Hypocretin/orexin and nociceptin/orphanin FQ coordinately regulate analgesia in a mouse model of stress-induced analgesia

Abstract

Stress-induced analgesia (SIA) is a key component of the defensive behavioral "fight-or-flight" response. Although the neural substrates of SIA are incompletely understood, previous studies have implicated the hypocretin/orexin (Hcrt) and nociceptin/orphanin FQ (N/OFQ) peptidergic systems in the regulation of SIA. Using immunohistochemistry in brain tissue from wild-type mice, we identified N/OFQ-containing fibers forming synaptic contacts with Hcrt neurons at both the light and electron microscopic levels. Patch clamp recordings in GFP-tagged mouse Hcrt neurons revealed that N/OFQ hyperpolarized, decreased input resistance, and blocked the firing of action potentials in Hcrt neurons. N/OFQ postsynaptic effects were consistent with opening of a G protein-regulated inwardly rectifying K+ (GIRK) channel. N/OFQ also modulated presynaptic release of GABA and glutamate onto Hcrt neurons in mouse hypothalamic slices. Orexin/ataxin-3 mice, in which the Hcrt neurons degenerate, did not exhibit SIA, although analgesia was induced by i.c.v. administration of Hcrt-1. N/OFQ blocked SIA in wild-type mice, while coadministration of Hcrt-1 overcame N/OFQ inhibition of SIA. These results establish what is, to our knowledge, a novel interaction between the N/OFQ and Hcrt systems in which the corticotropin-releasing factor and N/OFQ systems coordinately modulate the Hcrt neurons to regulate SIA.

Figure 1. N/OFQ-containing fibers innervate Hcrt neurons, and N/OFQ inhibits Hcrt neuronal activity.

(A) Left panel shows confocal image of N/OFQ-immunoreactive fibers in the vicinity of, and in putative contact with, Hcrt-immunoreactive neurons in the PLH of WT mice. N/OFQ (green) fibers are in close proximity to Hcrt-immunoreactive (red) cells. The arrow indicates the N/OFQ innervation of an Hcrt cell body. Middle panel shows light micrograph of a light brown immunolabeled Hcrt neuron contacted by a dark black bouton (arrow) representing immunolabeling for N/OFQ. Right panel shows electron micrograph taken from ultrathin sections of the same labeled terminal and dendrite shown in the light micrograph in the middle panel. Black arrow indicates the N/OFQ-immunolabeled axon terminal in synaptic contact (red arrowhead) with the dendrite of the Hcrt cell. Scale bars: 10 μm (left and middle panels); 1 μm (right panel). (B) Under current clamp, bath application of N/OFQ (1 μM) hyperpolarized Hcrt neurons, decreased input resistance, and blocked spontaneous firing of action potentials. The resting potential of this cell was –54 mV and was manually adjusted to –60 mV with DC current. Membrane resistance was monitored using hyperpolarizing current pulses (–0.3 nA, 800 ms) delivered every 5 seconds throughout the experiment. (C) Under voltage-clamp mode at a V_h of –60 mV, N/OFQ (1 μM) induced an outward current (–53 pA) in an Hcrt neuron. Notice that the frequency but not the amplitude of the miniature synaptic currents (inward currents) recorded in the presence of TTX (0.5 μM) was also reduced.

Figure 2. N/OFQ directly hyperpolarizes Hcrt neurons in a dose-depenedent manner.

(A) N/OFQ (1 μM) caused hyperpolarization and a decrease in input resistance in the presence of TTX (0.3 μM), which was used to block synaptic activity and action potentials in an Hcrt-containing cell. (B) In the same neuron, the NOP receptor antagonist SR14148 (10 μM) blocks the N/OFQ-induced hyperpolarization and input resistance decrease. The blockade was reversible after 20-minute washout of the antagonist (data not shown). (C) Since the N/OFQ-induced hyperpolarization apparently did not differ in the presence or absence of TTX, both data sets were pooled to construct a concentration-dependent response curve using the pharmacologic dose-response model-fitting function (OriginPro 7.5; OriginLab). The half-maximal effect of N/OFQ-induced hyperpolarization (EC₅₀) was calculated at 0.329 μM with a Hill coefficient of 1.1. Error bars represent SEM.

Figure 3. N/OFQ modulates membrane currents and depresses intracellular Ca²⁺ levels.

(A–C) N/OFQ activates a K⁺ conductance. The I-V relationship of neuronal responses in the presence (B) and absence (A) of N/OFQ (1 μM) indicates a reversal potential of –98 mV (C), which is close to the K⁺ equilibrium potential (approximately –110 mV) under our experimental conditions. Before N/OFQ application, membrane potential was adjusted to a resting level of –60 mV by DC current injection. (D) N/OFQ inhibits Ca²⁺ currents. From a V_h of –60 mV, membrane voltage was stepped to +20 mV, which elicited an inward current. N/OFQ (1 μM) inhibited this current. Partial recovery was obtained after washout of N/OFQ, and the recovered current was completely blocked by Cd²⁺ (200 μM, data not shown). (E) N/OFQ depresses cytoplasmic Ca²⁺ in Hcrt neurons. Representative trace demonstrating the effect of N/OFQ on Ca²⁺ fluorescence in transgenic orexin/YC2.1 mice in which Hcrt neurons express the calcium-sensing protein yellow cameleon 2.1. Ca²⁺ imaging from these mice revealed that N/OFQ inhibited approximately 65% of Hcrt neurons tested (18 of 28). (F) Concentration-dependent depression of cytoplasmic Ca²⁺ in Hcrt neurons induced by N/OFQ (mean ± SEM; n = 6–13 cells per concentration).

Figure 4. N/OFQ significantly decreases the frequency, but not the amplitude, of sEPSCs in Hcrt neurons.

(A) Representative traces demonstrating the effect of N/OFQ on sEPSCs (left). Average sEPSCs from the same cell in the presence and absence of N/OFQ (1 μM, right). (B) Cumulative probability distributions of inter-event interval and amplitude for the cell shown in A. (C) Average effect of N/OFQ on sEPSC frequency and amplitude (n = 4). Cells were voltage clamped at –60 mV using a CsCl internal solution. Error bars represent SEM. *P < 0.05.

Figure 5. N/OFQ significantly decreases the frequency, but not the amplitude, of sIPSCs in Hcrt neurons.

(A) Representative traces demonstrating the effect of N/OFQ on sIPSCs (left). Average sIPSCs from the same cell in the presence and absence of N/OFQ (1 μM, right). (B) Cumulative probability distributions for inter-event interval and amplitude for the cell shown in A. (C) Average effect of N/OFQ on sIPSC frequency and amplitude (n = 4). Cells were voltage clamped at –60 mV using a KCl internal solution. Error bars represent SEM. *P < 0.05.

Figure 6. N/OFQ reduces the frequency of both mEPSCs and mIPSCs.

(A) Representative traces showing mEPSCs in the presence and absence of N/OFQ (1 μM) and bar graphs summarizing the effects on mEPSC frequency and amplitude (mean ± SEM; n = 8). (B) Representative traces showing mIPSCs in the presence and absence of N/OFQ (1 μM) and bar graphs summarizing the effects on mIPSC frequency and amplitude (mean ± SEM; n = 8). *P < 0.05, **P < 0.01.

Figure 7. Inverse modulation of SIA by the Hcrt and N/OFQ systems.

(A) SIA occurs in WT but not orexin/ataxin mice. From –30 to 0 minutes, mice (n = 8) were restrained; this was followed immediately by a hot-plate test at 0 minutes and again at 30 and 60 minutes following restraint. (B) SIA occurs in WT but not in orexin/ataxin-3 mice. Post-hoc tests revealed a significant increase in hot-plate latency in WT mice after 30 minutes of restraint stress (P < 0.05) but not in orexin/ataxin-3 mice. (C) Hcrt-1 administration produced acute analgesia. Hcrt-1 (1.5 nmol/mouse, i.c.v.) caused acute analgesia in both unrestrained and restrained animals compared with vehicle groups (P ≤ 0.05), mimicking SIA in orexin/ataxin-3 mice. (D) SIA occurs in WT mice subjected to i.c.v. injections. WT mice (n = 8 per group) were subjected to 30-minute restraint immediately after i.c.v. injection of either vehicle or Hcrt-1. (E) N/OFQ blocks SIA, and coapplication of Hcrt-1 with N/OFQ restores it in WT mice. WT mice (n = 8 per group) were subjected to 30-minute restraint immediately after i.c.v. injection of vehicle alone, vehicle plus N/OFQ, or a combination of the 2 neuropeptides. N/OFQ (1 nmol/mouse) inhibited SIA compared with vehicle restraint (P = 0.044) to a latency that was no different (P = 0.503) than in the unrestrained i.c.v. vehicle-injected WT mice shown in D. Coadministration of Hcrt-1 (1.5 nmol/mouse) with N/OFQ (1 nmol/mouse) restored SIA relative to vehicle/unrestrained mice (P = 0.026). Significance of differences compared with vehicle/unrestrained WT mice was determined by Fisher’s protected least significant difference test. *P < 0.05 compared with respective controls. Error bars represent SEM.