous EPSCs in control conditions (black) or following GABA uncaging (red/grey) in the same cell. Pairs of EPSCs were evoked either 50 ms or 350 ms after GABA uncaging. Each plot consists of 10 sweeps.  C 2016 The Authors. The Journal of Physiology  C 2016 The Physiological Society

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Figure 2. Enhancement and inhibition of vesicle release are mediated by GABAA and GABAB receptors, respectively A, representative traces of EPSCs in control (black) or following GABA-uncaging (red/grey; 50 or 350 ms ISI) in the presence of picrotoxin, CGP 55845, or both. In the traces labelled +CGP(high

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uncaging with high RuBi-GABA inhibited EPSCs at 350 ms (52 ± 10% of control, n = 4, P = 0.02), but enhanced EPSCs in the presence of CGP 55845 (193.9 ± 41.6% of control, n = 8, P = 0.01). In the presence of both PTX and CGP 55845, there was no change in EPSC amplitude either 50 ms (102 ± 22% of control, n = 9, P = 0.2) or 350 ms (107 ± 15% of control, n = 10, P = 0.7; Fig. 2A (bottom) and B) after GABA uncaging (60 μM RuBi-GABA). Taken together, these data suggest that both GABAA and GABAB receptors are active for hundreds of milliseconds after GABA uncaging; however, GABAA receptor-mediated enhancement dominates over the first 100 ms and GABAB receptor-mediated inhibition dominates afterwards. Presynaptic GABAA receptors can enhance EPSCs by both increasing release probability at individual synapses and by increasing the excitability of parallel fibres such that more fibres are activated with each stimulus (Pugh & Jahr, 2011; Stell, 2011; Dellal et al. 2012). This raises the possibility that vesicle release is inhibited by GABAB receptors at all time points, but increased excitability of parallel fibres masks this inhibition over the first 100 ms. In order to distinguish between GABAA receptor effects on vesicle release and axon excitability, we evoked EPSCs 50 ms after GABA uncaging in the presence of the acetoxymethyl ester form of EGTA, EGTA-AM, a membrane-permeable calcium chelator. EGTA-AM will block or reduce calcium-dependent processes, such as enhancement of vesicle release by residual calcium, but have little or no effect on calcium-independent processes, such as the change in axon excitability due to depolarization (Chen & Regehr; 1999; Pugh & Jahr, 2011). In the presence of EGTA-AM, EPSCs were inhibited (probably due to GABAB receptor activation) rather than enhanced after GABA uncaging (81.2 ± 5.8% of control, n = 6, P = 0.03; Fig. 3A and B), suggesting GABAA receptors enhance EPSCs primarily through increasing vesicle release probability. EGTA-AM tended to reduce the PPR of control sweeps compared to recordings in the absence of EGTA-AM (P = 0.056), but GABA no longer had a significant effect on PPR (P = 0.1), consistent with chelating residual calcium in the presynaptic terminal. These data confirm that GABAA receptor-mediated effects on vesicle release dominate over the first 100 ms following GABA application. We have used full-field uncaging of GABA in these experiments to activate presynaptic GABA receptors; however, this technique is certain to activate postsynaptic receptors as well. In fact, we often observed a small

GABA), a higher concentration of RuBi-GABA (5 mM) was applied by pressure ejection. B, average change in EPSC amplitudes following GABA uncaging in control (cnt), or in the presence of picrotoxin and/or CGP 55845.  C 2016 The Authors. The Journal of Physiology  C 2016 The Physiological Society

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postsynaptic current despite voltage-clamping the cell near the chloride reversal potential (Fig. 1A). It is therefore possible that changes in postsynaptic input resistance and shunting are at least partially responsible for the observed changes in EPSC amplitude, despite the relatively good space-clamp of cerebellar stellate cells. In order to test this possibility, we estimated vesicular release probability by observing successes and failures of vesicle

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release using minimal electrical stimulation. Parallel fibres typically form only a single synapse with each postsynaptic cell (Napper & Harvey, 1988), and have large quantal responses in stellate cells (Nahir & Jahr, 2013). We could therefore stimulate a single or small number of parallel fibre synapses using minimal electrical stimulation, and easily distinguish failures and successes of vesicle release in the postsynaptic cell (Fig. 4A). We then compared

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Figure 3. GABA enhances EPSC amplitudes by increasing release probability A, diagram (top) showing the relative timing of light (LED) and electrical stimuli (stim) and current traces (bottom) of EPSCs evoked in control (black) or 50 ms after GABA uncaging (red/grey) in the presence of 20 µM EGTA-AM. B, average change in EPSC amplitudes 50 ms after GABA uncaging in control (black, same data as Fig. 2B) or in the presence of EGTA-AM (red/grey).

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Figure 4. GABA modulates response probability during minimal electrical stimulation A, 20 consecutive interleaved sweeps of EPSCs (3 stimuli at 50 Hz; arrows) evoked by minimal parallel fibre stimulation in control (left) or 50 ms after GABA uncaging (right). B, same as in A, with a 350 ms interval between GABA uncaging and parallel fibre stimulation. C, average EPSC success rate in control (black) or following GABA uncaging (red/grey) with an ISI of 50 ms or 350 ms.  C 2016 The Authors. The Journal of Physiology  C 2016 The Physiological Society

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the success rate (number of sweeps with a response as a proportion of all sweeps) of the first stimulus in control and GABA uncaging sweeps. The success rate was significantly increased when parallel fibre stimulation followed GABA uncaging by 50 ms (0.32 ± 0.05 versus 0.45 ± 0.06, n = 16, P = 0.008; Fig. 4A and C) and significantly decreased at 350 ms (0.44 ± 0.06 vs. 0.35 ± 0.06, n = 12, P = 0.003; Fig. 4B and C). There was no significant difference in control success rates at 50 ms or 350 ms (P = 0.12). These data confirm that activation of GABAA and GABAB receptors enhances and inhibits, respectively, EPSCs by modulating vesicle release probability rather than through changes in postsynaptic membrane properties. Effects of presynaptic GABA receptors on high-frequency trains of activity

Granule cells often fire in high frequency bursts of action potentials in vivo (Chadderton et al. 2004). We therefore tested how presynaptic GABAA and GABAB receptors modulate the profile of short-term plasticity during trains of parallel fibre EPSCs. Parallel fibre EPSCs were evoked at 20, 50 and 100 Hz and on alternating sweeps GABA was uncaged either 50 ms or 350 ms before the first stimulus. We found that uncaging GABA 50 ms prior to the first stimulus significantly altered the profile of short-term plasticity at all frequencies tested (20 Hz: n = 11, F(1,80) = 11.097, P = 0.008; 50 Hz: n = 8, F(1,126) = 7.612, P = 0.03; 100 Hz: n = 13, F(1,216) = 9.856, P = 0.009, two-way repeated measures ANOVA (ANOVA RM); Fig. 5B, circles and squares). Specifically, the first EPSC amplitude was generally increased (20 Hz, P = 0.16; 50 Hz, P = 0.08; 100 Hz, P = 0.01), probably due to a GABAA receptor-mediated increase of release probability, and subsequent EPSCs (stim 2–6) were generally reduced, both in absolute amplitude (20 Hz, P < 0.001; 50 Hz, P < 0.001; 100 Hz, P = 0.64; Fig. 5B, triangles) and when normalized to the first EPSC amplitude (20 Hz, P = 0.008; 50 Hz, P = 0.02; 100 Hz, P = 0.03; Fig. 5B, squares), indicating a reduction in short-term facilitation. This could be due to increased release probability at the beginning of the train (and therefore increased vesicle depletion) and/or slow activation of GABAB receptors during the train. In order to distinguish between these possibilities we plotted the cumulative EPCS amplitude vs. stimulus number for 100 Hz trains, a method commonly used to estimate vesicular release probability and size of the readily releasable pool (RRP, Fig. 5C, Schneggenburger et al. 1999; Moulder & Mennerick, 2005;Chu et al. 2012). We found that the total cumulative EPSC amplitude was reduced for all frequencies tested (20 Hz, P < 0.0001; 50 Hz, P = 0.003; 100 Hz, P = 0.008). This result is not consistent with a simple increase in release probability

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at the beginning of the train due to GABAA receptor activation. Previous studies using this analysis at a variety of synapses have found that increasing release probability (by increasing extracellular calcium or activating presynaptic glycine receptors) increases, not decreases, the cumulative EPSC amplitude (Schneggenburger et al. 1999; Chu et al. 2012; Thanawala & Regehr, 2013). Instead, the reduced cumulative EPSC we observed is consistent with GABAB receptor-mediated reduction in release probability and size of the effective RRP, as has been observed previously with presynaptic GABAB receptor activation (Thanawala & Regehr, 2013). In fact, plots of cumulative EPSCs during 100 Hz trains show that, on average, GABA uncaging increases the cumulative EPSC over the first five stimuli and reduces the cumulative EPSC thereafter (Fig. 5D). This pattern of activity is unlikely to arise from activation of either GABAA or GABAB receptors alone; rather, this pattern strongly suggests that the effects of both receptors work together to reduce facilitation at parallel fibre synapses. Taken together, these results indicate that immediately following GABA exposure parallel fibres have enhanced transmission of single action potentials (enhanced first EPSC), but reduced transmission of bursts of action potentials (reduced short-term facilitation/enhanced depression). We also tested the effects of uncaging GABA 350 ms before the first stimulus. Here, we again found a significant effect of GABA uncaging on short-term plasticity (20 Hz: n = 7, F(1,48) = 6.231, P = 0.047; 50 Hz: n = 7, F(1,108) = 9.003, P = 0.024; 100 Hz: n = 7, F(1,108) = 2.843, P = 0.143, two-way ANOVA RM; Fig. 5F, circles and squares). In this case the first EPSC was significantly reduced at all frequencies (20 Hz, P < 0.001; 50 Hz, P = 0.01; 100 Hz, P = 0.04), as would be expected from earlier results (Fig. 1). However, subsequent EPSCs (stim 2–6) were reduced in absolute amplitude (20 Hz, P < 0.001; 50 Hz, P = 0.002; 100 Hz, P = 0.03; Fig. 5F, triangles), but generally enhanced when normalized to the amplitude of the first EPSC (20 Hz, P = 0.06; 50 Hz, P = 0.03; 100 Hz, P = 0.17; Fig. 5F, squares). During 100 Hz trains cumulative EPSC amplitudes were reduced at every point, consistent with previous studies of presynaptic GABAB receptor activation (Thanawala & Regehr, 2013). These data suggest that with longer exposures GABA has the opposite effects on short-term plasticity, enhancing, instead of reducing, facilitation. This means transmission of single action potentials will be reduced (reduced first EPSC), but transmission of bursts will be maintained or enhanced (increased facilitation). Taken together, these data demonstrate that GABA can have opposite effects on short-term facilitation and the relative transmission of single or bursts of action potentials depending on the timing of GABA release and synaptic activity.  C 2016 The Authors. The Journal of Physiology  C 2016 The Physiological Society

Biphasic action of presynaptic GABAA and GABAB receptors

PTX + CGP 55845. We found that drug treatment had significant effects on the profile of short-term plasticity at each frequency (20 Hz: n = 14–21, F(2,459) = 12.405, P < 0.001; 50 Hz: n = 14–19, F(2,900) = 20.718, P < 0.001; 100 Hz: n = 16–19, F(2,960) = 22.973, P < 0.001; Fig. 6). Specifically, application of PTX reduced facilitation/increased depression (Bonferroni t test: P < 0.001 for all frequencies) with no difference in the first EPSC amplitude (P = 0.16–0.6). This suggests

One potential caveat to these experiments is the possibility that high-frequency parallel fibre stimulation itself activates MLIs, causing them to release GABA (Dittman & Regehr, 1997; Stell et al. 2007; Pugh & Jahr, 2011). This raises the possibility that GABA is also present during control sweeps when there is no GABA uncaging. We tested this possibility by evoking trains of EPSCs at 20, 50 and 100 Hz in control conditions, following bath application of PTX, and following bath application of 50 ms

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Figure 5. Effects of presynaptic GABA receptor activation on trains of synaptic responses A, current traces of EPSCs evoked at 20, 50 and 100 Hz in control (black) or 50 ms after GABA uncaging (red/grey) from the same cell (each trace is the average of 10 sweeps). B, average EPSC amplitudes normalized to the first EPSC in the train in control (circles) or following GABA uncaging (squares) for each stimulus frequency. For comparison, EPSC amplitudes following GABA uncaging are also plotted normalized to the first control EPSC amplitude (triangles). C, representative plot of cumulative EPSC amplitudes during 100 Hz train in control conditions (black) or following GABA uncaging (red/grey). Lines are fitted to last 15 points. D, summary of changes for EPSC trains at 100 Hz between control and GABA uncaging. E–H, same as A–D with a 350 ms interval between GABA uncaging and the onset of the stimulus train. Paired t test: ∗ P < 0.05; ∗∗ P < 0.01.  C 2016 The Authors. The Journal of Physiology  C 2016 The Physiological Society

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Figure 6. Release of endogenous GABA during high-frequency stimulation modulates the profile of short-term plasticity A, representative EPSC trains at 50 Hz under control (top), PTX (middle), or PTX + CGP (bottom) conditions. B, normalized EPSC trains at 20 Hz (top), 50 Hz (middle) and 100 Hz (bottom), in control (circles), PTX (triangles) and PTX + CGP (diamonds) conditions. ∗∗∗ Control differs from PTX at P < 0.001. ##,### Control differs from PTX-CGP at P < 0.005 and P < 0.001, respectively. +,+++ PTX differs from PTX-CGP at P < 0.05 and P < 0.001, respectively.

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Biphasic action of presynaptic GABAA and GABAB receptors

that GABA release is evoked by the trains and increases EPSC amplitudes during the train through activation of GABAA receptors. In the presence of PTX and CGP 55845, EPSC trains at 20 and 50 Hz showed less depression compared to trains in PTX alone (Bonferroni t test: 20 Hz, P = 0.044; 50 Hz, P < 0.001), suggesting that GABA release during the train also activates presynaptic GABAB receptors. These data indicate that high frequency parallel fibre stimulation alone is sufficient to activate MLIs and evoke GABA release. Furthermore, parallel fibres were probably exposed to GABA during the control trains in Fig. 5, suggesting the differences observed in short-term plasticity may be an underestimate of the presynaptic effects of GABA. GABA concentration dependence of presynaptic receptor effects

In addition to differences in activation kinetics, GABAA and GABAB receptors can also differ significantly in their affinity for GABA. Somatodendritic GABAB receptors typically have a higher GABA affinity (EC50 20–60 nM; Bowery, 1993; Galvez et al. 2000; Bowery et al. 2002) than GABAA receptors, which can vary greatly depending on subunit composition (EC50 0.5–15 μM; Hanchar et al. 2005; Mortensen et al. 2012). However, biophysical properties of presynaptic channels and receptors can differ significantly from those measured in postsynaptic compartments (Pennock et al. 2012; Huang et al. 2012). To measure the relative GABA concentrations required to produce enhancement or inhibition of parallel fibre EPSCs in stellate cells, pairs of EPSCs were again evoked by parallel fibre stimulation and on alternating sweeps RuBi-GABA was uncaged 50 ms or 350 ms before the first stimulus (see Fig. 1A top). We then measured the enhancement/inhibition of EPSCs for a range of uncaging light intensities from 1% of total LED power (the lowest power at which the LED was reliable) to 20% power (the value used in all other uncaging experiments). We found that EPSCs were inhibited 350 ms after the light pulse for all uncaging powers used (Fig. 7A (bottom) and B, grey), suggesting GABAB receptors are activated at all uncaging intensities (1% LED power: 76.4 ± 26.3% of control, n = 8, P = 0.04). However, enhancement of EPSCs 50 ms after the light pulse was observed only for LED powers of 5% or more (Fig. 7A (top) and B, black), indicating that the GABA concentration uncaged by 1% LED power did not sufficiently activate presynaptic GABAA receptors to enhance release (1% LED: 92.7 ± 30.8%, n = 10, P = 0.51). These data are consistent with presynaptic GABAB receptors having a higher GABA affinity than GABAA receptors, as has been observed for somatodendritic receptors (Bowery et al. 2002; Mortensen et al. 2012). In order to estimate the GABA concentrations required to activate presynaptic GABAA or GABAB  C 2016 The Authors. The Journal of Physiology  C 2016 The Physiological Society

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receptors, we calibrated the uncaging light intensity to GABA concentration. To do this, we made whole-cell patch-clamp recordings from cerebellar granule cells using a high chloride internal solution (ECl = 0 mV). During interleaved sweeps, GABA was applied by photolytic uncaging of RuBi-GABA or pressure ejection of a known concentration of GABA (2.5, 5, or 10 μM) from a pipette. In order to ensure that a maximal GABA concentration reached the granule cell during pressure ejection, the duration was relatively long (200 ms) compared to the uncaging light pulse (5 ms) and the tip of the pipette was placed in the slice near the granule cell soma (10 μm). For each cell we measured currents resulting from pressure ejection of GABA on the soma and from uncaging GABA at a range of light intensities (Fig. 7C). From these data we estimated the uncaging light intensity required to match the current amplitude produced by pressure ejection of GABA directly on the soma. Using this technique we estimated the LED power required to uncage GABA at three different concentrations (2.5, 5, 10 μM). These data were plotted and fitted with a linear function allowing us to estimate the GABA concentration for each uncaging light intensity (Fig. 7D). From this calibration we calculate that uncaging with 1% LED power results in a free GABA concentration of no more than 380 nM and 5% LED power results in a free GABA concentration of no more than 1.2 μM. This indicates that presynaptic GABAB receptors are able to inhibit synaptic transmission when exposed to sub-micromolar GABA, while GABAA receptors require micromolar levels of GABA to enhance vesicle release. Therefore, spillover of GABA from neighbouring inhibitory synapses or ambient GABA may be more effective at activating GABAB receptors than GABAA receptors, especially as the distance between synapses is increased. Discussion In this study we find that despite having opposing actions on vesicle release, presynaptic GABAA and GABAB receptors work in concert to temporally and spatially modulate synaptic transmission at parallel fibre synapses. Following exposure to GABA, rapidly activating GABAA receptors enhance synaptic transmission for 100 ms, after which slowly activating GABAB receptors inhibit transmission for hundreds of milliseconds, producing a brief window of enhanced synaptic transmission. During trains of synaptic stimuli, this has the net effect of reducing short-term facilitation by increasing the first EPSC and inhibiting subsequent EPSCs when GABA is uncaged just prior to the first stimulus. With longer intervals between GABA uncaging and the onset of the train, facilitation is enhanced due primarily to a GABAB receptor-mediated decrease in release probability. Furthermore, presynaptic GABAB receptors are able to

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respond to lower concentrations of GABA than GABAA receptors, and may therefore more effectively respond to GABA spillover from neighbouring inhibitory synapses or to spillover from synapses at a greater distance.

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onto parallel fibre synapses in the presence of GABAB receptor blockers (Pugh & Jahr, 2011). While it is not possible to determine the concentration of GABA reaching the parallel fibres during iontophoresis, it is probably much higher than the 5 μM free GABA produced by uncaging RuBi-GABA with 20% LED power. This suggests that GABAA receptor-mediated enhancement may require a higher GABA concentration in the presence of GABAB receptor antagonists. In fact, we found that when the RuBi-GABA concentration was increased to 1–5 mM, we saw robust enhancement of EPSCs 50 ms after uncaging, even with GABAB receptors blocked, and this enhancement was blocked by bicuculline and PTX, consistent with results in Pugh & Jahr (2011). Recent studies in thalamic relay neurons, dentate granule cells, and cerebellar granule cells have shown that

Interaction between GABAA and GABAB receptors

Consistent with earlier findings (Dittman & Regehr, 1997), we found that inhibition of EPSCs following GABA application was blocked by CGP 55845, indicating this effect is mediated by GABAB receptors. However, enhancement of EPSCs immediately after GABA uncaging was blocked by both picrotoxin and CGP 55845, making interpretation of these results difficult. This result appears to conflict with our previous results showing enhancement of EPSCs 50 ms after iontophoresis of 1 M GABA

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Figure 7. GABA concentration dependence of presynaptic GABAA and GABAB receptor effects A, representative traces of EPSCs evoked in control (black) and 50 ms (top) or 350 ms (bottom) after GABA uncaging (red/grey). GABA was uncaged at light intensities ranging from 1 to 20% of the total LED power. B, average change in EPSC amplitudes evoked 50 ms (black) or 350 ms (grey) after GABA uncaging as a function of LED power used to uncage GABA (n = 5–11). C, example current traces evoked by uncaging RuBi-GABA with 5, 10, or 20% LED power (grey) or pressure application of 5 µM GABA (black trace) recorded from a cerebellar granule cell. In this cell, the peak current evoked by 5 µM GABA closely matched the peak current produced by uncaging with 20% LED power. D, for each cell, the uncaging light intensity required to match the current produced by pressure application of GABA was calculated. These values are plotted against the GABA concentration ejected onto the soma and the average value for each GABA concentration is shown in red/grey. The average values were fitted with a linear function, which was used to convert uncaging light intensities to GABA concentration.

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activation of GABAB receptors enhances tonic currents mediated by extra-synaptic GABAA receptors (Connelly et al. 2013; Tao et al. 2013). While little is known about the molecular composition of presynaptic GABAA receptors, it is likely that they are similar to extrasynaptic receptors on the soma and dendrites of granule cells and are generally included in the broad category of extrasynaptic receptors (Dellal et al. 2012). Our data suggest that presynaptic GABAA receptor currents are also enhanced by GABAB receptor activation. Concentration dependence of GABA effects

Our data show that GABAB receptor-mediated inhibition of vesicle release can be elicited by lower concentrations of GABA (380 nM) than GABAA receptor-mediated enhancement of vesicle release (1.2 μM GABA). This could reflect differences in the location of receptor expression (for example, GABAB receptors may be near the active zone while GABAA receptors are in the axon between boutons). However, previous studies using electron microscopy have demonstrated that both receptor types are found at parallel fibre boutons and within the synaptic active zone (Kulik et al. 2002; Stell et al. 2007), suggesting a large overlap in the spatial distribution of presynaptic GABAA and GABAB receptors. Rather, the different responses to GABA probably reflect differences in the affinity of the receptors and/or differences in their relative expression level. Several factors may skew our estimate of GABA concentrations produced by RuBi-GABA uncaging. We calibrated photolytic uncaging of GABA by comparing the amplitudes of currents produced by flash photolysis with currents produced by pressure ejection of a known concentration of GABA onto the same cell. Despite our efforts to maximize the GABA concentration reaching the cell and coverage of the short granule cell dendrites during pressure ejection (pipette tip was placed within the slice near the cell and ejection duration was 200 ms), it is possible that the GABA concentration that reached the cell was less than that in the pipette at some locations. Furthermore, the rise time of the GABA current following uncaging was relatively rapid (10–90% rise time: 15–50 ms) compared to the rise time following puffing of GABA (140–180 ms) indicating a more rapid GABA concentration jump and more synchronous activation of GABAA receptors. Consequently, more receptors may desensitize before the peak GABA current is reached following a GABA puff resulting in an underestimate of the peak GABA current evoked by each concentration of GABA. These effects would both result in an overestimate of the GABA concentration produced by photolytic uncaging. However, the GABA concentrations produced by these calibrations are still valuable in establishing an upper bound on the GABA concentrations produced by  C 2016 The Authors. The Journal of Physiology  C 2016 The Physiological Society

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each uncaging light intensity, and an upper bound on the minimum GABA concentration necessary to elicit GABAA or GABAB receptor-mediated modulation of vesicle release. In several brain regions, including the cerebellar granule cell layer, ambient GABA in the extracellular fluid can tonically activate GABAA and/or GABAB receptors (Brickley et al. 1996; Jensen et al. 2003). Ambient GABA levels are estimated to be 150–250 nM (Santhakumar et al. 2006; Bright et al. 2011). Our data indicate that presynaptic GABAB receptors can be activated by GABA concentrations as low as 380 nM, and probably much lower, raising the possibility that presynaptic GABAB receptors are also tonically activated by ambient GABA. However, we did not see any change in evoked parallel fibre EPSC amplitudes after washing on CGP 55845 (data not shown) suggesting either ambient GABA is lower in the molecular layer, or there are mechanisms that prevent tonic activation of presynaptic GABAB receptors. Effects on signalling in the cerebellar circuit

Granule cells have been observed to fire single isolated action potentials or high-frequency bursts of four to six action potentials in vivo (Chadderton et al. 2004; van Beugen et al. 2013). The low release probability but highly facilitating nature of parallel fibre synapses suggests single action potentials are poorly transmitted while bursts of action potentials are robustly transmitted at parallel fibre synapses onto MLIs and Purkinje cells. This raises the possibility that parallel fibre synapses act as a high-pass filter in cerebellar signalling, removing the noise of random single action potentials (D’Angelo & De Zeeuw, 2009; van Beugen et al. 2013). However, our results suggest that the relative transmission of single and bursts of action potentials is modulated by activation of presynaptic GABA receptors. We hypothesize that GABA release and spillover onto parallel fibre synapses transiently increases transmission of single action potentials (by increasing the release probability) and decreases transmission of bursts (by reducing facilitation/enhancing depression, see Fig. 5B). The effects of GABA then reverse after 100 ms and transmission of single action potentials is reduced relative to transmission of bursts (facilitation is enhanced even though the absolute amplitudes of EPSCs during a burst are reduced, see Fig. 5F). Therefore, the timing of GABA release and spillover onto parallel fibre boutons provides a mechanism for modulating the level of filtering at parallel fibre synapses and determines the type of information that passes through the cortical loop of the cerebellum. Our measurements were made from parallel fibre synapses onto stellate cells; however, previous work has shown that parallel fibre synapses onto Purkinje cells also express presynaptic GABAA (Stell et al. 2007) and GABAB (Dittman & Regehr, 1996, 1997) receptors,

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suggesting these receptors also modulate filtering of signals onto Purkinje cells. Presynaptic GABA receptors are probably activated by spillover of GABA from neighbouring inhibitory synapses (Stell et al. 2007; Pugh & Jahr, 2011, 2013). We find that GABAB receptor-mediated inhibition of vesicle release can be elicited by lower concentrations of GABA than GABAA receptor-mediated enhancement of release. This means that release of GABA from an inhibitory synapse will result in (transient) enhancement of adjacent parallel fibre synapses receiving a relatively high GABA concentration and inhibition of more distant surrounding parallel fibre synapses receiving a lower GABA concentration. This will probably result in a centre–surround spatial pattern of enhanced–reduced parallel fibre synaptic transmission following GABA release. This pattern of modulation of parallel fibre transmission may reinforce centre–surround patterns of firing observed in the granule cell layer (Mapelli & D’Angelo, 2007; Gandolfi et al. 2014) and enhance pattern detection in the cerebellar cortex (Steuber et al. 2007; Solinas et al. 2010). Balance of excitation and inhibition

Proper functioning of neuronal circuits requires a precise balance of excitation and inhibition, while disruptions of this balance have been associated with several neurological conditions (Zhang & Sun, 2011). Presynaptic GABAA and GABAB receptors are co-expressed at many glutamatergic synapses in the central nervous system (Wu & Saggau, 1995; Turecek & Trussell, 2002; Ruiz et al. 2003; Sakaba & Neher, 2003; Jang et al. 2005; Cabezas et al. 2012; Soiza-Reilly et al. 2013), suggesting this may be a general mechanism by which inhibitory circuits control excitatory neurotransmission and maintain a balance of excitation and inhibition. Regulation of the relative expression levels of presynaptic GABAA and GABAB receptors could fine-tune the excitation–inhibition balance. For example, if a circuit experiences excessive inhibition, presynaptic GABAA receptors may be up-regulated at glutamatergic synapses while GABAB receptors are down-regulated, enhancing glutamatergic neurotransmission and restoring the balance of excitation and inhibition. Likewise, pathophysiological conditions may disrupt this balance by altering presynaptic GABA receptor expression. This has been observed in epilepsy, where seizures cause presynaptic GABAB receptors to be down-regulated at excitatory synapses in the hippocampus, resulting in enhanced excitatory neurotransmission (Chandler et al. 2003; Tsai & Leung, 2006; Thompson et al. 2006). The opposing actions of presynaptic GABAA and GABAB receptors make them ideally suited to maintain (or disrupt) the excitation–inhibition balance by dynamically regulating glutamate release.

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Additional information Competing interests None declared. Author contributions All experiments were performed in the laboratory of Jason Pugh at the University of Texas Health Science Centre San Antonio. R.D.H. and J.R.P. both contributed to the design of the work, acquisition, analysis and interpretation of data, and drafting of the manuscript. Both authors have approved the final version of the manuscript, agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved, and agree all persons designated as authors qualify for authorship, and all those who qualify for authorship are listed. Funding This work was supported by a NIH grant (092809-01) and a Voelker Fund Young Investigator Award to J.R.P. Acknowledgements We thank Adeline Orts-Del’Immagine for helpful discussion and comments on the manuscript.

 C 2016 The Authors. The Journal of Physiology  C 2016 The Physiological Society

Biphasic modulation of parallel fibre synaptic transmission by co-activation of presynaptic GABAA and GABAB receptors in mice.

Many excitatory synapses co-express presynaptic GABAA and GABAB receptors, despite their opposing actions on synaptic transmission. It is still unclea...
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