Swim stress enhances nociceptin/orphanin FQ-induced inhibition of rat dorsal raphe nucleus activity in vivo and in vitro: role of corticotropin releasing factor

Abstract

The effects of nociceptin/orphanin FQ on putative serotonin (5HT) neurons of the dorsal raphe nucleus (DRN), known to modulate the behavioral responses to stress, were investigated in vivo and in vitro. In DRN slices from unstressed rats, nociceptin/orphanin FQ concentration-dependently inhibited the firing rate of putative 5HT neurons (EC(50) = 21.6 +/- 1.21 nM) and the selective NOP receptor antagonist UFP-101 shifted the concentration-response curve to the right (estimated pA(2) 6.86). Nociceptin/orphanin FQ potency was enhanced in slices prepared from rats previously subjected to a 15 min swim stress (EC(50) = 1.98 +/- 0.11 nM). Swim stress did not change the number or affinity of NOP receptors in DRN. Stress-elicited potentiation involved corticotropin-releasing factor (CRF)(1) receptors, GABA signaling and protein synthesis, being attenuated by pre-treatment with antalarmin (20 mg/kg, i.p.), diazepam (2.4 mg/kg, i.p.) and cycloheximide (2.5 mg/kg, i.p.), respectively. In anesthetized unstressed rats, locally applied nociceptin/orphanin FQ (0.03 and 0.1 ng/30 nl) inhibited the firing rate of DRN neurons (to 80 +/- 7 and 54 +/- 10% of baseline, respectively). Nociceptin/orphanin FQ inhibition was potentiated both 24 h after swim stress and 1 h after CRF (30 ng/30 nl intra-DRN). Stress-induced potentiation was prevented by the selective CRF(1) receptor antagonist, NBI 30755 (20 mg/kg, i.p.). In contrast, the inhibitory response of DRN neurons to the 5HT(1A) agonist, 8OH-DPAT (1 microg/1 microl, intra-DRN) was not potentiated by swim stress, ruling out a non-specific enhanced permeability of GIRK channel. Together, these findings suggest that CRF and the nociceptin/orphanin FQ/NOP system interact in the DRN during stress to control 5HT transmission; this may play a role in stress-related neuropsychopathologies.

Figure 1

Single unit extracellular recordings in rat dorsal raphe nucleus slices from unstressed rats and from rats submitted to 15 min of forced swim (stressed rats). Concentration-response curve to Nociceptin/Orphanin FQ (N/OFQ), bath applied for 10 to 15 min. The spontaneous firing, facilitated by adding 10 μM phenylephrine, was sampled on-line in 10 s bins. No more than one neuron was recorded from each slice. Basal firing rate was 1.76 ± 0.16 Hz in dorsal raphe nucleus slices from unstressed rats (n=8) and 2.08 ± 0.22 Hz from stressed rats (n=8).

Figure 2

Saturation curves and Scatchard plot of [³H]-Nociceptin binding in dorsal raphe nucleus slices from unstressed rats and from rats submitted to 15 min of forced swim (stressed rats). Saturation binding experiments were performed as described in Materials and Methods. The points represent the means ± s.e.m. of 5 experiments.

Figure 3

In vivo single unit extracellular recordings in rat dorsal raphe nucleus (DRN). A: inhibitory effect of intra-raphe infusion of 0.03 ng/30 nl Nociceptin/Orphanin FQ (N/OFQ) in unstressed rats, in rats exposed to swim stress 24 h prior to recording, in rats treated with 30 ng/30 nl oCRF 1 h prior to recording. The histograms represent the means ± s.e.m. of the discharge rates of DRN units (Hz); in parentheses the number of experiments. * P<0.05, ** P< 0.01, significantly different vs. baseline, Student's t test for paired data; § P<0.05 vs. unstressed rats, Student's t test for unpaired data. B: Time course of the effect of intra-raphe infusion of 0.1 ng/30 nl N/OFQ on DRN neuronal activity in unstressed, stressed, vehicle-injected and NBI 30755-injected stressed rats. Either the CRF₁ antagonist NBI 30755 (20 mg/kg, i.p.) or its vehicle were administered 30 min prior to the swim stress. Abscissa: time after N/OFQ administration; ordinate: mean discharge rate, expressed as a percentage of the mean determined over 180 s prior to N/OFQ infusion (baseline); in parentheses the number of experiments. Repeated-measures analysis of variance indicated a statistically significant effect of group [F_(3,22) = 4.657, P= 0.0115] and time [F_(5,110) = 28.740, P < 0.0001]. Symbols indicate differences determined by Fisher post hoc test: * P < 0.05, **P <0 .01, stressed vs. unstressed; § P < 0.05, stressed + vehicle vs. unstressed; $ P < 0.05, $$ P <0 .01, stressed + NBI 30775 vs. stressed; # P < 0.05, stressed + NBI 30775 vs. stressed + vehicle.