GABAB Receptors Constrain Glutamate Presynaptic Release and Postsynaptic Actions in Substantia Gelatinosa of Rat Spinal Cord

doi:10.21203/rs.3.rs-976317/v1

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GABA_B Receptors Constrain Glutamate Presynaptic Release and Postsynaptic Actions in Substantia Gelatinosa of Rat Spinal Cord

https://doi.org/10.21203/rs.3.rs-976317/v1

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published 22 Mar, 2022

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The substantia gelatinosa (SG, lamina II of spinal cord gray matter) is pivotal for modulating nociceptive information from the peripheral to the central. γ-Aminobutyric acid type B receptors (GABA_BRs), the metabotropic GABA receptor subtype, are widely expressed in pre- and postsynaptic structures of the SG neurons. Activation of GABA_BRs by exogeneous agonists induces both pre- and postsynaptic inhibitions. However, the actions of endogenous GABA via presynaptic GABA_BRs on glutamatergic synapses, and the postsynaptic GABA_BRs interaction with glutamate, remain elusive. In the present study, first, using in vitro whole cell recordings and taking minimal stimulation strategies, we found that in rat spinal cord glutamatergic synapses, blockade of presynaptic GABA_BRs switched “silent” synapses into active ones and increased the probability of glutamate release onto SG neurons; increasing ambient GABA concentration mimicked GABA_BRs activation on glutamatergic terminals. Next, using holographic photostimulation to uncage glutamate on postsynaptic SG neurons, we found that postsynaptic GABA_BRs modified glutamate-induced postsynaptic potentials. Taken together, our data identify that endogenous GABA heterosynaptically constrains glutamate release via persistently activating presynaptic GABA_BRs; and postsynaptically, GABA_BRs modulate glutamate responses. The results give new clues for endogenous GABA in modulating nociception circuit in spinal dorsal horn and shed fresh light on postsynaptic interaction of glutamate and GABA.

Cellular & Molecular Neuroscience

Spinal cord

GABAB receptors

Minimal stimulation

Holographic photostimulation

Spinal dorsal horn plays a pivotal role in receiving and modulating nociception from the peripheral to the central nervous system (CNS) (Finnerup et al. 2021). Neuronal network in the dorsal horn is mainly composed of excitatory and inhibitory neurons where substantia gelatinosa (SG; lamina II of the spinal cord gray matter) is the main origination of the inhibitory neurotransmitter γ-aminobutyric acid (GABA) (Zeilhofer et al. 2012; Todd 2015). Since the proposal of “gate control theory of pain”, SG has become a target for analyzing nociception modulation (Merighi 2018).

GABA is the primary inhibitory neurotransmitter in the CNS acting on both ionotropic and metabotropic receptors, where GABA_B receptors (GABA_BRs) are the family of metabotropic G protein-coupled receptors (GPCRs). In the spinal dorsal horn, GABA_BRs are expressed in the presynaptic and postsynaptic structures (Yang et al. 2001b; Yang et al. 2002; Malcangio 2018). Maintaining certain concentrations at the synaptic clefts and extrasynaptic structures, GABA exerts an important role in neuronal homeostasis in the CNS (Bowery and Smart 2006). Extrasynaptic GABA (we refer this as “ambient” GABA) is regulated spatially and temporally by GABA transporter activity which is significant in neuronal diseases (Zeilhofer et al. 2012; Smirnova et al. 2020). GABA_BRs are found at both synaptic cleft and extrasynaptic membrane, making GABA_BRs as therapeutic targets for addiction, depression, epilepsy and pain (Goudet et al. 2009; Malcangio 2018).

On the other hand, glutamate is the principal excitatory neurotransmitter in the CNS, glutamatergic synapses onto SG neurons are originated from the primary afferents (Yoshimura and Jessell 1990), intrinsic interneurons (Kato et al. 2007) and the descending pain modulation system (Merighi 2018). Experiments using exogenous agonists revealed that GABA_BRs mediate pre- and postsynaptic inhibitions on glutamate release and action (Ataka et al. 2000; Iyadomi et al. 2000; Yang et al. 2001b). For the action of endogenous GABA, we have previously reported that blocking primary afferents GABA_BRs facilitates action-potential-driven glutamate release, suggesting that endogenous GABA may inhibit the synapses from primary fibers (Yang and Ma 2011). However, the function of endogenous GABA in the spinal dorsal horn circuit remains largely unknown. In specific, whether endogenous GABA constrains silent glutamatergic synapses, and whether there is an integration of GABA_BRs and glutamate actions at postsynaptic neurons, are poorly understood. In the present study, employing whole cell recordings and minimal stimulation strategies at acute rat spinal cord slices, we revealed that endogenous GABA modulates the excitatory glutamate presynaptic release; taking advantage of holographic photostimulation and uncaging glutamate, we found that postsynaptic GABA_BRs blunt glutamate responses.

Slice preparation and whole-cell recordings

All experiments were approved by the Animal Ethics Committee of the institute. Acute transverse spinal cord slices were obtained following methods described elsewhere (Yang et al. 2001a; Yang et al. 2021). In brief, male Sprague-Dawley rats (5-7 weeks old) were anesthetized by urethane (1.5 g/kg bodyweight, i.p.) and a laminectomy was carried out. The spinal cord trunk was quickly transferred to ice-cold “cutting solution” containing (in mM) sucrose 114, KCl 4, NaH₂PO₄ 1.2, MgSO₄ 8, NaHCO₃ 26, CaCl₂ 0.1, and glucose 11, oxygenated with mixed gas (95% O₂/5% CO₂). After removing the dura, arachnoid and pia mater, the lumbosacral trunk was mounted on a vibratome (7000smz-2, Campden Instruments Ltd., Leics, UK). The transverse slices (400 µm in thickness) were quickly transferred to an incubation chamber in artificial cerebrospinal fluid (aCSF) containing (in mM) NaCl 124, KCl 4, NaH₂PO₄ 1.2, NaHCO₃ 26, MgSO₄ 1.5, CaCl₂ 2.5, and glucose 11 (pH 7.2, mOsm 300-305) at ~35ºC with mixed gas saturation for 40 min. Slices were then kept in incubation solution at room temperature (23 ± 2ºC) for using up to 8 h.

Visualized whole-cell recordings were obtained by using glass pipettes with resistance 5-8 MΩ when filled with intrapipette solution composed of (in mM) K-gluconate 130, KCl 5, CaCl₂ 0.1, MgCl₂ 2, EGTA 5, HEPES 5, Na₃-GTP 0.3, and Mg-ATP 4 (adjusted by KOH to pH 7.3; mOsm 285-290). To exclude GABA_AR-mediated and glycine receptor-mediated actions, picrotoxin (100 µM) and strychnine (1 µM) were routinely added to the bath unless otherwise stated. In the presynaptic function experiments, 1 mM guanosine-5’-O-(2-thiodiphosphate) (GDP-β-S) was included in the internal solution to block the possible postsynaptic GABA_BRs’ effects (Yang et al. 2001a). Signals were amplified by Axopatch 200B, digitized by Digidata 1550A which was manipulated by software pClamp 10.2 (all from Molecular Devices, Sunnyvale, CA, USA). For presynaptic stimulation, a glass pipette filled with aCSF was preset 100–300 µm away from the recorded neuron. Stimulation intensity was set at the strength that induced detectable evoked excitatory postsynaptic currents (eEPSCs) which were sensitive to 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 µM), an α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor antagonist. All glutamatergic eEPSCs were recorded at a holding potential of –70 mV. Minimal stimulation protocols were similar to those described elsewhere (Dobrunz and Stevens 1997). Briefly, a minimal stimulation was accepted when the following criteria were satisfied: (1) eEPSCs latency between stimulation artifact and response onset remained stable (< 20% fluctuations); (2) lowering 20% stimulation intensity resulted in total failure of the events; and (3) elevating 20% stimulation intensity yielded no amplitude or shape change. For a subset experiment of synapse evaluation, the stimulation strength was maintained to that about half of all events were detectable. The failures of stimulation responses were estimated by visual discrimination. If the synapses did not respond to the first stimulus but exhibited occasional responses to the second stimulus with 80 ms delay, they were considered as “presynaptic silent synapses” (Voronin and Cherubini 2003; Safiulina and Cherubini 2009). Monosynaptic synapses were accepted if the eEPSCs had stable latency from stimulation artifact to the onset of eEPSCs (< 20% latency fluctuations) and stable amplitude of eEPSCs (< 20% amplitude fluctuations) (Yang et al. 2001a; Yang et al. 2021). To evaluate direct glutamatergic synapses onto SG neurons, only monosynaptic eEPSCs were accepted for further analysis. To evaluate possible postsynaptic GABA_BRs-induced K⁺ currents, we used a “ramp test” protocol as described before (Liu et al. 2012).

Holographic photostimulation and recordings

For three-dimensional (3-D) holographic stimulation, holographic beam was integrated into the optical axis of Olympus BX51WI upright microscope (Olympus, Tokyo, Japan) with a 60× lens (0.9 NA, Olympus), as described before (Tang 2006; Lutz et al. 2008; Yang et al. 2011). The output beam was expanded to a reflective spatial light modulator (SLM, LCOS Hamamatsu, Hamamatsu, Japan), split and controlled by a custom-made software, integrated to Clampex 10.2 electrophysiology acquisition software (Molecular Devices). The soma and dendrites of recorded neurons were illuminated by Alexa Fluor 594 (10 mM in recording pipette; ThermoFisher Scientific, Waltham, MA, USA) under fluorescence illumination (594 nm wavelength for excitation, 617 nm wavelength for emission). Then 10 mM caged glutamate (DNI-glutamate trifluoroacetate, FEMONICS, Budapest, Hungary) was freshly prepared and added to the superfusion medium. The selected spots as uncaging glutamate targets were aligned and the 3-D photostimulation spots were activated by uncaged glutamate at a resolution of 1 µm. The beam for uncaging glutamate was 405 nm wavelength controlled by a custom-made software (Yang et al. 2011). The uncaging excitatory postsynaptic potentials (uEPSPs) were recorded from neural soma at resting membrane potentials.

To investigate the integration of postsynaptic GABA_BRs with glutamate responses, one dendrite was stimulated by transient glutamate uncaging as “testing”, following a “priming” stimulation on a neighbor dendrite (60 ms interval). The “integration index” was calculated as the ratio of the testing uEPSPs amplitude to the priming uEPSPs amplitude. When applying baclofen, to rule out the possible voltage-dependent membrane response shift, membrane potential was maintained to the baseline value by injecting a direct current through the pipettes via “DC current injection” on the amplifier front panel (Liu et al. 2012).

Drugs and data analysis

CNQX, D-2-amino-5-phosphonopentanoic acid (AP-5), (±)-baclofen (baclofen), picrotoxin, strychnine and GDP-β-S were obtained from Sigma-Aldrich (St. Louis, MO, USA), CGP52432 and H89 dihydrochloride (H89) were obtained from Tocris Bioscience (Bristol, UK). Signals were digitized at 10 kHz and stored in a personal computer. Data were analyzed offline using Clampfit 10.2 (Molecular Devices). Results were expressed as mean ± S.E. The CGP52432 and baclofen actions were measured at the stable response. Statistical comparisons of drug effects were made with Student’s t-test for comparison of two groups and one-way ANOVA followed by a Bonferroni post hoc test for comparison of three or more groups with GraphPad Prism software (version 6.02, San Diego, CA, USA). Linear regression was performed using GraphPad Prism. Significance was determined as P < 0.05.

Blockade of presynaptic GABA_BRs unsilences glutamatergic synapses

Because GABA_BRs are ubiquitously expressed at both pre- and postsynaptic structures in the dorsal horn, in this subset of experiments, we nulled postsynaptic GABA_BRs by intracellular GDP-β-S dialysis (see Methods) to clarify the role of presynaptic GABA_BRs (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 GABA_BRs 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 GABA_BRs, 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 GABA_BRs on presynaptic glutamatergic terminals facilitates glutamate release, converts some silent synapses into functional ones. The possible mechanisms for GABA_BRs keeping glutamatergic synapse silent are shown in Fig. 1c (see Figure legends).

Blockade of presynaptic GABA_BRs 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 GABA_BRs being blocked, most synapses were activated (see Figure legends). Taken together, the results support an idea that presynaptic GABA_BRs, presumably bound and activated by endogenous GABA, constrain glutamate release.

High frequency stimulation facilitates glutamate release revealed by blocking presynaptic GABA_BRs

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 GABA_BRs 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 GABA_BRs 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 GABA_BRs (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 GABA_BRs in modulating glutamate responses. The recording pipette did not include GDP-β-S, making postsynaptic GABA_BRs available for investigation (Yang et al. 2001a). In the presence of baclofen (10 µM) which activated GABA_BRs, 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 GABA_BRs 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 GABA_BRs modulate postsynaptic glutamate responses.

We investigated whether ambient GABA induced a tonic postsynaptic inhibition via GABA_BRs 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 (E_K) as calculated by the Nernst equation (−89.8 mV) under our recording conditions, suggesting that GABA_BR-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 GABA_BRs by exogeneous agonist baclofen affects postsynaptic glutamate actions.

Postsynaptic GABA_BRs integrate glutamate actions with different arbors

Blocking endogenous GABA_BRs has little effect on postsynaptic glutamate action (see Fig. 5), we next investigated endogenous GABA_BR 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 GABA_BRs 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 GABA_BRs integrate glutamate-mediated uEPSPs (Fig. 6c).

Technical considerations for presynaptic and postsynaptic GABA_BRs studies

The present study first focused on the presynaptic GABA_BRs in controlling glutamate synapses. Although GABA_BRs are expressed both pre- and postsynaptically, the possible postsynaptic GABA_BR action was ruled out by intracellular GDP-β-S dialysis from the recording electrodes (Yang et al. 2001a). Under these given experimental conditions, the minimal stimulation and the perfusion of CGP52432 only affected presynaptic GABA_BRs, making it possible to clarify GABA_BRs’ presynaptic roles. Our study shows that ambient GABA is able to maintain the silence of some glutamatergic synapses, and that endogenous GABA constrains glutamate release probability onto SG neurons; these actions depend upon ambient GABA concentrations.

On the other hand, to study the postsynaptic actions, the presynaptic GABA_BRs’ role should be ruled out. We here used uncaging glutamate with a defined time and volume which approximated the transmitter release with a couple of micrometer diameter (Dodt et al. 1999; Yang et al. 2011; Yang and Yuste 2018). The present holographic photostimulation combined with whole cell recordings made it possible to precisely give repeated stimulation (Yang et al. 2011), allowing us to bypass the presynaptic terminals and directly activate the postsynaptic glutamate receptors. Our sophisticated holographic photostimulation resulting in glutamate uncaging allowed us to selectively study postsynaptic GABA_BRs, which revealed that the postsynaptic GABA_BRs modulate glutamate-mediated postsynaptic responses.

Presynaptic GABA_BRs’ actions on glutamatergic synapses

A synapse that connects two neurons can be “silent”. Conventional notion suggests that the postsynaptic silencing is attributed to the lack of AMPA receptors on the subsynaptic membrane, and the presynaptic silencing is thought of lacking or insufficient presynaptic neurotransmitter release (Voronin and Cherubini 2003; Kerchner and Nicoll 2008). If a synapse does not respond to the first stimulus but exhibits occasional responses to the second one 30-100 ms latter, it is considered as a “presynaptically” silent synapse (Voronin and Cherubini 2003; Safiulina and Cherubini 2009). In the present study, we used paired-pulses test and clarified that the postsynaptic AMPA receptors were in the presence and the failure of synaptic events was due to too few glutamate release (too weak presynaptic stimulation), this notion is in line with a previous report in the spinal dorsal horn (Yasaka et al. 2009). Occlusion of GABA_BRs switched silent glutamatergic synapses to functional ones, our present results suggest that ambient GABA silences glutamatergic synapses onto SG neurons (Fig. 7).

We also revealed that endogenous GABA constrained heterogeneous glutamatergic terminals by decreasing the release probability, and this process was under the control of presynaptic GABA_BRs by endogenous GABA. Taken together, we conclude that GABA heterogeneously constrains presynaptic glutamatergic synapses, as suggested in Fig. 7.

Certain frequency of train stimulation increases presynaptic neurotransmitter release (Zucker and Regehr 2002). The present data showed a frequency-dependent manner of eEPSCs revealed by CGP52432, suggest that the concentrations of ambient GABA affect GABA_BRs at the presynaptic glutamatergic terminals. The data further support the conclusion that ambient GABA modulates glutamate release.

It should be noted that the present “heterosynaptic hypothesis” is based on assumption that GABA is not co-released from glutamatergic terminals. It has been proposed that glutamate is released at GABAergic synapses together with GABA in the auditory system (Noh et al. 2010) and the lateral habenula (Shabel et al. 2014); this co-release idea, however, was not the case in many brain areas (Uchigashima et al. 2007). In the spinal cord, to our knowledge, there is no evidence showing that inhibitory GABA and excitatory glutamate co-release from the same terminal (Jonas et al. 1998; Jo and Schlichter 1999). Therefore, a logical explanation for the present results is that GABA_BR-mediated inhibition of glutamatergic synapses is due to ambient and/or spillover GABA which binds to GABA_B heteroreceptors at the neighboring glutamatergic terminals (Fig. 7). The common pathway for GABA_BRs modulation on glutamatergic terminals might be through calcium channels (Pfrieger et al. 1994; Yang and Ma 2011).

Postsynaptic GABA_BRs modulation on glutamate-mediated uEPSPs

GABA_BRs mediate calcium channels and potassium channels by intracellular interactions (Kanatamneni 2015; Bettler and Fakler 2017). Since these channels are proteins on the cell surface, it is conceivable that GABA_BRs may integrate glutamate receptors which are virtually membrane proteins. However, there is no complex formation or physical contact has been found between these two kinds of receptors (Bettler and Fakler, 2017). In the present study, we found integration between uEPSPs and GABA_BRs, three possibilities may underlie these results. First, GABA_BRs may modulate the number of excitatory synapses and membrane surface AMPA receptors (Terunuma et al. 2014), Ca²⁺ influx via NMDA receptors may also be inhibited by GABA_BRs (Terunuma 2018). Second, postsynaptic GABA_BRs suppress NMDA receptor responses via an intracellular signaling pathway (Lur and Higley 2015). It should be noted that some reports do not support this notion by showing that neither AMPA nor NMDA receptors are modulated by postsynaptic GABA_BRs (Chalifoux and Carter 2010). The intracellular PKA signaling pathway plays a certain role (Chalifoux and Carter 2010), however, our current results do not find this modulation pathway in SG neurons (Fig. 4c). And the third, in the present study, baclofen decreased the average amplitude of uEPSPs, these may be resulted by an increasing membrane conductance by GABA_BRs activation, as we reported before (Liu et al. 2012). Nevertheless, GABA_BRs’ activities do affect the function of the glutamate receptors and vice versa (Kantamneni 2015). A simplest explain is that different structures have different physiological properties and may interact with each other. In the present study, GABA_BRs are GPCRs and have a long extracellular N-terminal domain, a C-terminal intracellular tail and a transmembrane domain, with integration of other receptors, including glutamate receptors (Kantamneni 2015).

It is intriguing that uncaging glutamate activating from two separate dendrites has integration (Mueller and Egger, 2020; see Fig. 6 in the present study), although the underlying mechanism is still elusive, a “hold and read” theory may explain this. In specific, when one dendrite is activated, this dendrite “holds” a short term information storage by a small depolarization in the soma, the subsequent depolarization from another dendrite “reads out” this information and yields a stronger response (Santos et al. 2012). Single dendrite, and even single spine can serve as basic functional units of neuronal integration by individually detecting the temporal coincidence of postsynaptic activity (Yuste and Denk 1995; Mueller and Egger 2020). It is worth noting that in the present study, the integration is a postsynaptic event, not a presynaptic integration (Zucker and Regehr 2002). Baclofen, presumably activating postsynaptic GABA_BRs, impaired the “hold and read” (Santos et al. 2012), suggesting GABA_BRs not only contribute to postsynaptic glutamate response, but also affect information integration. The integration of ionotropic glutamate receptors and metabotropic GABA_BRs is still unsure (Fig. 7), more experiments are required to elucidate how GABA_BRs affect the postsynaptic integration.

Significance of the findings

GABAergic neurons constitute as many as one-third of neural cells in the brain (Bowery and Smart 2006). Studies on endogenous GABA inhibition are mainly focused on “tonic inhibition” which is mediated by ionotropic GABA_ARs (Kullmann et al. 2005). Enhancing GABAergic tone alleviates neuropathic and inflammatory pain at the spinal cord level via GABA_ARs (Eccles et al. 1963; Witschi et al. 2011; Hanack et al. 2015; Petitjean et al. 2015; Neumann et al. 2021) Relatively, little attention has been paid to the action of GABA_BRs, although they are the first targets for the released GABA in the spinal dorsal horn and mediate the majority of prolonged inhibitory signaling (Chéry and De Koninck 2000). Previous physiological and behavioral reports concerning mechanisms of GABA_BR-mediated antinociception were based on exogenous synthetic agonists (see Introduction), our present study focused on endogenous GABA in modulating glutamatergic synapses via GABA_BRs. Furthermore, this study also clarified the postsynaptic GABA_BRs interaction with glutamate.

Clinical use of GABA analogues is limited largely due to our insufficient understanding of its actions in the “pain circuit”. Ambient GABA modulates glutamatergic synapses, while ambient glutamate may also modulate GABA release from heterosynaptic GABAergic terminals (Mitchell and Silver 2000; Drew et al. 2008; Yang et al. 2015; Bonalume et al. 2021). The “net effect” of GABA_BRs functions depends on the dynamic balance of its actions on excitatory and inhibitory inputs (Malcangio 2018). Given that SG neurons in the superficial dorsal horn play pivotal roles in regulating nociception inflow from primary nociceptors and outflow from projection neurons, the significance of GABA_BR functions manipulated by GABA should be important in the pain circuit. Our data thus reveal an overall view of GABA_BRs on glutamatergic terminals and postsynaptic glutamate actions, paving a way for better understanding the whole scenario of GABA actions in the spinal dorsal horn.

Acknowledgements

The authors thank Prof. Scott M. Thompson, Prof. Cha-Min Tang and Dr. Sunggu Yang (University of Maryland School of Medicine, Baltimore, MD, USA) for support and inspiration; Prof. Eiichi Kumamoto (Saga Medical School, Saga University, Saga, Japan) for critical reading of the manuscript early version. This project was supported by the Jiangsu Province Education Department Grant to K.Y. and funds from the Graduate Research and Practice Innovation Program of Jiangsu Province to M.Z. (KYCX20_3088) and Q.C. (KYCX17_1800).

Author contributions

Mingwei Zhao, Jiaxue Dong, Caifeng Shao, Qian Chen: Investigation; Formal analysis. Rui Ma, Ping Jiang, Wei-Ning Zhang: Formal analysis. Kun Yang: Conceptualization; Methodology; Investigation; Supervision; Project administration; Resources; Writing-original draft; Writing-review & editing.

Competing interests

The authors declare no competing interests.

Additional information

Data Availability Statement: The data that supports the findings of this study are available from the corresponding author upon reasonable request.

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GABA_B Receptors Constrain Glutamate Presynaptic Release and Postsynaptic Actions in Substantia Gelatinosa of Rat Spinal Cord

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Materials And Methods

Slice preparation and whole-cell recordings

Holographic photostimulation and recordings

Drugs and data analysis

Results

Blockade of presynaptic GABA_BRs unsilences glutamatergic synapses

Blockade of presynaptic GABA_BRs enhances glutamate release

High frequency stimulation facilitates glutamate release revealed by blocking presynaptic GABA_BRs

Postsynaptic GABA_BRs alter glutamate responses

Postsynaptic GABA_BRs integrate glutamate actions with different arbors

Discussion

Technical considerations for presynaptic and postsynaptic GABA_BRs studies

Presynaptic GABA_BRs’ actions on glutamatergic synapses

Postsynaptic GABA_BRs modulation on glutamate-mediated uEPSPs

Significance of the findings

Declarations

References

Status:

Journal Publication

Version 1

GABAB Receptors Constrain Glutamate Presynaptic Release and Postsynaptic Actions in Substantia Gelatinosa of Rat Spinal Cord

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Materials And Methods

Slice preparation and whole-cell recordings

Holographic photostimulation and recordings

Drugs and data analysis

Results

Blockade of presynaptic GABABRs unsilences glutamatergic synapses

Blockade of presynaptic GABABRs enhances glutamate release

High frequency stimulation facilitates glutamate release revealed by blocking presynaptic GABABRs

Postsynaptic GABABRs alter glutamate responses

Postsynaptic GABABRs integrate glutamate actions with different arbors

Discussion

Technical considerations for presynaptic and postsynaptic GABABRs studies

Presynaptic GABABRs’ actions on glutamatergic synapses

Postsynaptic GABABRs modulation on glutamate-mediated uEPSPs

Significance of the findings

Declarations

References

Status:

Journal Publication

Version 1

GABA_B Receptors Constrain Glutamate Presynaptic Release and Postsynaptic Actions in Substantia Gelatinosa of Rat Spinal Cord

Blockade of presynaptic GABA_BRs unsilences glutamatergic synapses

Blockade of presynaptic GABA_BRs enhances glutamate release

High frequency stimulation facilitates glutamate release revealed by blocking presynaptic GABA_BRs

Postsynaptic GABA_BRs alter glutamate responses

Postsynaptic GABA_BRs integrate glutamate actions with different arbors

Technical considerations for presynaptic and postsynaptic GABA_BRs studies

Presynaptic GABA_BRs’ actions on glutamatergic synapses

Postsynaptic GABA_BRs modulation on glutamate-mediated uEPSPs