Presynaptic inhibition of gamma-aminobutyric acid release in the bed nucleus of the stria terminalis by kappa opioid receptor signaling

Abstract

Background: The kappa opioid receptor (KOR) and its endogenous agonist, the neuropeptide dynorphin, are a critical component of the central stress system. Both dynorphin and KOR are expressed in the bed nucleus of the stria terminalis (BNST), a brain region associated with anxiety and stress. This suggests that KOR activation in this region may play a role in the regulation of emotional behaviors. To date, however, there has been no investigation of the ability of KOR to modulate synaptic transmission in the BNST.

Methods: We used whole-cell patch-clamp recordings from acutely prepared mouse brain slices to examine the actions of KOR on inhibitory transmission in the BNST. Additionally, we used neurochemical and pathway-specific optogenetic manipulations to selectively stimulate gamma-aminobutyric acid (GABA)ergic fibers from the central nucleus of the amygdala (CeA) to the BNST.

Results: We found that activation of KOR reduced GABAergic transmission through a presynaptic mechanism. Furthermore, we examined the signal transduction pathways that mediate this inhibition and provide the first functional information implicating extracellular signal-regulated kinase in KOR-mediated presynaptic modulation. Moreover, we found that at KOR signaling robustly reduced inhibitory synaptic transmission in the CeA to BNST pathway.

Conclusions: Together, these results demonstrate that KOR provides important inhibitory control over presynaptic GABAergic signaling within the BNST and provides the first direct functional demonstration of KOR-sensitive long-range GABAergic connections between the CeA and the BNST.

Figure 1. Kappa Opioid Receptor (KOR) activation reduces synaptic inhibition in the BNST

A) Representative experiment demonstrating the ability of Dynorphin A (300 nM) to reduce the peak amplitude of eIPSCs in the BNST. Inset: average traces from the representative experiment showing the effects before (black) and after (red) application of Dynorphin A. B) Pooled data demonstrating that the ability of 300 nM dynorphin to reduce eIPSC peak amplitude is blocked by pre-application of the KOR receptor antagonist, nor-BNI (100 nM). C) Pooled data demonstrating that 100 nM nor-BNI reverses the Dynorphin A effect on eIPSC amplitude. D) Pooled data demonstrating that the ability of the KOR agonist, U69593, to inhibit eIPSC amplitude is blocked by the KOR antagonist nor-BNI in the BNST. E) Pooled data demonstrating no tonic activation of KORs by endogenous dynorphin. F) Summary of magnitude of maximal inhibition produced by bath-application of drugs in A–E.

Figure 2. KOR inhibition is increased in reduced external calcium

A) Pooled data demonstrating that reducing the external concentration of calcium to 1.0 mM (n = 5) leads to an increased effect of U69593 (2.0 mM calcium data replotted from Figure 1 for comparison). B) Bar chart demonstrating the significantly increase in U69593-mediated inhibition in 1.0 mM calcium.

Figure 3. KOR activation reduces sIPSC and mIPSC frequency but not amplitude

A & B) Representative baseline (A) and post-drug (B) traces demonstrating the effect of U69593 on spontaneous GABAergic transmission in the BNST. C & D) Cumulative probability distribution of peak amplitude (C) and frequency (D) before (black) and after (red) application of U69593, demonstrating no effect on amplitude but a reduction in frequency. E) Pooled data demonstrating that U69593 reduces sIPSC frequency but not amplitude. F) Pooled data demonstrating that U69593 reduces mIPSC frequency but not amplitude.

Figure 4. Evaluation of CV/PPR as means to determine presynaptic versus postsynaptic changes in synaptic transmission in the BNST

A) Representative paired pulse traces at baseline and following application of 100 nM baclofen (red). B) The same traces from panel A normalized to highlight the effect of baclofen on the paired pulse ratio. C & D) Pooled results demonstrating the effect of baclofen on eIPSC amplitude (C) and PPR (D). E & F) Pooled results demonstrating the effect of reduced stimulation on eIPSC amplitude (E) and PPR (F). G & H) Pooled results demonstrating the effect of sub-saturating picrotoxin (5 μM) on eIPSC amplitude (G) and PPR (H). I & J) Summary bar graphs demonstrating the effects drug treatments on PPR (I) and coefficient of variation (J).

Figure 5. Effect of KOR activation on PPR/CV in the BNST

A) Pooled data demonstrating the lack of an effect of Dynorphin A on PPR. (B) Pooled data demonstrating the lack of an effect of U69593 on PPR. C) Summary bar chart demonstrating the lack of an effect of either KOR agonist on PPR in the BNST. D) Bar chart demonstrating the significant effects of Dynorphin A and U69593 on CV in the BNST.

Figure 6. ERK, but not p38, signaling is required for KOR-mediated inhibition of GABA release in the BNST

A) Pre-incubation of brain slices with the p38 map kinase inhibitor SB204580 (20 μM) did not alter the ability of U69593 to reduce eIPSC peak amplitude in the BNST, while pre-incubation of the MEK inhibitors, U0126 (20 μM) and SL327 (10 μM) led to significant impairment in the ability of U69593 to modulate GABA transmission in the BNST. B) Summary of the impact of MAPK inhibitors on KOR-mediated inhibition. C) Both MEK inhibitors, U0126 (20 μM) and SL327 (10 μM), bath-applied alone increased eIPSC amplitude in the BNST. D) Summary of the effects of MEK inhibitors on GABA transmission in the BNST. E) Pre-incubation of brain slices with the MEK inhibitor, U0126 (20 μM), did not alter the ability of baclofen to modulate GABA transmission in the BNST. F) Summary of the impact of U0126 on baclofen-mediated inhibition of GABA transmission in the BNST.

Figure 7. KOR activation inhibits CeA to BNST GABAergic transmission

A & B) Injection of an AAV-expressing, Cre-inducible GFP in the CeA resulted in GFP positive neurons in the CeA (A) and a large number of GFP positive fibers in the dorsolateral BNST. C) A brief flash of blue-light leads to a depolarization of GABAergic cell bodies in the CeA (i), and an inhibitory post-synaptic current (IPSC) in a BNST neuron (ii), which was completely blocked by the GABA_A-R receptor antagonist picrotoxin (25 μM), demonstrating that this current is mediated via GABA_A receptors. D) Pooled data demonstrating that the KOR agonist, U69593, inhibits light-evoked IPSC amplitude in the CeA-BNST pathway. E) Model summarizing the results of this study.