Prefrontal Cortical Kappa Opioid Receptors Attenuate Responses to Amygdala Inputs

Abstract

Kappa opioid receptors (KORs) have been implicated in anxiety and stress, conditions that involve activation of projections from the basolateral amygdala (BLA) to the medial prefrontal cortex (mPFC). Although KORs have been studied in several brain regions, their role on mPFC physiology and on BLA projections to the mPFC remains unclear. Here, we explored whether KORs modify synaptic inputs from the BLA to the mPFC using in vivo electrophysiological recordings with electrical and optogenetic stimulation. Systemic administration of the KOR agonist U69,593 inhibited BLA-evoked synaptic responses in the mPFC without altering hippocampus-evoked responses. Intra-mPFC U69,593 inhibited electrical and optogenetic BLA-evoked synaptic responses, an effect blocked by the KOR antagonist nor-BNI. Bilateral intra-mPFC injection of the KOR antagonist nor-BNI increased center time in the open field test, suggesting an anxiolytic effect. The data demonstrate that mPFC KORs negatively regulate glutamatergic synaptic transmission in the BLA-mPFC pathway and anxiety-like behavior. These findings provide a framework whereby KOR signaling during stress and anxiety can regulate the flow of emotional state information from the BLA to the mPFC.

Figure 1

BLA-evoked synaptic responses in the mPFC are blocked by pressure-ejected glutamate receptor antagonists. (a) Time course of the effects of pressure-ejected aCSF (white squares; n=11 sites in seven rats) or CNQX/AP-5 (black squares; n=7 sites in five rats) on the BLA-evoked fEPSP slope. Horizontal bar depicts the period of CNQX/AP-5 pressure ejection. (b) Mean fEPSP slope expressed as a percentage of baseline in the 5-min window after the last puff of aCSF or CNQX/AP-5. (***) reflects a significant difference from aCSF controls. Representative traces of BLA-evoked fEPSPs at baseline (black trace) and after aCSF or CNQX/AP-5 (gray trace) are shown above their corresponding bars. (c) Mean fEPSP slope expressed as a percentage of baseline in the window following picrotoxin pressure ejection. Right, representative traces of BLA-evoked fEPSPs at baseline and (black trace) and after picrotoxin (gray trace). Note that picrotoxin modifies later components of the fEPSP without affecting the slope. Scale bars: 0.5 mV and 20 ms. In this and subsequent figures, data points depict mean±SEM. aCSF, artificial cerebrospinal fluid; BLA, basolateral amygdala; fEPSP, field excitatory postsynaptic potential; mPFC, medial prefrontal cortex.

Figure 2

Systemic administration of a KOR agonist inhibits glutamatergic fEPSPs in a pathway-specific manner. (a) Time course of the effects of systemic U69,593 on fornix-evoked (white circles; n=11 rats) and BLA-evoked (black circles; n=10 rats) fEPSP slope. (b) Mean fEPSP slope expressed as a percentage of baseline in the 30-min window after systemic U69,593 injection. (**) reflects a significant difference from fornix-evoked synaptic responses. Representative traces of fornix- and BLA-evoked fEPSPs at baseline (black trace) and after systemic U69,593 administration (gray trace) are shown above their corresponding group. Scale bars: 0.5 mV and 20 ms. BLA, basolateral amygdale; fEPSP, field excitatory postsynaptic potential; KOR, kappa opioid receptor.

Figure 3

mPFC KORs negatively modulate glutamatergic synaptic transmission in the BLA–mPFC pathway. (a) Time course of the effects of pressure-ejected artificial cerebrospinal fluid (aCSF) (white circles; n=11 sites in seven rats), U69,593 (black circles; n=13 sites in seven rats), or U69,593 in nor-BNI pre-treated rats (gray circles; n=12 sites in seven rats) on the BLA-evoked fEPSP slope. (b) Mean fEPSP slope expressed as a percentage of baseline in the 5-min window after the last puff of aCSF or U69,593 in rats pretreated with saline or nor-BNI ~24 h prior to electrophysiological recordings. (**) reflects a significant difference from aCSF controls. (*) reflects a significant difference between saline- and nor-BNI-pretreated rats that were challenged with U69,593 pressure ejection. Representative traces of BLA-evoked fEPSPs at baseline (black trace) and after aCSF or U69,593 (gray trace) are shown above their corresponding bars. Scale bars: 0.5 mV and 20 ms. (c) Mean BLA-evoked fEPSP normalized to maximal response during baseline at different stimulation intensities (n=10 sites in 10 rats). (*) reflects a significant difference from baseline post U69,593 pressure ejection. Scale bars: 0.5 mV and 20 ms. Representative traces of BLA-evoked fEPSPs in the mPFC with increasing BLA stimulation intensity (0.2–1.0 mA) at baseline (black trace) or after U69,593 administration (gray trace) are shown above the corresponding points. BLA, basolateral amygdale; fEPSP, field excitatory postsynaptic potential; KOR, kappa opioid receptor; mPFC, medial prefrontal cortex.

Figure 4

Optically evoked synaptic responses are blocked by CNQX/AP-5 pressure ejection. (a) Photomicrograph of ChR2-YFP expression (green) and DAPI-stained nuclei (blue) in the BLA. The dotted line depicts the boundaries of the BLA. Inset scale bar: 500 μm. (b) ChR2-YFP expression (green) in the mPFC. (c) Time course of the effects of pressure-ejected aCSF (white squares; n=8 sites in four rats) or CNQX/AP-5 (black squares; n=5 sites in three rats) on the slope of BLA optically evoked fEPSP. (d) Top, representative traces of BLA-evoked fEPSPs at baseline (black traces) and after aCSF or CNQX/AP-5 (red traces). In gray is a representative trace of a site where blue-light pulses did not evoke a significant fEPSP response. Vertical blue bars depict 1-ms blue-light pulses. Bottom, mean fEPSP slope expressed as a percentage of baseline in the 5-min window after the last puff of aCSF or CNQX/AP-5. (***) reflects a significant difference from aCSF controls. Black bar depicts the period of CNQX/AP-5 pressure ejection. (e) Time course of the effects of pressure ejected aCSF (white circles; n=8 sites in four rats) or U69,593 (black circles; n=9 sites in six rats) on the BLA optically evoked fEPSP slope. (f) Mean fEPSP slope expressed as a percentage of baseline in the 5-min window after the last puff of aCSF or U69,593. (***) reflects a significant difference from aCSF controls. Black bar depicts the period of U69,593 pressure ejection. Representative traces of BLA-evoked fEPSPs at baseline (black traces) and after aCSF or U69,593 (gray traces) are shown above their corresponding group. Blue lines depict 1-ms blue-light pulses. Scale is the same as in d. aCSF, artificial cerebrospinal fluid; BLA, basolateral amygdale; ChR2-YFP, channelrhodopsin-2-YFP; DAPI, 4′,6-diamidino-2-phenylindole; fEPSP, field excitatory postsynaptic potential; mPFC, medial prefrontal cortex.

Figure 5

Effects of intra-mPFC and systemic administration of nor-BNI on locomotor activity. (a) The lower dose of intra-mPFC nor-BNI (2 ng) significantly increased center time, whereas the higher dose (2 μg) as well as the systemic dose (10 mg/kg) had no effect. (b) Nor-BNI (2 ng, 2 μg, or 10 mg/kg) had no effect on total distance traveled. (c) Vertical activity was also not affected by nor-BNI (2 ng, 2 μg, or 10 mg/kg). Bars represent the mean (n=10–12)±SEM **p<0.01 vs vehicle. mPFC, medial prefrontal cortex.