Highly Specific Role of Hypocretin (Orexin) Neurons: Differential Activation as a Function of Diurnal Phase, Operant Reinforcement versus Operant Avoidance and Light Level

Diagram of the training schedules for nonoperant conditions for WT and KO animals. Nonoperant conditions: 1, expected and unexpected reward; 2, unavoidable footshock; 3, chamber control; VI-4, variable interval-4 min (food delivery with no contingency on lever pressing); FI-5 fixed interval-5 min.

Operant performance of WT and KO mice on PR5 food reinforcement paradigm. Hcrt KO mice are unable to sustain bar pressing for food in the light phase, in contrast to WT mice. a, b, Representative cumulative records of the performance of a WT animal (a, 2817 total presses) and an Hcrt KO animal (b, 456 total presses). The downward pips on the cumulative record denote food deliveries. The KO mouse session was terminated because it ceased pressing the lever for 15 min. c, The final ratios reached by KO were significantly lower than those reached by the WT in the light phase (WT, n = 8; KO, n = 8; mean ± SEM in all figures). No significant difference in performance was found between groups in the dark phase (WT, n = 7; KO, n = 7). d, Performance of WT (open triangles, n = 8) and KO (filled diamonds, n = 8) were analyzed in 10 min intervals during the first 40 min of the light-phase sessions. Performance deteriorates in the KO. e, Comparison of interpress intervals in the light phase for WT (n = 8) and KO (n = 8) animals. Values represent the percentage of presses falling under each interval. KO mice show more long intervals between presses. f, Example of the cumulative records of the same KO animal performing the operant task during the light phase (red line, 351 total presses) and the dark phase (blue line, 2837 total presses). KOs are unimpaired on the same task in the dark phase. **p < 0.01, *p < 0.05, Newman–Keuls post hoc test.

EEG, EEG power spectrum, and EMG of KO mice during operant tasks. There is EEG slowing during pauses in KO mice working for positive reinforcement, indicating that these are sleep attacks, not cataplexy. a, EEG, EMG, and spectral power of the theta rhythm of a KO exhibiting short pauses (1, 2, 3) during a PR5 food reward operant task in the light phase. There is an increase of theta power in the EEG during these short pauses, but no complete EMG atonia is seen, indicating that no cataplexy or REM sleep occurred. Magnified displays of the three short pauses are shown at the bottom. L, Lever press; F, food pellet delivery. b, The same KO animal exhibited a long pause that led to NREM sleep and REM sleep during the operant task. NREM sleep is evident with increase of delta power and reduced EMG, while REM sleep is characterized by increased theta power and EMG atonia. Active waking (AW) is characterized by lower power in all analyzed bands. c, EEG power spectrum, expressed in relative power of REM, of the Hcrt KO animal during the long behavioral pauses seen during positive reinforcement in a PR5 food operant condition in the KOs (pause), during NREM sleep, REM sleep, AW, and shock avoidance. EEG power <9 Hz is increased during pauses. Low voltage spectrum during AW and shock avoidance indicates that the animals had a high degree of EEG activation. WT mice do not have pauses during these tasks and show active waking EEG during both positive reinforcement and shock avoidance.

Effect of the extent of food restriction on food operant performance. a, WT and KO animals tested in the light phase at 100%, 85–90%, and 70–75% of their initial body weight. Performance of WT mice improves substantially and linearly with weight restriction, but performance of Hcrt KO mice improves to a much lesser extent, with no improvement after reduction to 85–90% of baseline weight but significant improvement only at 70–75% of baseline weight. Even at 70–75%, KO animals had a significantly lower BP/final ratio than WT animals (**p < 0.01, relative to WT; ^#p < 0.02 for KO relative to weight at 85–90%; ⁺p < 0.05, ⁺⁺p < 0.01 for WT relative to weight at 85–90%; Newman–Keuls post hoc test). b, Difference in performances of WT and KO animals in the dark phase under two weight conditions. WT increased their bar press performance at 85–90% ad libitum condition (⁺p < 0.05, Newman–Keuls post hoc test). At 100% of weight, the performance of KO was not significantly different from WT. The break point ratios of the WT at 85–90% and at 100% do not significantly differ between light and dark phases. We did not run mice at the 70–75% weight conditions in the dark phase because of the light-phase results. c, Average amount of food consumed by WT and KO in 120 min in the home cage when food is freely available (11:00 A.M. to 1:00 P.M.) during the light phase. There is no significant difference in the consumption of food by the two groups (p > 0.05, t test; n = 7 for each genotype).

Distribution of Hcrt⁺ and cFos⁺/Hcrt⁺ neurons in the hypothalamus of WT mice under different behavioral conditions. Hcrt neurons express Fos during a food-motivated task in the light phase. Neither food nor shock avoidance tasks increase Fos expression in the dark phase. a, Diagrams of coronal sections of the hypothalamus stained for Hcrt and cFos of six animals each under one of six different experimental conditions during the light and the dark phase: L1, PR food, light phase; L2, shock avoidance, light phase; L3, chamber control, light phase; D1, PR food, dark phase; D2, shock avoidance, dark phase; D3, chamber control, dark phase. Red dots indicate double-labeled cFos⁺/Hcrt⁺ neurons; blue triangles correspond to Hcrt⁺ neurons. Fx, Fornix; 3V, third ventricle. Scale bar, 150 μm. b, Photomicrographs of the same hypothalamic region in a section processed for Hcrt and cFos. LH, Lateral hypothalamus; MH, medial hypothalamus. Scale bar, 150 μm. The rectangular region in the LH is magnified in the insert at the lower left. Scale bar, 20 μm. The double-labeled neurons (red arrows) show the characteristic black nucleus due to the presence of cFos protein and a brown precipitate in the cytoplasm, indicating their hypocretinergic nature. These cells are easily distinguishable from single-labeled hypocretin neurons (blue arrows) and single-labeled cFos cells (black arrowheads).

Percentage of Hcrt neurons expressing Fos in the hypothalamus of WT mice under different behavioral conditions. Activation of Hcrt neurons was maximal when bar pressing for food in the light phase, but not when given expected or unexpected food under the same conditions, not during shock avoidance, and not during bar pressing for food when light was absent in the subjective light phase. a, Comparison of the percentage of hypocretin neurons expressing Fos in the PR food, shock avoidance, and chamber control conditions during the light and dark phases (**p < 0.01, Newman–Keuls post hoc test comparing food task during the light phase with all other conditions; n = 4 in each condition). There is no significant difference between the light and dark phases in shock avoidance and chamber control conditions. b, Comparison of the percentage of hypocretin neurons expressing Fos under PR food condition with other behavioral conditions during the light phase (**p < 0.01, Newman–Keuls post hoc test compared with all other conditions; n = 4 in each condition). c, Percentage of the double-labeled cFos⁺/Hcrt⁺ cells in the LH, MH, and PFA in different behavioral conditions during the light phase (**p < 0.01, Newman–Keuls post hoc test comparing PR food with all other conditions within each subdivision; ⁺⁺p < 0.01, Newman–Keuls post hoc test comparing PFA with lateral subdivision in PR food condition; ^##p < 0.01 Newman–Keuls post hoc test comparing PFA with lateral and medial subdivisions in chamber control condition; ^xxp < 0.01 Newman–Keuls post hoc test comparing PFA with lateral and medial subdivisions in subjective day condition; ^†p < 0.05 Newman–Keuls post hoc test comparing with unexpected food condition within PFA; ^‡p < 0.05, Newman–Keuls post hoc test comparing PFA with lateral subdivision in shock avoidance condition). d, Comparison of percentage increase of double-labeled cFos⁺/Hcrt⁺ cells among the three subdivisions in PR food condition relative to chamber control. There is no significant difference.

Operant performance on PR3 water reinforcement paradigm in the light phase. KOs are as deficient in bar pressing for water as they are in bar pressing for food. a, b, Typical examples of the cumulative records showing the difference in performance of a WT (a, 2494 total presses) and a KO (b, 164 total presses). The downward pips on the cumulative record denote water deliveries. c, KO mice showed a significant deficit in the final ratio completed compared with the WT (n = 5 for each genotype; ***p < 0.005, t test). d, There is no difference in the amount of water consumed by the WT and KO mice during the 60 min in the home cage when water is readily available (p > 0.15, t test).