Blockade of presynaptic GABABRs unsilences glutamatergic synapses
Because GABABRs are ubiquitously expressed at both pre- and postsynaptic structures in the dorsal horn, in this subset of experiments, we nulled postsynaptic GABABRs by intracellular GDP-β-S dialysis (see Methods) to clarify the role of presynaptic GABABRs (Liu et al. 2013). Silent synapses were found in the spinal dorsal horn (Merrill and Wall, 1972) and then other areas of the CNS (Isaac et al. 1995). We here used minimal stimulation technique to investigate the possible roles of presynaptic GABABRs in keeping glutamatergic synapses silent. In 9 neurons where the stimulation strength virtually yielded no detectable response in the first (1st) stimulus, bath application of CGP52432 (1 µM, 5 min), presumably blocking presynaptic GABABRs, switched silent synapses into functional ones (Fig. 1a). In specific, CGP52432 perfusion reliably increased the amplitude of the 1st eEPSCs (control: 0.44 ± 0.24 pA, in CGP52432: 5.89 ± 1.18 pA; P < 0.01, paired t-test, n = 9) and the success rates (control: 1.22 ± 0.81%, in CGP52432: 62.33 ± 6.00%; P < 0.01, paired t-test, n = 9) (Fig. 1b). In the control tests, the synaptic strength of 4 neurons without CGP52432 treatment showed no significant change in 30 min, excluding a drift possibility under our present recording conditions (data not shown). The results suggest that blocking heterosynaptic GABABRs on presynaptic glutamatergic terminals facilitates glutamate release, converts some silent synapses into functional ones. The possible mechanisms for GABABRs keeping glutamatergic synapse silent are shown in Fig. 1c (see Figure legends).
Blockade of presynaptic GABABRs enhances glutamate release
Bath application of CGP52432 (1 µM, 5 min) increased the glutamate release probability. As shown in Fig. 2a, in a paired-pulse stimulation where the 1st stimulation yielded ~50% events detectable (see Methods), CGP52432 perfusion increased the 1st eEPSCs amplitude (control: 6.63 ± 1.21 pA, in CGP52432: 15.00 ± 2.54 pA; P < 0.05, paired t-test, n = 8), enhanced the events (eEPSCs) success ratio (control: 50.38 ± 2.87%, in CGP52432: 89.63 ± 4.25%; P < 0.01, paired t-test, n = 8), and altered the paired pulse ratio (PPR; the ratio of the 2nd eEPSCs amplitude over the 1st eEPSCs amplitude; control: 2.42 ± 0.36, in CGP52432: 1.33 ± 0.27; P < 0.05, paired t-test, n = 8) (Fig. 2a, 2b). The second stimulation also resulted in higher eEPSCs amplitude than that under control conditions (Fig. 2a).
The decreased PPR indicates that this synaptic strength alteration occurs at presynaptic loci (Zucker and Regehr 2002). As suggested in Fig. 2c, the first stimulation only pushed the synapses to “halfway” (~50% events were successful), while under the condition of GABABRs being blocked, most synapses were activated (see Figure legends). Taken together, the results support an idea that presynaptic GABABRs, presumably bound and activated by endogenous GABA, constrain glutamate release.
High frequency stimulation facilitates glutamate release revealed by blocking presynaptic GABABRs
Presynaptic stimulation initiates action potentials and induces GABA release and subsequent spillover from the synaptic cleft; certain range of stimulation frequency induces more GABA release with frequency increasing (Isaac et al. 1995; Zucker and Regehr 2002). In the present study, a train of presynaptic stimulation (5 pulses, 0.1 ms each shock, 5-100 Hz) induced a slow excitatory membrane current that followed the stimulation volley. This membrane current was mediated by presynaptic glutamate release because it was sensitive to a specific AMPA receptor antagonist, CNQX (10 µM). As shown in a representative neuron, a train of presynaptic stimulation at a certain frequency induced a slow membrane current (Fig. 3a1); CGP52432 perfusion (1 µM, 5 min) increased the amplitude of the slow current, with a frequency-dependent manner. In 9 neurons tested, the average amplitude of 100 Hz stimulation-induced current was increased to 293 ± 51% of that induced by 40 Hz train with CGP52432 perfusion (P < 0.01, unpaired t-test, n = 9; Fig. 3b), indicating that the functional presynaptic GABABRs on glutamatergic terminals were activated by endogenous GABA. Compared to that of 40 Hz test, CGP52432 induced smaller “net” effects at lower frequency stimuli (P < 0.05 or P < 0.01, one-way ANOVA; Fig. 3a2).
GABA in the extrasynaptic structures is regulated partially by GABA transporters (GATs) (Isaacson et al. 1993; Sem’yanov 2005). In all 10 neurons tested, tiagabine (30 µM), a GABA transporter 1 (GAT-1) inhibitor, decreased the amplitude of eEPSCs. As shown in a representative neuron in Fig. 3B, further perfusing CGP52432 (1 µM) rescued the eEPSCs; CNQX (10 µM) completely blocked the amplitude, indicating that eEPSCs were mediated by glutamate AMPA receptors.
Postsynaptic GABABRs alter glutamate responses
Holographic photostimulation and whole cell recordings are shown in Fig. 4a. We first verified the methodology of holographic photostimulation. Recording pipette with 10 mM Alexa Fluor 594 revealed the soma and dendrites morphology (Fig. 4b, insert picture). Under the high magnification of microscope, the uncaging spots were firstly aligned as described before (Tang 2006; Lutz et al. 2008; Yang et al. 2011). Increasing the photostimulation light “flashing” pulse width which uncaged more glutamate increased the amplitude of uEPSPs and finally triggered action potentials (Fig. 4b). The subthreshold uEPSPs showed a linear relationship between uncaging light beam duration and amplitude (R2 = 0.9967; P < 0.001). uEPSPs were reversibly inhibited by CNQX (10 µM) and AP-5 (100 µM) to 19.25 ± 4.53% of the control, revealing their ionotropic glutamate receptor-mediated nature (Fig. 4c; control: 14.29 ± 1.23 mV, in the presence of CNQX/AP-5: 2.75 ± 0.65 mV; P < 0.01, one-way ANOVA, n = 8). Intracellular cAMP-dependent protein kinase A (PKA) signaling pathway was indicated in postsynaptic glutamate interaction with GABABRs (Chalifoux and Carter 2010), we thus tested the PKA roles in the present study. The uEPSPs were not significantly changed in the presence of H89 (10 µM, perfused to the slices with superfusion medium), a specific PKA blocker, indicating that the postsynaptic PKA pathway did not mediate the interaction (90.13 ± 6.80% of the control; P > 0.05, one-way ANOVA, n = 8; Fig. 4d).
Taking advantage of holographic stimulation and uncaging glutamate, we compared the effects of postsynaptic GABABRs in modulating glutamate responses. The recording pipette did not include GDP-β-S, making postsynaptic GABABRs available for investigation (Yang et al. 2001a). In the presence of baclofen (10 µM) which activated GABABRs, the average amplitude of uncaged glutamate responses (uEPSPs) decreased to 59.47 ± 11.58% of the control (P = 0.011, paired t-test, n = 10), indicating that postsynaptic GABABRs activation blunted glutamate responses (Fig. 5a). Uncaged glutamate-evoked action potentials were depressed by baclofen, in a reversible manner after washout (Fig. 5b). These results suggest that postsynaptic GABABRs modulate postsynaptic glutamate responses.
We investigated whether ambient GABA induced a tonic postsynaptic inhibition via GABABRs to study the role of endogenous GABA. A voltage ramp protocol (from −155 mV to −25 mV with 800 ms ramp) induced a membrane current and perfusion of CGP52432 (1 µM) shifted the curve with a reversal potential of −85.3 ± 2.5 mV (n = 8; Fig. 5c), which was close to the K+ equilibrium potential (EK) as calculated by the Nernst equation (−89.8 mV) under our recording conditions, suggesting that GABABR-mediated actions via potassium channels. We also tested possible endogenous GABA effects on uEPSPs. However, perfusing CGP52432 (1 µM, 5 min) did not change uEPSPs amplitude (control: 13.55 ± 0.71 mV, in CGP52432: 12.28 ± 0.78 mV; P = 0.18, paired t-test, n = 6), suggesting that endogenous GABA had little effect on postsynaptic glutamate actions (Fig. 5d). Taken together, the results suggest that although endogenous GABA affects postsynaptic membrane K+ current, it contributes little to glutamate responses; however, activating postsynaptic GABABRs by exogeneous agonist baclofen affects postsynaptic glutamate actions.
Postsynaptic GABABRs integrate glutamate actions with different arbors
Blocking endogenous GABABRs has little effect on postsynaptic glutamate action (see Fig. 5), we next investigated endogenous GABABR agonist interaction with glutamate responses. A neuron receives released glutamate at multiple dendrites, but how the inputs at different dendrites are integrated is not clear. In the present study, we took advantage of the holographic photostimulation and whole cell recordings to study the postsynaptic glutamate actions from different arbors. Activating different dendrites from the same soma (Fig. 6a) resulted in uEPSPs integration. In specific, activating a neighbor dendrite facilitated another dendrite uEPSP to the photostimulation (Fig. 6b). We termed this phenomenon as “integration index” (the amplitude ratio of the “testing” uEPSP to the “priming” uEPSP; see Methods). In the presence of baclofen, the integration index significantly decreased (Fig. 6c), indicating that GABABRs mediated integration of glutamate responses between two different arbors (control: 8.83 ± 1.14, in the presence of baclofen: 4.39 ± 0.90; P = 0.02, paired t-test, n = 10), further supporting the idea that postsynaptic GABABRs integrate glutamate-mediated uEPSPs (Fig. 6c).