Orexin A/hypocretin-1 selectively promotes motivation for positive reinforcers

Abstract

Orexin A/hypocretin-1 (oxA/hcrt-1) is known to be a modulator of dopamine-dependent neuronal activity and behaviors. However, the role of this system in driving motivated behaviors remains poorly understood. Here, we show that orexin/hypocretin receptor-1 (ox/hcrt-1R) signaling is important for motivation for highly salient, positive reinforcement. Blockade of ox/hcrt-1R selectively reduced work to self-administer cocaine or high fat food pellets. Moreover, oxA/hcrt-1 strengthened presynaptic glutamatergic inputs to the ventral tegmental area (VTA) only in cocaine or high fat self-administering rats. Finally, oxA/hcrt-1-mediated excitatory synaptic transmission onto VTA neurons was not potentiated following an arousing, aversive stimulus, suggesting that oxA/hcrt-1-mediated glutamatergic synaptic transmission was potentiated selectively with highly salient positive reinforcers. These experiments provide evidence for a selective role of oxA/hcrt-1 signaling in motivation for highly salient reinforcers and may represent a unique opportunity to design novel therapies that selectively reduce excessive drive to consume positive reinforcers of high salience.

Figure 1.

Ox/hcrt-1R signaling is involved in work for highly salient reinforcers. A, SB334867 (10 mg/kg) significantly reduced the breakpoint for cocaine (blue bars, n = 12, p < 0.05), high fat chocolate food pellets (red bars, n = 12, p < 0.05), but SB334867 [10 mg/kg (hatched green bars, n = 10) or 20 mg/kg (green bars, n = 12)] did not reduce the breakpoint for regular food self-administration (p > 0.05). B, Cumulative presses for cocaine were significantly reduced after SB334867 (10 mg/kg; blue line) compared with cumulative presses after a vehicle injection (black line) on the previous day (n = 12, p < 0.05 at 150 min). C, Cumulative presses for regular food were not significantly altered after animals were injected with SB334867 (20 mg/kg; green line) compared with vehicle (black line; n = 12; p > 0.05 at 175 min). D, Cumulative presses for high-fat chocolate food pellets were significantly reduced after SB334867 (10 mg/kg; red line) compared with cumulative presses after a vehicle injection (black line) on the previous day (n = 12, p < 0.05 at 20 min). Bars represent mean ± SEM. **p < 0.001.

Figure 2.

SB334867 blocks cocaine-potentiated food seeking. Rats learned to lever press for cocaine (0.5 mg/infusion), regular, or high fat chocolate food on a fixed ratio schedule. Each reinforced lever press on the active lever was associated with a tone and a light cue. Once stability criteria on Fixed Ratio 5 were met, rats were switched to a progressive ratio schedule of reinforcement for 4 consecutive days. Rats received i.p. injections of vehicle (Veh) and/or cocaine (Coc) (15 mg/kg) or GBR 12909 (GBR) (5 mg/kg) on days 2 and 3 and of the ox/hcrt-1R antagonist, SB334867 (SB) on day 4. A, Cocaine injections (15 mg/kg, i.p.) on days 2 and 3 significantly increased the breakpoint for regular food (^#p < 0.05). SB334867 (10 mg/kg) injected 15 min before cocaine (15 mg/kg) significantly reduced the breakpoint for regular food (shaded bar) compared with that after vehicle injections (n = 11, **p < 0.01). B, GBR injections (5 mg/kg, i.p.) on days 2 and 3 significantly increased the breakpoint for regular food (^#p < 0.05). SB334867 (10 mg/kg) injected 15 min before GBR (5 mg/kg) significantly reduced the breakpoint for regular food (black bar) compared with that after vehicle injections (n = 12, *p < 0.05). C, Rats received a vehicle injection (i.p.) on days 2 and 3 and an injection of SB334867 (10 mg/kg, i.p) with cocaine (15 mg/kg, i.p.) on day 4. In the presence of SB334867, a single cocaine exposure did not potentiate the breakpoint in regular food self-administering rats (p > 0.05). Bars represent mean and SEM of 11 rats. Repeated measures ANOVA was used.

Figure 3.

SB334867 reduces the effort, but not cue-learning for palatable reinforcers. In the effort based task, rats had free access to one arm on the T-maze baited with 3–45 mg of pellets of regular food or could climb a barrier to obtain equal pellets of high fat chocolate food. A, Timeline depicting the habituation, training and testing procedures used. B, C, Rats must climb (B) a 15 cm barrier (n = 8 rats) or (C) a 30 cm barrier (n = 9 rats) to obtain high fat food. Bars represent mean and SEM of the ratio of high fat chocolate food arm entries on the last day of untreated training (shaded bars), 15 min after a vehicle injection (open bars), or SB334867 (filled bars). Treatment of SB334867 significantly reduces the preference for the high fat chocolate food arm (*p < 0.05, **p < 0.001, repeated measures ANOVA). D, Schematic of the cue-conditioning paradigm. Delivery of a sucrose pellet was contingent on a tone/light conditioned stimulus (CS+) during a daily 30 min session. E, Conditioned-approach behavior, measured as the ratio of nosepokes during the CS+ to the nosepokes 30 s following CS+ presentation, was not different between rats having daily injections of vehicle (i.p., n = 11) or SB334867 (15 mg/kg, i.p., n = 11, p > 0.05).

Figure 4.

High fat chocolate food and cocaine self-administration selectively potentiates oxA/hcrt-1-mediated NMDAR plasticity in VTA neurons. eEPSCs were recorded while neurons were voltage-clamped at +40 mV before and after a 5-min bath application of oxA/hcrt-1 (100 nm). A, OxA/hcrt-1 potentiated NMDAR eEPSCs in age-matched non-food-restricted naive rats (n = 7; open circles). Food restriction did not alter oxA/hcrt-1-mediated potentiation of NMDAR eEPSCs in FR rats (n = 7; open squares). OxA/hcrt-1 (100 nm, 5 min) potentiated NMDAR eEPSCs in regular food self-administering animals (n = 7; green squares). Potentiation of NMDAR eEPSCs by oxA/hcrt-1 was significantly enhanced after rats self-administered cocaine (n = 9; blue circles) or high fat chocolate food (n = 8; red triangles). B, Example traces of NMDAR eEPSCs before (black trace) and 15 min after oxA/hcrt-1 in naive (gray), FR (gray), food (green), cocaine (blue), or high fat chocolate food (red) self-administering rats. Stimulus artifact has been removed for clarity. Calibration: 50 ms, 50 pA. C, Maximal oxA/hcrt-1-mediated potentiation of NMDAR eEPSCs is significantly greater in cocaine and high fat chocolate food self-administering rats compared with naive, FR and regular food self-administering rats (p < 0.05). Each bar represents the mean and SEM of EPSCs over a period of 2 min, 15 min after a 5 min application of oxA/hcrt-1 (100 nm) as shown by the shaded bar in A. One-way ANOVA with a Bonferroni correction for multiple comparisons was used. D, The lateral hypothalamus was taken from horizontal midbrain slices used for electrophysiology 20–24 h after the final self-administration session. The tissue was “snap frozen” and processed later using a radio-immuno assay for determination of oxA/hcrt-1 protein concentrations from naive (n = 11, open bar), FR (n = 6; shaded bar), regular food (n = 9; green bar), cocaine (n = 9; blue bar) or high-fat chocolate food (n = 12; red bar) self-administering rats. There were no significant differences between each group (p > 0.05, one-way ANOVA). Each bar represents the mean and SEM.

Figure 5.

Cocaine or high fat self-administration increases presynaptic oxA/hcrt-1 signaling. mEPSCs were recorded while VTA neurons were voltage-clamped at –70 mV before and 10 min after a 5 min bath application of oxA/hcrt-1 (100 nm). A–C, Example recordings of AMPAR mEPSCs before (i) and 15 min after (ii) oxA/hcrt-1 application in VTA slices of rats self-administering (A) regular food, (B) cocaine, or (C) high-fat chocolate food. Calibrations: 50 pA, 200 ms. D, AMPAR mEPSC amplitude (i) or frequency (ii) was not significantly different after oxA/hcrt-1 application to VTA neurons from regular food self-administering rats (n = 8, p > 0.05). E, F, In contrast, mEPSC frequency (ii), but not amplitude (i), was significantly increased after oxA/hcrt-1 application in (E) cocaine (n = 8, p < 0.05) or (F) high fat chocolate food self-administering rats (n = 6, p < 0.05). Each bar represents the mean and SEM. Paired t tests. D–F, Cumulative probability plots of amplitude (i) or interevent interval (ii) before (black) and after (colored) oxA/hcrt-1 application in example VTA neurons from (D) regular food, (E) cocaine, or (F) high fat chocolate food self-administering rats.

Figure 6.

Orexin/hypocretin receptor signaling is increased after cocaine or high fat chocolate food self-administration. Rats self-administered regular food, cocaine or high fat chocolate food pellets on a fixed ratio 1 schedule. Horizontal midbrain sections were prepared 24 h after the last session. eEPSCs were recorded while neurons were voltage-clamped at +40 mV before and after a 5-min bath application of oxA/hcrt-1 (1 nm). Peak NMDAR current was measured 20 ms after the artifact. A, Application of 1 nm oxA/hcrt-1 (5 min) did not potentiate NMDAR eEPSCs in VTA neurons of naive (open circles, n = 7) or regular food self-administering rats (green squares, n = 8). However, in rats self-administering cocaine (blue circles, n = 7) or high fat chocolate food (red triangles, n = 8), 1 nm oxA/hcrt-1 significantly potentiated NMDAR eEPSCs. B, Example traces of eEPSCs evoked at +40 mV before (black) and 10 min after (colored) oxA/hcrt-1 (1 nm, 5 min) from naive rats (i, gray) or rats self-administering regular food (ii, green), cocaine (iii, blue) or high fat chocolate food (iv, red). Stimulus artifacts have been removed for clarity. Calibrations: 50 pA, 50 ms.

Figure 7.

Footshock stress does not potentiate oxA/hcrt-1-mediated excitatory synaptic transmission in VTA neurons. Rats received 30 min of random footshocks for 7 consecutive days. Cardiac blood was drawn from FR (n = 6) or stressed (n = 6) rats immediately before aCSF perfusion and decapitation 24 h after the final footshock session. A, ACTH levels in stressed rats (filled bar) were significantly increased compared with FR controls (open bar; p < 0.05). Bars represent mean and SEM. Unpaired Student's t test. Evoked EPSCs were recorded while neurons were voltage clamped at +40 mV before and after a 5 min bath application of oxA/hcrt-1 (100 nm). B, NMDAR eEPSCs of VTA neurons were potentiated by 100 nm oxA/hcrt-1 in stressed rats (n = 10). Inset is an example eEPSC evoked at +40 mV before (black) and 10 min after (purple) a 5 min application of oxA/hcrt-1. Calibration: 50 pA, 50 ms. C, mEPSCs were recorded while VTA neurons were voltage clamped at –70 mV before and 10 min after a 5-min bath application of oxA/hcrt-1 (100 nm). Example recordings of AMPAR mEPSCs before (i) and 15 min after (ii) oxA/hcrt-1 application in VTA slices of stressed rats. AMPAR mEPSC amplitude or frequency was not significantly different after oxA/hcrt-1 application to VTA neurons of stressed rats (n = 8, p > 0.05). Each bar represents the mean and SEM of 8 neurons from at least 3 rats. Paired t tests.