GABA(B) receptors inhibit dendrodendritic transmission in the rat olfactory bulb

Abstract

In the mammalian olfactory bulb, mitral cell dendrites release glutamate onto the dendritic spines of granule cells, which in turn release GABA back onto mitral dendrites. This local synaptic circuit forms the basis for reciprocal dendrodendritic inhibition mediated by ionotropic GABA(A) receptors in mitral cells. Surprisingly little is known about neurotransmitter modulation of dendrodendritic signaling in the olfactory bulb. In this study, we examine whether metabotropic GABA(B) receptors modulate dendrodendritic signaling between mitral and granule cells. We find that the selective GABA(B) agonist baclofen reduces mitral cell recurrent inhibition mediated by dendrodendritic synapses. GABA(B) receptor activation causes only a weak inhibition of field EPSCs in the external plexiform layer and only slightly reduces glutamate-mediated mitral cell self-excitation. Although GABA(B) receptors depress mitral cell glutamate release only weakly, baclofen causes a marked reduction in the amplitude of granule-cell-evoked, GABA(A)-mediated IPSCs in mitral cells. In addition to reducing the amplitude of granule-cell-evoked IPSCs, baclofen causes a change from paired-pulse depression to paired-pulse facilitation, suggesting that GABA(B) receptors modulate GABA release from granule cells. To explore the mechanism of action of GABA(B) receptors further, we show that baclofen inhibits high-voltage-activated calcium currents in granule cells. Together, these findings suggest that GABA(B) receptors modulate dendrodendritic inhibition primarily by inhibiting granule cell calcium channels and reducing the release of GABA. Furthermore, we show that endogenous GABA regulates the strength of dendrodendritic inhibition via the activation of GABA(B) autoreceptors.

Fig. 1.

GABA_B receptor activation reduces mitral cell recurrent inhibition. Top, Individual records from one mitral cell (V_m = −70 mV, KCl internal solution). A brief train of depolarizing voltage steps (2 msec, +70 mV, 7 pulses) elicits action currents, followed by a long-lasting barrage of synaptic currents (Control). The synaptic response is reduced after bath application of the GABA_B agonist baclofen (50 μm). Switching to a solution containing the GABA_B antagonist CGP 55845 (5 μm) reverses the action of baclofen. Subsequent application of the GABA_Aantagonist bicuculline methiodide (20 μm) abolishes the synaptic response (+BMI). Bottom, Summary plot of the results for 11 cells. Points represent the average of two consecutive responses evoked every 30 sec. The dashed line represents unity. Inset, Recording configuration. MC, Mitral cell; GC, granule cell.

Fig. 2.

GABA_B receptor activation reduces mitral cell dendrodendritic inhibition. Top, Individual records from one cell (CsCl internal solution) in the presence of TTX (1 μm). A brief (25 msec) voltage step to 0 mV evokes a calcium current (blanked) followed by a long-lasting barrage of IPSCs (Control). Baclofen (50 μm) reduces dendrodendritic inhibition (DDI), and the response recovers after the application of CGP 55845 (5 μm). Bottom, Summary of the results for 10 cells. Points represent the average of two consecutive responses evoked every 30 sec. The dashed line represents unity.Inset, Recording configuration.

Fig. 3.

GABA_B receptors weakly modulate mitral cell glutamatergic transmission. A, Baclofen (25–50 μm) application causes a small and reversible reduction in the slope of fEPSPs recorded in the external plexiform layer.Bottom, Results from nine slices. Top, fEPSPs from one representative experiment. B, Baclofen causes a small and reversible reduction in mitral cell self-excitation. Mitral cells (V_m= −50 mV) were recorded in the presence of TTX (1 μm) and picrotoxin (100 μm) in Mg²⁺-free aCSF. Self-excitation was evoked by brief (5–25 msec) voltage steps to 0 mV.Top, Representative traces from one experiment.Bottom, Summary of results (n= 7). Self-excitation was abolished at the end of each experiment by APV, confirming that the current was mediated by NMDARs. The dashed lines represent unity.

Fig. 4.

GABA_B receptor activation inhibits IPSCs evoked in mitral cells by granule cell stimulation.A, Summary plot (n = 9) of the action of baclofen (25–50 μm) on IPSC amplitude. After the washout of baclofen, CGP 55845 (5 μm) caused a recovery of IPSC amplitude. Top, Traces from a representative experiment using paired-pulse stimulation (50 msec interstimulus interval). At the end of the experiment, bicuculline methiodide (+BMI; 20 μm) abolished the IPSC, confirming that it was mediated by GABA_A receptors. Thedashed line represents unity. B, Summary of results from cells in which paired-pulse stimulation was applied (n= 4, each point is the average of 6 consecutive IPSCs). Baclofen caused a change from paired-pulse depression to paired-pulse facilitation that was reversed after washout into CGP 55845. Top, Traces from one cell showing the IPSCs before (Control) and after baclofen and after the traces are scaled to the peak of the first IPSC (Scaled). The dashed line represents the average ratio during control.

Fig. 5.

GABA_B receptor activation causes a reduction in HVA calcium current in granule cells. A, Current–voltage relationship of calcium current in granule cells (V_m = −100 mV; n = 8).Inset, Currents in one cell in response to the voltage steps indicated to the leftof the sweeps.B, Voltage steps to −50 and 0 mV evoke LVA and HVA calcium currents, respectively, in a granule cell (V_m = −100 mV). Application of Ni²⁺(100 μm) almost completely abolishes LVA current and partially blocks HVA current. Subsequent application of Cd²⁺(100 μm) abolishes the remaining HVA current.C₁, Response to voltage steps to −50 and 0 mV in one cell before (Control) and after the application of baclofen (50 μm). Baclofen reduced the HVA but not the LVA calcium current; the action was reversed after washout into CGP 55845.C₂, Time course of the experiment shown in C₁. Filled circles, HVA current; open circles, LVA current.C₃, Summary of the actions of baclofen on HVA and LVA current in granule cells (n= 10).

Fig. 6.

Endogenous activation of GABA_Breceptors limits the strength of mitral cell dendrodendritic inhibition (DDI). Summary plot of the actions of the GABA_B antagonist CGP 55845 (5–10 μm) applied alone to slices (n = 11). The GABA_Bantagonist enhances dendrodendritic inhibition in naive slices.Top traces are from one cell (50 msec step) before (Control) and after CGP 55845. The dashed line represents unity.

Fig. 7.

Enhancing excitatory drive to granule cells increases the activation of GABA_B receptors by endogenous GABA. A, The action of baclofen is partially occluded when dendrodendritic inhibition (DDI) is evoked in Mg²⁺-free aCSF and NBQX (10 μm).Bottom, Summary of results (n = 10).Top, Representative results from one experiment.B, Baclofen has no effect when dendrodendritic inhibition is enhanced in the presence of cyclothiazide (CTZ, 100 μm). The aCSF was supplemented with APV (25 μm) and MK-801 (40 μm) to block NMDARs. Bottom, Summary of results (n = 8). Top, Representative responses from one experiment. The dashed lines represent unity.