Inactivation of hypocretin receptor-2 signaling in dopaminergic neurons induces hyperarousal and enhanced cognition but impaired inhibitory control

Abstract

Hypocretin/Orexin (HCRT/OX) and dopamine (DA) are both key effectors of salience processing, reward and stress-related behaviors and motivational states, yet their respective roles and interactions are poorly delineated. We inactivated HCRT-to-DA connectivity by genetic disruption of Hypocretin receptor-1 (Hcrtr1), Hypocretin receptor-2 (Hcrtr2), or both receptors (Hcrtr1&2) in DA neurons and analyzed the consequences on vigilance states, brain oscillations and cognitive performance in freely behaving mice. Unexpectedly, loss of Hcrtr2, but not Hcrtr1 or Hcrtr1&2, induced a dramatic increase in theta (7-11 Hz) electroencephalographic (EEG) activity in both wakefulness and rapid-eye-movement sleep (REMS). DA^Hcrtr2-deficient mice spent more time in an active (or theta activity-enriched) substate of wakefulness, and exhibited prolonged REMS. Additionally, both wake and REMS displayed enhanced theta-gamma phase-amplitude coupling. The baseline waking EEG of DA^Hcrtr2-deficient mice exhibited diminished infra-theta, but increased theta power, two hallmarks of EEG hyperarousal, that were however uncoupled from locomotor activity. Upon exposure to novel, either rewarding or stress-inducing environments, DA^Hcrtr2-deficient mice featured more pronounced waking theta and fast-gamma (52-80 Hz) EEG activity surges compared to littermate controls, further suggesting increased alertness. Cognitive performance was evaluated in an operant conditioning paradigm, which revealed that DA^Hcrtr2-ablated mice manifest faster task acquisition and higher choice accuracy under increasingly demanding task contingencies. However, the mice concurrently displayed maladaptive patterns of reward-seeking, with behavioral indices of enhanced impulsivity and compulsivity. None of the EEG changes observed in DA^Hcrtr2-deficient mice were seen in DA^Hcrtr1-ablated mice, which tended to show opposite EEG phenotypes. Our findings establish a clear genetically-defined link between monosynaptic HCRT-to-DA neurotransmission and theta oscillations, with a differential and novel role of HCRTR2 in theta-gamma cross-frequency coupling, attentional processes, and executive functions, relevant to disorders including narcolepsy, attention-deficit/hyperactivity disorder, and Parkinson's disease.

Fig. 1. Generation of mice with selective disruption of Hcrtr2 in dopamine neurons.

a Homologous recombination of the Hcrtr2 gene with the targeting vector creates the Hcrtr2-flox allele. The 5’loxP site was inserted in Exon1 5’-untranslated-region. 3’loxP was inserted within Intron1. In Dat-IRES-Cre-expressing neurons, CRE/lox recombination creates the Hcrtr2^del-GFP allele, with genomic deletion of DNA encoding 74 aa, encompassing HCRTR2 signal peptide, N-terminal domain, and most transmembrane region 1, and replacement of the Hcrtr2 coding sequence with a promoterless Gfp cassette. The endogenous Hcrtr2 promoter now drives Gfp instead of Hcrtr2 in DA neurons, marking cells having lost Hcrtr2 expression with GFP. TSS, Transcription start site. pA, polyadenylation signal. Chrm9, Chromosome 9. Hcrtr2^flox is Hcrtr2^tm1.1Ava (MGI:5637402), and Hcrtr2^KO-Gfp is Hcrtr2^tm1.2Ava (MGI: 5637403). b Schematic representation of DA^OxR2-KO mice. c Evidence of tissue-specific genomic recombination. DNA was isolated from various tissues and subject to PCR. Unrecombined HcrtR2-flox diagnostic band (Flox, 2,145 bp) is observed in cortex, TMN, VTA and ear from DA^OxR2-CT and DA^OxR2-KO mice, while the knockout diagnostic band (KO; 792 bp) is only observed in VTA of DA^OxR2-KO mice. The 792 bp recombined fragment was fully sequenced, confirming exact recombination event (n = 2). d GFP and Tyrosine Hydroxylase (TH) immunostaining demonstrates efficiency and specificity of Hcrtr2 Exon1 deletion in DA cells of the ventral midbrain (−2.92 to −3.88 mm from bregma) of DA^OxR2-KO mice. (Left) Quantification in several midbrain subregions. (Right) Overall penetrance (% of TH⁺ neurons co-expressing GFP) was 87.2 ± 1.5% (n = 12 sections, 2 mice). Specificity (% of GFP⁺ cells co-expressing TH) was 73.2 ± 2.4% (n = 12 sections, 2 mice). VTA, ventral tegmental area; PBP, parabrachial pigmented nucleus; PN paranigral nucleus, SNc substantia nigra pars compacta. e Representative confocal images depicting TH and GFP co-localization in ventral midbrain of DA^OxR2-KO, but not DA^OxR2-CT, mice. Coronal 20-µm brain sections at −3.08 mm from bregma. Scale bar: low magnification, 100 µm; high magnification, 20 µm. f Electrophysiological demonstration that Hcrtr2 Cre/lox recombination inactivates HCRTR2. (Left) Voltage trace recordings from putative histaminergic neurons in TMN of C57BL/6 J, Hcrtr2^flox/flox, and Hcrtr2^del/del mice. Cells were held at −50 mV in current-clamp mode. Voltage traces represent 5-min continuous recordings, before, during (green horizontal line), and after OXB-AL (200 nM) application. OXB-AL triggers a long train of action potentials in neurons from C57BL/6J and Hcrtr2^flox/flox, but not Hcrtr2^del/del, mice. (Right) Percentage of neurons responding to different treatments in each genotype. Treatments were as follows: OXB (100 nM); OXB-AL (200 nM); TCS (5 μM) + OXB (100 nM); TCS (10 μM) + OXB (100 nM); TCS (10 μM) + OXB-AL (200 nM). Number of neurons per treatment: C57BL/6J: 19/17/17/8/5; Hcrtr2^flox/flox: 4/3/6/5/6; Hcrtr2^del/del: 10/11/7/5/5). OXB-AL: [Ala¹¹, D-Leu¹⁵]-Orexin B; TCS: TCS-OX2-029; TMN: tuberomammillary nucleus.

Fig. 2. Dopaminergic Hcrtr2-ablated mice exhibit theta and fast-gamma enriched wakefulness.

a Schematic representation of DA^OxR1-KO, DA^OxR2-KO, and DA^OxR1&2-KO mice. b EEG power spectral density (PSD) analysis of wakefulness in the 3 mutant and control groups averaged across 2 baseline days. PSD values are expressed as percentage of a baseline total power density reference value calculated for each mouse (see SI Methods). Red lines indicate frequency ranges with significant differences. Note logarithmic Y-scales. Insets magnify EEG spectra across 0.75–15 Hz with linear Y-axis. DA^OxR2-KO mice show lower delta power (across 3.25–4.75 Hz), but higher theta power (across 6.75–9.75 Hz) compared to DA^OxR2-CT mice (two-way ANOVA; genotypeXfrequency interaction F(280,4502) = 1.858, P < 0.001; Tukey post-hoc test, P < 0.05). Wakefulness of DA^OxR1&2-KO mice also display lower delta (2–4 Hz), but diminished power extends through inter-delta/theta and slow-theta frequencies (4–7.25 Hz). Only a narrow fast-theta band (8.5–9.75 Hz) shows increased power (two-way ANOVA; genotypeXfrequency interaction F(286,4879) = 1.175, P = 0.026; Tukey post-hoc test, P < 0.05). c Time-frequency heatmaps on the left depict dynamics of the waking EEG in the 3 KO lines across the 3-day recordings as schematized on top. Color-coding represents EEG power calculated across time for each 0.25-Hz frequency bin and expressed relative to mean baseline (BSL) wakefulness during the light phase last 4 h (ZT8-12). Heatmaps on the right depict differential power between KO and controls (CT values are subtracted from KO values, KO-CT). d Time-course analysis of waking delta (1–4 Hz), inter-delta/theta (4–7 Hz), theta (7–11 Hz), and fast-gamma (52.5–80 Hz) band powers. Depicted are EEG powers during wakefulness across time in (averaged) 2 baseline days, 6-h SD and 18 h of recovery, expressed relative to their mean values in baseline ZT8-12 wakefulness (two-way ANOVA; Delta: DA^OxR2-KO: baseline: genotype effect F(1,17) = 39.615, P < 0.001; Inter-delta/theta: DA^OxR2-KO: baseline: genotype effect F(1,17) = 20.667, P < 0.001; SD: genotype effect F(1,23) = 17.107, P < 0.001; Theta: DA^OxR2-KO: baseline: genotype effect F(1,17) = 33.896, P < 0.001, genotypeXtime interaction F(17,288) = 2.159, P = 0.005; SD: genotype effect F(1,23) = 84.493, P < 0.001, genotypeXtime interaction F(23,384) = 1.896, P = 0.008; DA^OxR1&2-KO: SD: genotype effect F(1,23) = 10.676, P < 0.001; Fast-gamma: DA^OxR2-KO: baseline: genotype effect F(1,17) = 31.217, P < 0.001, genotypeXtime interaction F(17,288) = 1.693, P = 0.043; SD: genotype effect F(1,23) = 73.561, P < 0.001, genotypeXtime interaction F(23,384) = 2.085, P = 0.003; DA^OxR1&2-KO: baseline: genotype effect F(1,17) = 6.551, P = 0.011; SD: genotype effect F(1,23) = 5.922, P = 0.015; Bonferroni post-hoc test, *P < 0.05). n = 9 mice per group, except n = 10 for DA^OxR1&2-CT.

Fig. 3. Dopaminergic Hcrtr2-ablated mice upregulate a theta-dominated waking state uncoupled from locomotion.

a Total time spent in theta-dominated wakefulness (TDW) in DA^OxR1-KO, DA^OxR2-KO, and DA^OxR1&2-KO mice, normalized to their controls. DA^OxR2-KO mice show profound increases in TDW time during dark and SD periods (baseline dark: P = 0.027; SD: P = 0.002 recovery dark: P = 0.028; independent t-test). b Time-courses of wakefulness, TDW, TDW-to-wakefulness ratio (TDW/W), and locomotor activity in DA^OxR2-KO and DA^OxR2-CT mice. Wakefulness of DA^OxR2-KO mice is profoundly enriched in a theta-dominated state uncoupled from locomotion, during dark and SD periods (two-way ANOVA, genotype effect; baseline TDW: F(1,23) = 52.804, P < 0.001; SD TDW: F(1,23) = 97.222, P < 0.001; baseline TDW/W: F(1,23) = 127.572, P < 0.001; SD TDW/W: F(1,23) = 133.609, P < 0.001; Bonferroni post-hoc test, *P < 0.05). Locomotor activity was unchanged between DA^OxR2-KO mice and controls (two-way ANOVA, genotype effect; baseline: F(1,23) = 0.863, P = 0.354; SD: F(1,23) = 0.110, P = 0.740). c Number and duration of TDW bouts in DA^OxR2-KO and DA^OxR2-CT mice. Mean duration of TDW bouts was increased in DA^OxR2-KO mice during baseline dark, SD, and recovery dark periods (DA^OxR2-KO vs DA^OxR2-CT; baseline dark: P = 0.0028; SD: P < 0.001; recovery dark: P = 0.0083; independent t-test). The number of TDW bouts however did not differ between genotypes. d TDW bout duration distributions during 48 h baseline reveals that DA^OxR2-KO mice display less (Top) and a smaller fraction of total TDW time (Bottom) in bouts lasting ≤64 s, but more and a larger fraction of total TDW in >1024s-long bouts (two-way ANOVA; distribution of bout number: genotypeXduration interaction F(8,144) = 4.646, P < 0.001; distribution of relative time: genotypeXduration interaction F(8,144) = 4.049, P < 0.001; Bonferroni post-hoc test, *P < 0.05). Only the lower bin limit of bout durations is indicated for 4, 8–12, 16–28, 32–60, 64–124, 128–252, 256–508, 512-1020, >1024 s long bouts. e Baseline TDW spectra (averaged across 2 days). Power spectral density (PSD) values expressed as in Fig. 2b. Red lines indicate frequency ranges with significant differences. DA^OxR1-KO mice show lower delta (1-2.75 Hz) and slow-theta (6.75-8 Hz), but higher fast-theta (8.5–10.75 Hz) powers compared to controls (two-way ANOVA; genotypeXfrequency interaction F(288,4624) = 1.419, P < 0.001; Tukey post-hoc test, P < 0.05). DA^OxR1&2-KO mice show similar spectra with a more pronounced decrease in slow-theta (two-way ANOVA; genotypeXfrequency interaction F(286,4879) = 1.302, P < 0.001; Tukey post-hoc test, P < 0.05). In contrast, DA^OxR2-KO mice show increased theta power density across 7.75-10.25 Hz. (two-way ANOVA; genotypeXfrequency interaction F(280,4496) = 1505, P < 0.001; Tukey post-hoc test, P < 0.05). f TDW spectra during SD reveal an even further increase in theta power in DA^OxR2-KO compared to DA^OxR2-CT mice (two-way ANOVA; genotypeXfrequency interaction F(280,4496) = 3.514, P < 0.001; Tukey post-hoc test, P < 0.05). Bar graphs depict mean ± SEM. n = 9 mice per group, except n = 10 for DA^OxR1&2-CT.

Fig. 4. Dopaminergic Hcrtr2-ablated mice exhibit a prolonged and theta-enriched REMS.

a Total time spent in REMS during baseline light period for DA^OxR1-KO, DA^OxR2-KO, and DA^OxR1&2-KO mice, normalized to their respective controls. DA^OxR2-KO mice spent more time in REMS in baseline light period than controls (P = 0.0341; independent t-test). b Time-course of REMS amount in DA^OxR2-KO and DA^OxR2-CT mice (two-way ANOVA; genotype effect F(1,23) = 14.208, P < 0.001; Bonferroni post-hoc test, *P < 0.05). c Number and duration of REMS bouts, and bout-duration distribution, during baseline light in DA^OxR2-KO and DA^OxR2-CT mice. Top, While number of REMS episodes did not differ between groups, mean duration of REMS bouts was increased in DA^OxR2-KO mice in baseline light (P = 0.0455, independent t-test). Bottom, Bars show the relative contribution of bout categories to total REMS time (%). DA^OxR2-KO mice spent a smaller fraction of REMS in bouts lasting 32–64 s, while they spent a larger fraction in bouts lasting 128-252 s, compared to controls (two-way ANOVA; genotypeXduration interaction F(8,153) = 5.165, P < 0.001; Tukey post-hoc test, *P < 0.05). Only the lower bin limit of bout durations is indicated for 8–12, 16–28, 32–60, 64–124, 128–252, >256 s long bouts. d Mean duration of phasic REMS events is longer in DA^OxR2-KO mice compared to controls (P = 0.0423, independent t-test). Number of phasic REMS events however is unchanged in all DA^KO models. e During recovery following SD, DA^OxR2-KO mice show increased REMS latency (P = 0.0167, independent t-test, Top), and a slower and less extensive REMS rebound (Bottom). REMS latency is unchanged in DA^OxR1-KO and DA^OxR1&2-KO mice. Time-course of REMS recovery after SD, calculated as accumulated excess time spent in REMS compared to baseline, shows that DA^OxR2-KO mice regain only ~40% as much REMS time compared to controls by end of the recovery dark phase (P = 0.007, independent t-test). f Baseline spectral profiles of REMS in DA^OxR1-KO, DA^OxR2-KO, and DA^OxR1&2-KO mice and respective controls. Power density values are expressed as % of total EEG power density. Red lines indicate significant differences. DA^OxR2-KO mice display enhanced REMS theta power (6.0–8.25 Hz) compared to controls (two-way ANOVA; genotypeXfrequency interaction F(266,4272) = 1.600, P < 0.001; Tukey post-hoc test, P < 0.05). In contrast DA^OxR1&2-KO mice show reduced theta power (5.5-8.75 Hz) compared to controls (two-way ANOVA; genotypeXfrequency interaction F(291,4964) = 1.251, P = 0.003; Tukey post-hoc test, P < 0.05). Bar graphs depict mean ± SEM. n = 9 mice/group, except n = 10 for DA^OxR1&2-CT.

Fig. 5. Dopaminergic Hcrtr2-ablated mice exhibit enhanced theta-gamma phase-amplitude coupling during both wakefulness and REMS.

a Representative dynamics of theta-gamma coupling across vigilance states in DA^OxR1-KO, DA^OxR2-KO, and DA^OxR1&2-KO mice and respective controls during a 90-min interval in dark phase. Traces on top show the modulation index (MI) between theta (7–11 Hz) and fast-gamma (52–80 Hz) oscillations, calculated using a 4-s moving window. Heatmaps color-code the distribution of gamma amplitudes across the theta phase, i.e. depict phase-amplitude histograms of 4-s windows. Hypnogram is depicted below. b Heatmaps show the comodulogram analysis of phase-amplitude coupling for representative mice of each group during REMS (12-h baseline light). DA^OxR1-KO and DA^OxR1&2-KO mice show similar levels of theta-gamma coupling compared to their controls, while DA^OxR2-KO mice display enhanced coupling. Right panels show the phase-amplitude histograms of representative mice. c Pair-wise statistical comparisons between theta-gamma coupling values of KO and CT mice in wakefulness and TDW of baseline dark phase, and REMS of baseline light phase. Theta-gamma coupling significantly increased in DA^OxR2-KO mice compared to controls during all three states (DA^OxR2-KO vs DA^OxR2-CT: wake: P = 0.0172; TDW: P = 0.0397; REMS: P = 0.0412; independent t-test). Bar graphs depict mean ± SEM. n = 9 mice/group, except n = 10 for DA^OxR1&2-CT.

Fig. 6. Dopaminergic Hcrtr2-ablated mice learn faster but show compulsive and impulsive-like behaviors.

a Behavioral procedure by which mice perform a three-choice serial reaction time task (3-CSRTT), divided in training and test phases, and then undergo an attention and a motivation probe. The latter two probes are described in Fig. 7. b Training phase time-course. DA^OxR2-KO needed fewer days of training to reach the next stage of task acquisition compared to DA^OxR2-CT mice (two-way ANOVA, training effect: F(2.349, 44.63) = 27.15, P < 0.001, *genotype effect: F(1, 19) = 6.618, P = 0.0186, # interaction F(3, 57) = 3.708, P = 0.0166, with Sidak post-hoc test, P < 0.05). c Bar graphs depict the total number of sessions required to reach the next training stage. DA^OxR2-KO needed fewer sessions to reach the next stage of task acquisition compared to DA^OxR2-CT mice (P = 0.0404, independent t-test). During the test phase, DA^OxR2-KO mice displayed a higher number of correct responses (P = 0.0150, d), but no differences in incorrect responses (P = 0.1188, e), nor in response accuracy (P = 0.6737, f), or omissions (P = 0.0767, g) relative to DA^OxR2-CT mice. DA^OxR2-KO mice exhibited a higher number of premature responses, an index of impulsivity (P = 0.0286, h), and a higher number of perseverative responses, an index of compulsivity (P = 0.0203, i) compared to DA^OxR2-CT mice. Response accuracy was calculated as “correct responses/(correct+incorrect responses)x100”. Bar graphs depict mean ± SEM. n = 8 DA^OxR2-KO, n = 13 DA^OxR2-CT.

Fig. 7. Dopaminergic Hcrtr2-ablated mice display higher choice accuracy under increasingly demanding task contingencies, but no alterations in motivational drive.

Shown are results of the attention and motivation probes (see timeline in Fig. 6). In the attention probe, stimulus duration (i.e., cue light in apertures) varied from 3 to 1 s, thus exposing mice to contingencies of increasing attentional demand. a When the stimulus duration was 3 s, no differences between DA^OxR2-KO and DA^OxR2-CT mice were observed in correct and incorrect responses, omissions, and response accuracy (correct: P = 0.8213, incorrect: P = 0.5496, omission: P = 0.4297, accuracy: P = 0.5141, independent t-test). b Similarly, no differences were observed when the stimulus duration was 2 s (correct: P = 0.0739, incorrect: P = 0.7905, omission: P = 0.2969, accuracy: P = 0.8025, independent t-test). c When the stimulus duration was reduced to 1 s, no differences were observed between genotypes regarding correct responses and omissions (correct: P = 0.5486, omission: P = 0.4516, independent t-test). However, DA^OxR2-KO mice displayed fewer incorrect responses, and a higher response accuracy (incorrect: P = 0.0133, accuracy: P < 0.001, independent t-test). In the motivation probe, a single aperture was illuminated and only this choice was rewarded. Mice were challenged on a fixed ratio on one day, and on a progressive ratio on the next day. DA^OxR2-KO and DA^OxR2-CT mice did not show significant differences in number of nosepokes in the active aperture under a fixed ratio (P = 0.3926, independent t-test, d), nor the next day under a progressive ratio (P = 0.5083, independent t-test, e). Bar graphs depict mean ± SEM. n = 8 DA^OxR2-KO, n = 12 DA^OxR2-CT.