GABA_B receptor modulation of excitatory and inhibitory synaptic transmission onto rat CA3 hippocampal interneurons

Abstract

Hippocampal stratum radiatum inhibitory interneurons receive glutamatergic excitatory innervation via the recurrent collateral fibers of CA3 pyramidal neurons and GABAergic inhibition from other interneurons. We examined both presynaptic- and postsynaptic-GABA_B receptor-mediated responses at both synapse types. Postsynaptic GABA_B receptor-mediated responses were absent in recordings from young (P16-18) but present in recordings from older animals (≥P30) suggesting developmental regulation. In young animals, the GABA_B receptor agonist, baclofen, inhibited the amplitude of evoked EPSCs and IPSCs, an effect blocked by prior application of the selective antagonist CGP55845. Baclofen enhanced the paired-pulse ratio and coefficient of variation of evoked EPSCs and IPSCs, consistent with a presynaptic mechanism of regulation. In addition, baclofen reduced the frequency of miniature IPSCs but not mEPSCs. However, baclofen reduced the frequency of KCl-induced mEPSCs; an effect blocked by Cd²⁺, implicating presynaptic voltage-gated Ca²⁺ channels as a target for baclofen modulation. In contrast, although Cd²⁺ prevented the KCl-induced increase in mIPSC frequency, it failed to block baclofen's reduction of mIPSC frequency. Whereas N- and P/Q-types of Ca²⁺ channels contributed equally to GABA_B receptor-mediated inhibition of EPSCs, more P/Q-type Ca²⁺ channels were involved in GABA_B receptor-mediated inhibition of IPSCs. Finally, baclofen blocked the frequency-dependent depression of EPSCs and IPSCs, but was less effective at blocking frequency-dependent facilitation of EPSCs. Our results demonstrate that presynaptic GABA_B receptors are expressed on the terminals of both excitatory and inhibitory synapses onto CA3 interneurons and that their activation modulates essential components of the release process underlying transmission at these two synapse types.

GABA_B receptors belong to the larger super family of G-protein coupled heptahelical transmembrane receptors (Couve et al. 2000). Binding of GABA to GABA_B receptors results in GDP/GTP exchange in the associated G-protein and diffusion of G_α and G_βγ subunits, which couple to a wide variety of intracellular targets including adenylyl cyclase, inwardly rectifying K⁺ channels and voltage-gated Ca²⁺ channels (Mott & Lewis, 1994). GABA_B receptor-mediated activation of K⁺ channels typically produces postsynaptic hyperpolarization and inhibits neuronal excitability (Gage, 1992). Activation of GABA_B receptors on presynaptic terminals reduces GABA and glutamate release at numerous inhibitory and excitatory synapses throughout the mammalian central nervous system mainly by inhibiting presynaptic Ca²⁺ channels (Misgeld et al. 1995; Doze et al. 1995), although other mechanisms have been implicated (Capogna et al. 1996; Jarolimek & Misgeld, 1997). GABA_B receptor activation is involved in numerous neuronal processes, including regulation of long term potentiation induction (Davies et al. 1991; Mott & Lewis, 1991; Vogt & Nicoll, 1999) and modulation of rhythmic activity in the hippocampus (Scanziani, 2000).

GABAergic inhibitory interneurons in CA3 stratum (st.) radiatum receive both excitatory synaptic innervation from the recurrent collateral fibres of CA3 pyramidal neurons and inhibitory synaptic transmission from other GABAergic inhibitory interneurons distributed across numerous subfields (Freund & Buzsaki, 1996). Whereas GABA_B receptor-mediated inhibitory postsynaptic potentials and/or currents (IPSP(C)s) have been described in interneurons throughout the hippocampus, including CA1 st. pyramidale (Lacaille, 1991), CA1 st. lacunosum-moleculare (Khazipov et al. 1995) and dentate-hilus border (Mott et al. 1999), to our surprise, GABA_B receptor-mediated modulation of presynaptic transmitter release at either excitatory or inhibitory synapses onto CA3 interneurons has not been elucidated.

In the present paper, we examine the effects of GABA_B receptor activation on excitatory and inhibitory synaptic transmission onto interneurons located in the CA3 st. radiatum. Our results indicate that activation of GABA_B receptors inhibits both excitatory and inhibitory synaptic transmission at CA3 interneuron synapses via presynaptic mechanisms.

Methods

Hippocampal slice preparation

Transverse hippocampal slices (300 μm) were obtained from 16-18-day-old Sprague-Dawley rats, unless otherwise stated in the text, as described previously (Toth & McBain, 1998; Lei & McBain, 2002). Rats were deeply anaesthetized with isoflurane, rapidly decapitated, and the brain dissected out in ice-cold saline solution that contained (mm): 130 NaCl, 24 NaHCO₃, 3.5 KCl, 1.25 NaH₂PO₄, 1.0 CaCl₂, 5.0 MgCl₂, and 10 glucose, saturated with 95 % O₂ and 5 % CO₂, pH 7.4. All animal procedures conformed to the National Institutes of Health animal welfare guidelines.

Electrophysiology

Whole-cell patch-clamp recordings were made from visually identified interneurons located in the st. radiatum of CA3 by the use of an Axopatch 1D amplifier (Axon Instruments, Foster City, CA, USA) in voltage-clamp mode. Unless otherwise stated, recording electrodes were filled with the following (mm): 100 caesium gluconate, 0.6 EGTA, 5 MgCl₂, 8 NaCl, 2 ATP₂Na, 0.3 GTPNa, 40 Hepes, and 1 QX-314, pH 7.2-7.3. Biocytin (0.2 %) was routinely added to the recording electrode solution to allow post hoc morphological processing of recorded cells (Toth & McBain, 1998). In experiments where postsynaptic GABA_B responses were investigated, caesium gluconate was replaced by equimolar potassium gluconate in the internal solution. The extracellular solution comprised the following (mm): 130 NaCl, 24 NaHCO₃, 3.5 KCl, 1.25 NaHPO₄, 1.5 MgCl₂, 2.5 CaCl₂ and 10 glucose, saturated with 95 % O₂ and 5 % CO₂, pH 7.4.

Synaptic responses were evoked by placing a patch electrode filled with oxygenated extracellular solution in the st. radiatum to stimulate recurrent collateral fibres from CA3 pyramidal neurons (for EPSCs) and axons from other GABAergic interneurons (for IPSCs) via a constant-current isolation unit (A360; World Precision Instrument, Sarasota, FL, USA). With this method, contamination from mossy fibres could be excluded because synaptic currents were not sensitive to DCG-IV (102.9 ± 5.5 % of control, n = 5, Toth & McBain 2000; Lei & McBain, 2002). Unless stated otherwise, stimulation frequency was usually 0.333 Hz. Synaptic responses were included in the analysis if the rise times and decay time constants were monotonic and possessed no apparent multiple or polysynaptic waveforms. AMPA receptor-mediated EPSCs were evoked at a holding potential of −65 mV and were isolated by including bicuculline methobromide (20 μm) in the extracellular solution to block GABA_A responses. To record GABA_A receptor-mediated IPSCs, the external solution was supplemented with dl-APV (100 μm) and DNQX (10 μm) to block NMDA and AMPA receptors, respectively. Under these conditions, evoked inhibitory currents had a reversal potential of ≈-30 mV and were completely blocked by bicuculline methobromide (20 μm, Fig. 1B) confirming that they were mediated by GABA_A receptors. Usually IPSCs were evoked at a holding potential of +40 mV.

Figure 1. Baclofen inhibits both evoked EPSCs and IPSCs.

A, bath application of baclofen (20 μm) inhibited AMPA receptor-mediated EPSCs at an excitatory synapse. Upper panel, traces averaged from 10 EPSCs taken at the time points indicated in the lower panel. The stimulation artifact was blanked for each trace. Lower panel, time course of baclofen-mediated inhibition of EPSCs at an excitatory synapse. Note that the evoked EPSCs were completely inhibited by 10 μm DNQX at the end of the experiment indicating that they were mediated by AMPA receptors. B, GABA_A receptor-mediated IPSCs were inhibited by baclofen (20 μm). Upper panel, traces averaged from 10 IPSCs taken at the time points indicated in the lower panel. The holding potential was +40 mV. The stimulation artifact was blanked for each trace. Lower panel, time course of baclofen-mediated inhibition of IPSCs at an inhibitory synapse. Note that IPSCs were completely inhibited by application of 20 μm bicuculline at the end of the experiment indicating that they were mediated by GABA_A receptors. C, summarized mean data. **P < 0.01. D, concentration-response curve for baclofen-mediated inhibition of EPSCs. Numbers in parentheses are the number of cells recorded at each concentration. The concentration-response curve was fitted by the Hill equation (see Methods). IC₅₀ of baclofen-mediated inhibition of EPSCs is 8.5 μm. E, concentration-response curve for baclofen-mediated inhibition of IPSCs. Numbers in parenthesis are the number of cells recorded at each concentration. IC₅₀ fitted from the Hill equation is 1.7 μm.

Interneurons were identified on the basis of somata shape and position in the CA3 st. radiatum subfield using infrared video microscopy and differential interference contrast optics. Despite marked cell heterogeneity, all cells were treated as a single pool because GABA_B receptor-mediated modulation was uniform throughout the recorded cell population. Recordings were made at room temperature (≈24 °C). Series resistance was rigorously monitored by the delivery of 5 mV voltage steps after each evoked response. Experiments were discontinued if the series resistance changed by >15 %.

Spontaneous or miniature synaptic currents were initially recorded using pCLAMP 8 and subsequently analysed by Axograph 4.7 (Axon Instruments). A semi-automated sliding template for event detection was used (Clements & Bekkers 1997). The template was slid across the data one point at a time and optimally scaled and offset to fit the data at each position. The event detection was calculated from a scaled template and from the goodness of fit between the scaled template and the data. The threshold for detection was set to 3 times the standard deviation of the noise as recorded in an event-free stretch of data (Clements & Beckers 1997). Spontaneous events were recorded in the absence of TTX, and miniature synaptic currents were recorded in the presence of TTX (1 μm). To record depolarization-induced miniature synaptic currents the [KCl]_o concentration was elevated by 10 mm in the TTX-containing extracellular solution and where applicable CdCl₂ (100 μm) was added to the above solution to block depolarization-induced opening of Ca²⁺ channels (Doze et al. 1995).

Data analysis

Data are presented as the means ± s.e.m.. The paired-pulse ratio (PPR) was calculated as the meanP2/meanP1 (Kim & Alger 2001), where P1 was the amplitude of first evoked current and P2 was the amplitude of the second synaptic current, measured after subtraction of the remaining P1 ‘tail’ current. The coefficient of variation (CV) of synaptic currents was calculated as the standard deviation (s.d.) of current amplitude divided by the mean (x) of the current amplitude (CV = s.d./x). The concentration-response curves for baclofen-mediated inhibition of EPSCs and IPSCs were fitted by the Hill equation: I = I_max{1/[1+(IC₅₀/[ligand])^nH]}, where I_max is the maximum response, IC₅₀ is the concentration of ligand producing a half-maximal response, and n_H is the Hill coefficient. Student's paired or unpaired t test or analysis of variance (ANOVA) was used for statistical analysis as appropriate; P values are reported throughout the text.

Chemicals

NO711 and CGP55845 were purchased from Tocris (Ellisville, MO, USA). The following chemicals are products of Sigma (St Louis, MiO, USA): baclofen, 6,7-dinitroquinoxaline-2,3(1H, 4H)-dione (DNQX), bicuculline methobromide, dl-2-amino-5-phosphonovaleric acid (dl-APV), tetrodotoxin (TTX), ω-conotoxin GVIA, ω-agatoxin TK.

Results

Postsynaptic GABA_B receptor-mediated responses in CA3 interneurons

GABA_B receptor-mediated slow IPSP(C)s and responses to exogenously applied GABA_B-receptor agonists have been observed in interneurons of the CA1 st. pyramidale (Lacaille, 1991), lacunosum-moleculare (Khazipov et al. 1995) and dentate-hilus border (Mott et al. 1999). We initially examined whether postsynaptic activation of GABA_B receptors could be detected in CA3 interneurons of the st. radiatum. As GABA_B receptor-mediated postsynaptic responses are typically mediated by activation of a potassium conductance (Gage, 1992), application of baclofen, a GABA_B receptor agonist, would be expected to generate an outward current recorded at −60 mV with a K⁺-containing intracellular solution.

In hippocampal slices prepared from animals of postnatal age range 16-18, the holding current was not significantly affected by bath application of baclofen (100 μm, 100.8 ± 12.9 % of control, n = 5, P > 0.05) at a holding potential of −60 mV. Recordings were performed in the presence of dl-APV (100 μm), DNQX (10 μm) and bicuculline (20 μm) to block NMDA-, AMPA- and GABA_A-receptor-mediated currents, respectively. Since GABA_B-receptor-mediated synaptic transmission in the CA1 hippocampus is observed only at later developmental stages (>22 postnatal day, Nurse & Lacaille, 1999), we repeated these experiments in slices from older animals. In slices from >P30 animals, baclofen (100 μm) generated an outward current of 44.5 ± 10.1 pA (n = 6, P < 0.01; data not shown). Prior application of CGP55845 (2 μm), a specific GABA_B receptor antagonist, blocked the baclofen-induced outward current (103.2 ± 5.5 % of control, n = 5, P >0.05). These results are consistent with the observation that postsynaptic GABA_B receptor-mediated conductances are developmentally regulated and not expressed on interneurons of CA3 st. radiatum until later ages. Postsynaptic GABA_B responses on st. radiatum interneurons were not studied further. To exclude potential influences of postsynaptic GABA_B receptor-mediated responses for the remainder of experiments, which examine modulation of presynaptic GABA_B receptors at CA3 interneuron synapses, recordings were made from 16-18-day-old animals using caesium-based intracellular recording electrodes (see Methods).

GABA_B receptor-mediated inhibition of excitatory and inhibitory synaptic transmission

CA3 st. radiatum interneurons receive glutamatergic excitation from recurrent collateral fibres of CA3 pyramidal neurons and GABAergic inhibition from interneurons located in numerous subfields (Freund & Buzsaki 1996). Bath application of baclofen (20 μm) reversibly inhibited evoked AMPA receptor-mediated EPSCs to 51.0 ± 4.6 % of control (n = 14, P < 0.01, Fig. 1A and C) and evoked GABA_A receptor-mediated IPSCs to 20.5 ± 3.9 % of control (n = 8, P < 0.01, Fig. 1B and C). To verify the involvement of GABA_B receptors in the baclofen-mediated inhibition of EPSCs and IPSCs, we repeated the above experiments in the presence of the specific GABA_B receptor antagonist, CGP55845. Prior application of CGP55845 (2 μm) alone had no significant effect on AMPA receptor-mediated EPSCs (104.9 ± 6.9 % of control, n = 8, P = 0.5; Fig. 2A and B), but completely blocked the baclofen-mediated inhibition of EPSCs (107.4 ± 7.1 % of control, n = 8, P = 0.34) confirming that the effects of baclofen were mediated by activation of GABA_B receptors (Fig. 2A and B). Similarly, in the presence of CGP55845, baclofen failed to significantly inhibit IPSCs (91.0 ± 4.1 % of control, n = 8, P = 0.06, Fig. 2C and D). However, IPSC amplitudes were significantly enhanced by application of CGP55845 alone (135.8 ± 11.7 % of control, n = 8, P = 0.02) suggesting that activation of GABA_B receptors by ambient GABA exerted a tonic inhibitory effect on inhibitory synapses onto st. radiatum interneurons (Fig. 2C and D).

Figure 2. Baclofen-mediated inhibition of EPSCs and IPSCs occurs via GABA_B receptor activation.

A, prior application of CGP 55845 (2 μm) blocks the inhibition of baclofen (20 μm). Note CGP 55845 failed to enhance the EPSC amplitude. B, summarized mean data. C, application of CGP 55845 enhanced IPSC amplitudes and prevented their inhibition by baclofen. D, summarized mean data. *P < 0.05.

Construction of concentration-response curves for baclofen inhibition of EPSCs and IPSCs revealed an IC₅₀ of baclofen of 8.5 μm for EPSCs (Fig. 1D) and 1.7 μm for IPSCs (Fig. 1E), suggesting that baclofen was more potent at inhibitory synapses. However, we cannot rule out that the lower IC₅₀ for baclofen at inhibitory synapses results in part from a leftward shift in the concentration-response curve caused by tonic activation of GABA_B receptors as described above. Alternatively, molecularly distinct GABA_B receptors may be present on inhibitory versus excitatory terminals or the downstream targets that couple GABA_B receptors to their effector mechanisms may differ at the two synapse types.

Effect of GABA diffusion

A simple explanation for the differential tonic inhibition by endogenous GABA of inhibitory versus excitatory terminals, may be that the ambient GABA concentration reaching excitatory synapses is significantly lower than that at inhibitory synapses. This would not be surprising given that GABA probably only reaches excitatory synapses by diffusion from other inhibitory synapses. Since the concentration of GABA in the synaptic cleft can be enhanced by NO711, an inhibitor of both glial and neuronal GABA uptake (Suzdak et al. 1992), we next tested if GABA_B receptor-mediated inhibition by endogenously released GABA was observed at excitatory synapses if the GABA concentration at these synapses was elevated by the GABA transporter inhibitor. Bath application of NO711 (100 μm) significantly reduced EPSC amplitude to 72.9 ± 5.0 % of control (n = 6, P < 0.01 Fig. 3A); an effect blocked by co-application of CGP55845 (99.8 ± 4.4 % of control, n = 5, P > 0.05) suggesting that GABA_B receptors were involved in NO711-mediated inhibition of EPSCs. NO711 also reduced IPSCs to 64.3 ± 4.9 % of control (n = 5, P < 0.01). However, NO711-mediated inhibition was not completely prevented by CGP55845 (80.2 ± 6.0 %, n = 6, P < 0.05; Fig. 3B). The incomplete block may be due to the GABA-induced desensitization of postsynaptic GABA_A receptors by the elevated levels of persistent endogenous GABA (Overstreet et al. 2000).

Figure 3. Elevation of GABA concentration in the synaptic cleft by a GABA transporter inhibitor inhibits both EPSCs and IPSCs.

A, upper traces, EPSCs recorded from a synapse before (left) and during (right) the application of NO711 (100 μm). Lower traces, EPSCs recorded from another synapse during the application of CGP 55845 (2 μm) alone (left) and during the co-applications of both CGP 55845 and NO711 (right). Bottom, summarized EPSC data. Note that co-application of CGP 55845 completely blocked the inhibition mediated by NO711. B, upper traces, IPSCs recorded from a cell before (left) and during (right) application of NO711 (100 μm). Lower traces, IPSCs recorded from another cell during application of CGP55845 (2 μm) alone (left) and during the co-application of both CGP 55845 and NO711 (right). Bottom, summarized IPSC data. Note that co-application of CGP 55845 incompletely blocked the inhibition mediated by NO711.

GABA_B inhibition occurs via presynaptic mechanisms

We next examined whether the baclofen-mediated inhibition of EPSCs or IPSCs was mediated by a reduction of presynaptic transmitter release. We tested this possibility by monitoring the paired-pulse ratio (PPR) and the coefficient of variation (c.v.) of evoked EPSC and IPSC amplitudes. While changes in c.v. do not unequivocally reflect a presynaptic mechanism (Silver et al. 1998), these two parameters are widely used to evaluate changes in neurotransmitter release probability (Malinow & Tsien, 1990; McAllister & Stevens, 2000; Zucker & Regehr, 2002).

Bath application of baclofen (10 μm) significantly increased the PPR (calculated as meanP2/meanP1, Kim & Alger, 2001) of both EPSCs evoked at stimulus intervals between 25 and 100 ms (Fig. 4A) and IPSCs at stimulus intervals between 10 and 200 ms (Fig. 4B). The c.v. of both EPSCs and IPSCs was also reversibly enhanced by baclofen (Fig. 5). Taken together these two results suggest that the inhibition of both excitatory and inhibitory transmission by baclofen is mediated by activation of presynaptic GABA_B receptors.

Figure 4. Baclofen increases the paired pulse ratio of both EPSCs and IPSCs.

A, baclofen (10 μm) increases the PPR of EPSCs. Upper traces averaged from 20 EPSCs recorded by a paired-pulse stimulation at an interval of 100 ms before (thin) and in the presence of baclofen (thick). Lower traces, EPSCs recorded in the absence and presence of baclofen were scaled to the first EPSC. Note the second EPSC in the presence of baclofen is larger than control. Bottom, PPRs recorded at different intervals before and during the application of baclofen. Note that only the PPRs recorded between the intervals of 25 and 100 ms were significantly larger in the presence of baclofen than those in the absence of baclofen. *P < 0.05; **P < 0.01. B, baclofen (10 μm) increases the PPR of IPSCs. Upper traces averaged from 20 IPSCs recorded by a paired-pulse stimulation at an interval of 50 ms before (thin) and in the presence of baclofen (thick). Lower traces, IPSCs recorded in the absence and presence of baclofen were scaled to the first IPSC. Note the second IPSC in the presence of baclofen is larger than control. Bottom, PPRs recorded at different intervals before and during the application of baclofen. Note that the PPRs recorded at each interval were significantly larger in the presence of baclofen.

Figure 5. Baclofen increases the c.v. of both EPSCs and IPSCs.

A, baclofen increases the CV of EPSCs. Upper traces, ten consecutive EPSCs recorded in the absence (left) and presence (right) of baclofen at the time points indicated in the lower panel. Plots, time course of change in c.v. from an excitatory synapse before, during and after application of baclofen. Each point represents the c.v. calculated from 10 consecutive EPSCs. B, c.v. of EPSCs calculated from 14 neurons before, during and after application of baclofen. C, baclofen increases the c.v. of IPSCs. Upper traces, ten consecutive IPSCs recorded in the absence (left) and presence (right) of baclofen at the time points in the plots. Plots, time course of change in c.v. from an inhibitory synapse before, during and after application of baclofen. Each point represents the c.v. calculated from 10 consecutive IPSCs. D, c.v. of IPSCs calculated from 8 neurons before, during and after the application of baclofen.

We next tested the involvement of presynaptic Ca²⁺ channels in GABA_B receptor-mediated inhibition of transmitter release. We examined the effects of baclofen on spontaneous (in the absence of TTX, i.e. Ca²⁺-dependent transmitter release) and miniature (in the presence of TTX, Ca²⁺-independent transmitter release) synaptic currents. Baclofen (10 μm) significantly reduced both the amplitude (control, 18.1 ± 3.9 pA; baclofen, 12.0 ± 2.7 pA; n = 5, P < 0.05) and frequency (control, 4.1 ± 0.9 Hz; baclofen, 1.7 ± 0.6 Hz; n = 5, P < 0.01) of spontaneous (s)EPSCs (Fig. 6A). However, neither the amplitude (control, 22.7 ± 1.3; baclofen, 20.9 ± 1.3 pA, n = 5, P > 0.05) nor the frequency (control, 8.3 ± 3.9; baclofen, 6.1 ± 2.7 Hz, n = 5, P > 0.05) of miniature excitatory synaptic currents (mEPSCs), recorded in the presence of TTX, was significantly affected by baclofen (Fig. 6B). These results suggest that GABA_B receptor-mediated inhibition of EPSCs is related to Ca²⁺ influx, presumably through presynaptic Ca²⁺ channels. To confirm further the involvement of presynaptic Ca²⁺ channels, we elevated the concentration of KCl in the extracellular solution by 10 mm (from 3.5 to 13.5 mm) to depolarize the presynaptic membrane and activate Ca²⁺ channels in the continued presence of TTX. Elevation of [KCl]_o significantly increased the frequency of mEPSCs (control, 4.7 ± 1.1 Hz; KCl, 8.3 ± 2.2 Hz; n = 6, P < 0.05). Under these conditions baclofen significantly reduced the frequency of mEPSCs (KCl, 8.3 ± 2.2 Hz; KCl + baclofen, 4.2 ± 1.4 Hz, n = 6, P < 0.01) with no effect on the mEPSC amplitude (KCl, 17.8 ± 2.3 pA; KCl + baclofen, 16.8 ± 2.0 pA, n = 6, P > 0.05, Fig. 6C). If the inhibitory effect of baclofen in the presence of KCl is via inhibition of presynaptic Ca²⁺ channels, inhibition of Ca²⁺ channels should block the effects of baclofen. Bath application of Cd²⁺ (100 μm), a non-specific Ca²⁺ channel blocker, blocked the increase in mEPSC frequency induced by KCl (Cd²⁺, 5.4 ± 1.1; Cd²⁺ + KCl, 5.8 ± 0.9 Hz, n = 5, P > 0.05) and the decrease in mEPSC frequency induced by baclofen (Cd²⁺ + KCl, 5.8 ± 0.9 Hz; Cd²⁺ + KCl + baclofen, 5.9 ± 1.1 Hz, n = 5, P > 0.05, data not shown). These results indicate that GABA_B receptor activation reduces glutamate release primarily by inhibiting, Cd²⁺-sensitive, presynaptic Ca²⁺ channels involved in the transmitter release mechanism (Doze et al. 1995).

Figure 6. Modulation of spontaneous or miniature synaptic currents by GABA_B receptor activation.

Both the amplitude (A) and the frequency (F) of spontaneous EPSCs recorded in the absence of TTX were inhibited by baclofen (10 μm). Open bars, control; filled bars, baclofen. B, neither the amplitude nor the frequency of the miniature EPSCs recorded in the presence of TTX (1 μm) was inhibited by baclofen (10 μm). C, baclofen reduced the frequency of the miniature EPSCs recorded in the combined presence of TTX (1 μm) and elevated KCl (by 10 mm) without an effect on their amplitudes. D, both the amplitude and the frequency of the spontaneous IPSCs recorded in the absence of TTX were inhibited by baclofen (10 μm). E, only the frequency of miniature IPSCs was inhibited by baclofen (10 μm). F, baclofen reduced the frequency of miniature IPSCs recorded in the presence of both TTX (1 μm) and elevated KCl (by 10 mm) without inhibiting their amplitudes. For all the symbols in the figure, open bars represent control and filled bars represent the values in the presence of baclofen.

Bath application of baclofen reduced both the amplitude (control, 21.8 ± 1.5pA; baclofen, 14.3 ± 1.3 pA; n = 5, P < 0.01) and frequency (control, 5.9 ± 0.2 Hz; baclofen, 3.4 ± 0.2 Hz; n = 5, P < 0.01) of spontaneous inhibitory synaptic currents (sIPSCs; Fig. 6D). Unlike its effect on mEPSCs, baclofen also inhibited the frequency of miniature inhibitory synaptic currents (mIPSCs) recorded in the presence of TTX (control, 2.8 ± 1.1; baclofen, 1.5 ± 0.7 Hz; n = 5, P < 0.05, Fig. 6E) with no effects on their amplitude (control, 20.1 ± 3.1 pA; baclofen, 17.7 ± 2.9 pA; n = 5, P > 0.05), suggesting a Ca²⁺-independent effect of baclofen. Similar to mEPSCs, elevation of [KCl]_o increased mIPSC frequency (control, 3.1 ± 0.5 Hz; KCl, 4.7 ± 0.7 Hz; n = 5, P < 0.01). Baclofen subsequently reduced mIPSC frequency recorded in the presence of both TTX and elevated KCl (KCl, 4.7 ± 0.7 Hz; KCl + baclofen, 2.3 ± 0.4 Hz; n = 5, P < 0.05, Fig. 6F) with no effects on their amplitude (KCl, 21.9 ± 1.8 pA; KCl + baclofen, 19.6 ± 2.2 pA; n = 5, P > 0.05; Fig. 6F). Application of Cd²⁺ blocked the effects of KCl (Cd²⁺, 3.6 ± 0.8 Hz; Cd²⁺ + KCl, 3.9 ± 0.9 Hz; n = 6, P > 0.05) while only slightly inhibiting the effect of baclofen (Cd²⁺ + KCl, 3.9 ± 0.9 Hz; Cd²⁺ + KCl + baclofen, 2.7 ± 0.7 Hz; n = 6, P < 0.05; data not shown). Taken together these data suggest that GABA_B receptor-mediated modulation of inhibitory transmission onto interneurons may occur by both inhibiting presynaptic Ca²⁺ channels and by directly impairing transmitter exocytosis.

Differences in Ca²⁺ channel coupling to inhibitory and excitatory transmitter release

A variety of specific presynaptic Ca²⁺ channel types are downstream targets for GABA_B receptor modulation. For example, at associational-commisural synapses onto CA3 pyramidal cells, GABA_B receptors are coupled to both N- and P-type channels whereas P-type channels are primarily the target at mossy fibre inputs to CA3 pyramids (Castillo et al. 1994). We next compared the involvement of specific Ca²⁺ channels in the GABA_B receptor modulation of transmission at the two synapse types by using ω-conotoxin GVIA (CTX), an N-type Ca²⁺ channel inhibitor, and ω-agatoxin TK (AGTX), a P/Q-type Ca²⁺ channel inhibitor. N- and P/Q-types of Ca²⁺ channels are the two major channel types implicated in transmitter release at mammalian central synapses (Castillo et al. 1994; Dunlap et al. 1995). We initially examined whether glutamate or GABA release was mediated solely by activation of N- and P/Q-types of Ca²⁺ channels. Co-application of CTX (250 nm) and AGTX (250 nm) blocked evoked EPSCs and IPSCs by 95.7 ± 4.3 % (n = 5) and 97.6 ± 5.3 % (n = 6), respectively, suggesting that transmitter release is mediated exclusively by activation of N- and P/Q-types of Ca²⁺ channels at both excitatory and inhibitory synapses. We next determined the contribution of N- and P/Q-types of Ca²⁺ channels to the GABA_B receptor-mediated inhibition of both glutamate and GABA release. Because transmitter release at CA3 interneuron synapses is exclusively mediated by either N- or P/Q-types of Ca²⁺ channels, we reasoned that following the inhibition of N-type Ca²⁺ channels by CTX, the contribution of P/Q-type Ca²⁺ channels to GABA_B receptor-mediated inhibition of glutamate or GABA release could be resolved and vice versa. At excitatory synapses, bath application of AGTX (250 nm) alone irreversibly inhibited EPSCs by 38.0 ± 6.3 % (n = 4) and subsequent application of baclofen reduced EPSCs by a further 53.6 ± 8.9 % of the initial EPSC amplitude (n = 4, Fig. 7A and C). Application of CTX alone blocked EPSCs by 46.4 ± 5.8 % (n = 6) and the remaining EPSCs were further inhibited by 42.8 ± 5.2 % of the initial EPSC amplitude (n = 6, Fig. 7B and C). These results suggest that N- and P/Q-types of Ca²⁺ channels contribute approximately equally to glutamate release at recurrent collateral synapses onto interneurons and are both targets for GABA_B receptor-mediated inhibition of glutamate release at excitatory synapses (Fig. 7C).

Figure 7. Different proportions of N- and P/Q-type Ca²⁺ channels contribute to GABA_B receptor-mediated inhibition of EPSCs and IPSCs.

A, time course of EPSCs during sequential application of ω-agatoxin TK (AGTX, 250 nm) and baclofen (20 μm). Current traces in the upper panel are averages of 10 consecutive EPSCs recorded at the time points shown in the dot plot. B, time course of EPSCs recorded from a cell during sequential application of ω-conotoxin GVIA (CTX, 250 nm) and baclofen (20 μm). C, summarized EPSC data. Note that N- and P/Q-type Ca²⁺ channels contribute approximately equally to GABA_B receptor-mediated inhibition of EPSC. D, time course of IPSCs during sequential applications of AGTX (250 nm) and baclofen (20 μm). Traces in the upper panel are averages of 10 consecutive IPSCs recorded at the time points shown in the dot plot. E, time course of IPSCs recorded during sequential applications of CTX (250 nm) and baclofen (20 μm). F, summarized IPSC data. Note that P/Q-type Ca²⁺ channels contribute more to GABA_B receptor-mediated inhibition of IPSCs.

At inhibitory synapses, application of AGTX reduced IPSCs by 88.5 ± 3.2 % (n = 3), which was significantly larger than that produced by CTX alone (42.3 ± 8.2 %, n = 5, P < 0.01). Although this data again suggests the presence of more than one channel type on inhibitory terminals, more P/Q-type Ca²⁺ channels are involved in the GABA release mechanism (Fig. 7D-F). The observation that the algebraic sum of the effects of CTX and AGTX (≈130 %) exceeds 100 % is similar to that observed at GABAergic synapses onto CA1 pyramidal neurons (Wilson et al. 2001) and may be attributable to the supralinear relationship between calcium influx and neurotransmitter release (Dodge & Rahamimoff, 1967). Baclofen reduced IPSC amplitudes by only a further 10.5 ± 3.0 % (n = 3) of the initial IPSC amplitude following AGTX application, and following CTX application, baclofen inhibited the remaining IPSCs by 32.7 ± 5.7 % of their initial amplitude (n = 5, P < 0.05, Fig. 7F) suggesting that more P/Q-type Ca²⁺ channels were coupled to GABA_B receptor-mediated inhibition of GABA release.

Frequency dependence of GABA_B receptor modulation

Frequency-dependent modulation of EPSCs by GABA_B receptor activity has been observed at auditory glutamatergic synapses (Brenowitz et al. 1998) and in cerebellar glomeruli (Mitchell & Silver, 2000). At both synapses, activation of GABA_B receptors replaces frequency-dependent depression in response to short trains of stimuli with a non-decremental form of transmission. Whether GABA_B receptor activation is associated with a similar phenomenon at inhibitory synapses has not been reported.

Next, we examined the frequency dependence of GABA_B receptor modulation of both evoked EPSCs and IPSCs at CA3 hippocampal interneuron synapses. EPSCs or IPSCs were evoked at five different stimulation frequencies ranging from 0.1 to 10 Hz (Fig. 8 and Fig. 9). To prevent potential frequency-induced long-term alterations in synaptic strength, only ≈20 events were collected for each stimulation frequency. Frequency-dependent adaptation usually reached a steady state following 3-6 stimuli, accordingly the averaged data point was calculated from the final 10 events at each frequency.

Figure 8. GABA_B receptor activation blocks frequency-dependent depression but not frequency-dependent facilitation of EPSCs.

A, GABA_B receptor activation blocks the frequency dependence of depression of EPSCs. Upper traces, averaged from 20 EPSCs recorded from the same cell at different frequencies before (thin) and during (thick) application of baclofen (20 μm). Plots, amplitudes of 20 consecutive EPSCs evoked at different frequencies before and during application of baclofen. Note the frequency-dependent depression in control, and lack of frequency-dependent depression in the presence of baclofen. B, summarized data (n = 5). For each cell, the averaged EPSC amplitude recorded at each frequency in the absence or presence of baclofen was normalized to the averaged EPSC amplitude recorded at 0.1 Hz in control. C, GABA_B receptor activation was less effective at blocking frequency-dependent facilitation of EPSCs. Upper traces, averaged from 20 EPSCs recorded at different frequencies before (thin) and during (thick) application of baclofen (20 μm). Plots, amplitudes of 20 consecutive EPSCs evoked at different frequencies before and in the presence of baclofen. D, summarized data (n = 5). For each cell, the averaged EPSC amplitude recorded at each frequency, in the absence and presence of baclofen, was normalized to the averaged EPSC amplitude recorded at 0.1 Hz in control. Note that in the presence of baclofen, the EPSC amplitude at 0.1 Hz is significantly smaller than that at 3 and 10 Hz. (*P < 0.05; **P < 0.01).

Figure 9. GABA_B receptor activation blocks the frequency-dependent depression of IPSCs.

A, upper traces averaged from 20 IPSCs evoked at different frequencies before (thin) and during (thick) application of baclofen (20 μm). Plots, 20 consecutive IPSCs recorded at different frequencies. B, summarized data (n = 5). For each cell, the averaged IPSC amplitude at each frequency was normalized to the averaged IPSC amplitude observed at 0.1 Hz in control.

Consistent with the results of Toth et al. (2000), under control conditions either frequency-dependent depression (Fig. 8A and B) or facilitation (Fig. 8C and D) of EPSCs was observed. To allow for comparison, we normalized the averaged event, evoked at different stimulation frequencies in the presence or absence of baclofen, to the averaged event recorded at 0.1 Hz under control conditions. At synapses displaying frequency-dependent depression, the EPSC amplitude was reduced by 59.9 ± 3.2 % at 10 Hz compared with events evoked at 0.1 Hz (n = 5, P < 0.01, Fig. 8A and B). Activation of GABA_B receptors by baclofen prevented this frequency-dependent depression and normalized transmission across all frequencies tested (Fig. 8A and B). In the presence of baclofen, the averaged EPSC amplitude evoked at 0.1 Hz was reduced to 24.6 ± 3.6 % of that seen at 0.1 Hz in control. However increasing the stimulation frequency up to 10 Hz was without effect on the event amplitude (EPSC amplitude evoked at 10 Hz was 25.5 ± 3.5 % of that evoked at 0.1 Hz in control conditions, n = 5, P > 0.05, Fig. 8B).

At those synapses showing frequency-dependent facilitation (Fig. 8C and D), increasing the stimulation frequency from 0.1 Hz to frequencies up to 10 Hz significantly increased the EPSC amplitude (Fig. 8D). EPSC amplitude was increased by 68.8 ± 10.9 % (n = 6, P < 0.01) at 3 Hz and 98.3 ± 12.4 % (n = 6, P < 0.01) at 10 Hz (Fig. 8D). In the presence of baclofen, EPSC amplitude evoked at 0.1 Hz was reduced to 31.4 ± 6.3 % of that evoked at 0.1 Hz in control (n = 6, P < 0.01; Fig. 8D). Increasing the stimulation frequency failed to enhance EPSC amplitude until the stimulation frequency reached 3 Hz (Fig. 8D). EPSC amplitude was increased by 77.8 ± 23.2 % (n = 6, P < 0.05) at 3 Hz and 183.1± 41.6 % (n = 6, P < 0.01) at 10 Hz compared with that recorded at 0.1 Hz in the presence of baclofen (Fig. 8D). These results suggest that baclofen blocked the frequency-dependent facilitation at the lower range of frequencies tested, whereas it failed to block the frequency-dependent facilitation when stimulation frequencies were >3 Hz. One possible explanation for this observation is the high frequency-induced voltage-dependent relief of G-protein inhibition on presynaptic Ca²⁺ channels (see Discussion).

At inhibitory synapses, only frequency-dependent depression of evoked synaptic events was observed (Fig. 9A and B). Increasing the stimulation frequency from 0.1 to 10 Hz reduced the IPSC amplitude by 68.9 ± 3.9 % (n = 5, P < 0.01). Baclofen prevented this frequency-dependent depression. In the presence of baclofen, IPSC amplitude evoked at 0.1 Hz was 17.1 ± 5.3 % of control measured at 0.1 Hz in the absence of baclofen; IPSCs evoked at 10 Hz show no further depression (16.7 ± 4.9 % of control measured at 0.1 Hz, n = 5, P > 0.05; Fig. 9B).

Discussion

Our results demonstrate that both AMPA receptor-mediated excitatory and GABA_A receptor-mediated inhibitory synaptic transmission onto hippocampal CA3 interneurons of the CA3 st. radiatum are inhibited by presynaptic GABA_B receptor activation. Modulation by presynaptic GABA_B receptors was observed at all ages tested. In contrast GABA_B receptor-mediated postsynaptic responses in interneurons were developmentally regulated, being only observed in animals older than P30, consistent with previous reports from the CA1 hippocampus (Nurse & Lacaille, 1999). As expression of GABA_B receptor-mediated postsynaptic responses requires the co-operation of many signalling proteins including GABA_B receptors, G-proteins and K⁺ channels, it is unknown presently which protein(s) is(are) missing in CA3 interneurons of young animals.

While both excitatory and inhibitory transmission onto interneurons were inhibited by GABA_B receptor activation, the IC₅₀ value of baclofen modulation of IPSCs was lower than that of EPSCs. This might suggest that the subtypes of GABA_B receptors, G-proteins and Ca²⁺ channels expressed on the presynaptic terminals at excitatory synapses are different from those expressed at inhibitory synapses. Interestingly, application of CGP55845 alone, a GABA_B receptor antagonist, enhanced only the IPSC amplitude, suggesting that GABA_B receptors on inhibitory terminals are tonically activated by endogenous GABA. Consequently, it is possible that tonic activation of GABA_B receptors may, in part, account for the apparent lower IC₅₀ for baclofen: activation of GABA_B receptors by both ambient GABA and exogenous baclofen would result in a leftward shift in the concentration- response curve for baclofen.

Experiments targeted toward elucidating the Ca²⁺ channel types involved in presynaptic GABA_B receptor modulation demonstrated that N- and P/Q-channel types differentially contribute to the release of glutamate and GABA at the two synapse types. Co-application of N- and P/Q-type Ca²⁺ channel blockers CTX and AGTX produced >96 % inhibition of transmission, consistent with a role for both these channel types in determining transmitter release at both excitatory and inhibitory synapses onto st. radiatum interneurons. At excitatory synapses, N- and P/Q-types of Ca²⁺ channels contribute approximately equally to mediate glutamate release, whereas at inhibitory synapses more than 80 % of GABA release is mediated by P/Q-type Ca²⁺ channel activation. Consistent with this observation, approximately equal proportions of N- and P/Q-type Ca²⁺ channels were involved in the GABA_B receptor-mediated inhibition of glutamate release, whereas more P/Q-type Ca²⁺ channels were involved in GABA_B receptor-mediated inhibition of GABA release. Differences in coupling of Ca²⁺ channels to transmitter release at excitatory and inhibitory synapses may also underlie the observed differences in sensitivity of baclofen. One important caveat to these data is that the effects of specific Ca²⁺ channel toxins do not necessarily provide direct evidence for the molecular targets of baclofen. For example if a given terminal releases transmitter via activation of a single Ca²⁺ channel type then any antagonist of this channel will obscure the effects of baclofen on release, even if baclofen acted only to increase presynaptic K⁺ conductances. We think this unlikely, however, since effects of baclofen on postsynaptic K⁺ conductances were absent in the age range of animals studied; of course whether this developmental regulation extends to K⁺ conductances on the presynaptic terminal is completely unexplored.

Consistent with previous reports, the algebraic sum of the effects of CTX and AGTX alone was considerably greater than 100 % (≈130 %) at inhibitory synapses, while the additive effects at excitatory synapses did not exceed 100 %. While the reason for this discrepancy is not clear, differences in the interaction of Ca²⁺ transients originating from N- and P/Q-type Ca²⁺ channels or a linear interaction between Ca²⁺ influx and neurotransmitter release at excitatory synapses onto interneurons might be involved (Dodge & Rahamimoff, 1967; Castillo et al. 1994; Wilson et al. 2001). The observation that N- and P/Q channels contribute equally at recurrent collateral excitatory synapses onto interneurons is consistent with the observations of Castillo et al. (1994) who also reported approximately equal contributions of both channel types at recurrent collateral synapses onto CA3 pyramidal neurons. These data suggest that release mechanisms, at least at the level of Ca²⁺ channel types, may be similar at both recurrent collateral synapse types.

Previous reports have demonstrated that release of GABA from inhibitory terminals onto excitatory principal cells is mediated by either N- or P-type calcium channels (Poncer et al. 1997, Wilson et al. 2001). In paired recordings, N-type Ca²⁺-channels controlled transmitter release from interneurons located in the CA3 st. radiatum, whereas P/Q Ca²⁺-channel antagonists abolished IPSPs generated by CA3 st. lucidum and oriens interneurons (Poncer et al. 2001). Similar observations were made at inhibitory synapses made onto CA1 pyramidal neurons (Wilson et al. 2001). In this latter study, however, it was noted that interneurons with somata in st. oriens-alveus and axons in the st. lacunosum-moleculare, i.e. the so-called O-LM cell, use both P/Q- and N-type Ca²⁺ channels for neurotransmitter release (Wilson et al. 2001). Our data demonstrate that IPSCs onto st. radiatum interneurons are generated by both P/Q- and N-type Ca²⁺ channels. Assuming that inhibitory terminals share the same release mechanisms as inhibitory terminals onto principal cells (and this is not a safe assumption, Toth & McBain, 2000) we would speculate that the vast majority of IPSPs observed in our study arise from activation of interneurons with their axons deep in st. radiatum and lacunosum moleculare, perhaps O-LM cells. We cannot exclude however that extracellular stimulation of inhibitory terminals activates a mosaic of axon terminals, primarily from cells using P/Q-type channels with a smaller contribution of cells using N-type channels to control transmitter release.

Throughout the mammalian central nervous system, the downstream mechanisms of presynaptic GABA_B receptor modulation of synaptic transmission appear to be highly heterogeneous and differ from synapse type to synapse type. In the present experiments baclofen inhibited mIPSC frequency, whereas no effect of baclofen was observed on mEPSC frequency. Since mIPSCs and mEPSCs recorded in the presence of TTX are generally considered to be Ca²⁺ independent these results suggest that inhibition of presynaptic Ca²⁺ channels may not be the only mechanism by which GABA_B receptor activation inhibits inhibitory transmitter release. Consistent with this hypothesis was the observation that Cd²⁺ failed to block the baclofen-mediated inhibition of mEPSC frequency evoked by elevating [KCl]_o. Other potential mechanisms involved in GABA_B receptor-mediated modulation of inhibitory transmission may be activation of presynaptic K⁺ channels to produce hyperpolarization, direct interaction with transmitter exocytosis (Capogna et al. 1996) and phosphorylation (Pitler & Alger, 1994; Jarolimek & Misgeld, 1997).

GABA_B receptor-mediated inhibition of mIPSC frequency has also been shown in neurons of rat nucleus reticularis thalami (Ulrich & Huguebard, 1996), rat ventro-basal thalamocortical neurons (Le Feuvre et al. 1997), substantia gelatinosa neurons of the adult rat spinal dorsal horn (Iyadomi et al. 2000), cultured rat midbrain neurons (Jarolimek & Misgeld, 1992) and CA1 hippocampal pyramidal neurons of guinea-pig and Wistar rats (but not Sprague-Dawley rats, Jarolimek & Misgeld, 1997). However, baclofen failed to alter mIPSC frequency in rat CA1 pyramidal neurons (Cohen et al. 1992; Doze et al. 1995) and CA3 pyramidal neurons (Scanziani et al. 1992). The latter result (Scanziani et al. 1992) is of particular interest because it would appear that the mechanism by which presynaptic GABA_B receptors located on GABAergic terminals impinging onto CA3 interneurons inhibit release differs from the mechanism at inhibitory terminals impinging onto CA3 pyramidal neurons. Although it is impossible to assume that the terminal types being studied in the present investigation and those of Scanziani et al. (1992) arose from common inhibitory interneuron types it could suggest a target-specific transduction mechanism for GABA_B-mediated regulation of inhibitory transmission. This is a possibility that will be investigated in future studies.

The opposite appears to be true for mEPSCs. In the present study baclofen failed to inhibit mEPSC frequency. Furthermore, activation of GABA_B receptors inhibited mEPSCs recorded in elevated [KCl]_o in a calcium-dependent manner. Taken together these data suggest that GABA_B receptor-mediated inhibition of glutamate release occurs mainly, if not exclusively, through inhibition of presynaptic Ca²⁺ channels. This observation contrasts with other studies where GABA_B-receptor activation inhibited mEPSC frequency in CA3 pyramidal neurons of cultured rat hippocampal slices (Scanziani et al. 1992), substantia gelatinosa neurons of the adult rat spinal dorsal horn (Iyadomi et al. 2000), rat supraoptic magnocellular neurons (Kabashima et al. 1997; Kombian et al. 2001) and granule cells in the cerebellum (Mitchell & Silver, 2000). This apparent discrepancy between our data and those obtained from mEPSCs onto CA3 pyramidal cells might also suggest a target-specific modulation of excitatory transmission. However, in the study of Scanziani et al. (1992) no attempt was made to identify the origin of the mEPSCs i.e. whether they arose from mossy fibre- or recurrent collateral synapses. In the present study we can rule out any influence of mossy fibre inputs given that the cells were typically located deep in the st. radiatum, where few mossy fibre axons ramify.

At synapses showing marked short-term depression in response to repetitive stimuli, activation of GABA_B receptors reduced synaptic depression and ‘normalized’ transmission at both inhibitory and excitatory synapses. Similar results have been observed at auditory glutamatergic synapses (Brenowitz et al. 1998) and in the cerebellar glomerulus (Mitchell & Silver, 2000; see also Pananceau et al. 1998; Selig et al. 1999; Hanse & Gustafsson 2001). GABA_B receptor activation at these depressing synapses eliminates short-term plasticity and may provide a mechanism for sustaining synaptic transmission albeit at a lower peak amplitude. High frequency stimulus-induced depression has been suggested to arise via activation of presynaptic autoreceptors (Davies et al. 1990; Barnes-Davies & Forsythe, 1995; von Gersdorff et al. 1997), depletion of the readily releasable pool of synaptic vesicles (Debanne et al. 1996; Dobrunz & Stevens, 1997; Tsodyks & Markram, 1997), or desensitization of postsynaptic receptors (Jones & Westbrook, 1995; Otis et al. 1996). Thus, one possible mechanism for the observed GABA_B receptor-mediated blockade of frequency-dependent depression is that GABA_B receptor activation acts to reduce the initial presynaptic transmitter release probability and consequently reduces exhaustion of the ready releasable pool (Tsodyks & Markram, 1997; Valera et al. 1997; Dittman et al. 2000) and/or reduces postsynaptic receptor desensitization following each presynaptic action potential.

Whereas the frequency-dependent facilitation of EPSCs was similarly inhibited by baclofen at stimulation frequencies < 1 Hz, baclofen failed to block frequency-dependent facilitation at higher stimulation frequencies (> 3 Hz, Fig. 7C and D). This ‘escape’ from GABA_B normalization of transmission may be related to the voltage-dependent relief of G-protein inhibition of presynaptic Ca²⁺ channels observed in a number of systems (Bean, 1989; Brody et al. 1997; Dolphin, 1998; Park & Dunlap, 1998; Brody & Yue, 2000). Depolarization of presynaptic terminals by high-frequency stimulation may act to relieve G-protein-mediated inhibition of Ca²⁺ channels and render GABA_B receptor activity ineffective. Therefore, in this scenario EPSCs evoked at higher frequencies would be less susceptible to GABA_B receptor modulation.

In conclusion, we provide evidence indicating that GABA_B receptor-mediated postsynaptic responses undergo developmental regulation and presynaptic GABA_B receptor activation inhibits both excitatory and inhibitory synaptic transmission onto CA3 interneurons in st. radiatum. Activation of GABA_B receptors normalizes frequency-dependent depression at both excitatory and inhibitory synapses, while having a modest impact on frequency-dependent facilitation. Our results suggest that GABA_B receptor activation may be an essential component of short-term plasticity at both excitatory and inhibitory synapses onto hippocampal interneurons.