Research ArticleNew Research, Integrative Systems

Locus Coeruleus and Tuberomammillary Nuclei Ablations Attenuate Hypocretin/Orexin Antagonist-Mediated REM Sleep

Michael D. Schwartz, Alexander T. Nguyen, Deepti R. Warrier, Jeremiah B. Palmerston, Alexia M. Thomas, Stephen R. Morairty, Thomas C. Neylan and Thomas S. Kilduff
eNeuro 28 February 2016, 3 (1) ENEURO.0018-16.2016; DOI: https://doi.org/10.1523/ENEURO.0018-16.2016

Abstract

Hypocretin 1 and 2 (Hcrts; also known as orexin A and B), excitatory neuropeptides synthesized in cells located in the tuberal hypothalamus, play a central role in the control of arousal. Hcrt inputs to the locus coeruleus norepinephrine (LC NE) system and the posterior hypothalamic histaminergic tuberomammillary nuclei (TMN HA) are important efferent pathways for Hcrt-induced wakefulness. The LC expresses Hcrt receptor 1 (HcrtR1), whereas HcrtR2 is found in the TMN. Although the dual Hcrt/orexin receptor antagonist almorexant (ALM) decreases wakefulness and increases NREM and REM sleep time, the neural circuitry that mediates these effects is currently unknown. To test the hypothesis that ALM induces sleep by selectively disfacilitating subcortical wake-promoting populations, we ablated LC NE neurons (LCx) or TMN HA neurons (TMNx) in rats using cell-type-specific saporin conjugates and evaluated sleep/wake following treatment with ALM and the GABAA receptor modulator zolpidem (ZOL). Both LCx and TMNx attenuated the promotion of REM sleep by ALM without affecting ALM-mediated increases in NREM sleep. Thus, eliminating either HcrtR1 signaling in the LC or HcrtR2 signaling in the TMN yields similar effects on ALM-induced REM sleep without affecting NREM sleep time. In contrast, neither lesion altered ZOL efficacy on any measure of sleep–wake regulation. These results contrast with those of a previous study in which ablation of basal forebrain cholinergic neurons attenuated ALM-induced increases in NREM sleep time without affecting REM sleep, indicating that Hcrt neurotransmission influences distinct aspects of NREM and REM sleep at different locations in the sleep–wake regulatory network.

Significance Statement

The hypocretin/orexin (Hcrt) system powerfully regulates arousal in part by excitatory projections to wake-promoting cell groups in the posterior hypothalamus and brainstem. Cell-type-specific ablations of the locus coeruleus norepinephrine (LC NE) neurons or the tuberomammillary histamine (TMN HA) neurons decreased the hypnotic efficacy of the dual Hcrt receptor antagonist Almorexant, while having no effect on sleep promotion by the GABA receptor modulator zolpidem. Lesioning the LC or TMN attenuated almorexant-induced REM sleep without affecting NREM sleep time. Lesions exerted similar effects independently of the Hcrt receptor type expressed in each region, suggesting that the site of action, not just the specific receptor or receptors targeted, is a key determinant of how Hcrt receptor antagonism facilitates sleep.

Introduction

Hypocretin-1 and -2 (Hcrts; also known as orexin-A and -B), excitatory neuropeptides synthesized in neurons located in the tuberal hypothalamus, are involved in metabolism, feeding, reward, addiction, and sleep–wake control (Ohno and Sakurai, 2008). Hcrt neurons are wake-active (Estabrooke et al., 2001; Lee et al., 2005). Hcrt administration (Bourgin et al., 2000; Morairty et al., 2011) or optogenetic stimulation of Hcrt neurons (Adamantidis et al., 2007; Carter et al., 2010) is wake-promoting. Deficient Hcrt signaling underlies narcolepsy (Chemelli et al., 1999; Lin et al., 1999; Thannickal et al., 2000), a sleep disorder characterized by fragmented sleep, degraded sleep–wake rhythms, and profound dysregulation of REM sleep. Hcrt signaling thus plays a critical role in the organization and consolidation of sleep–wake states.

Hcrt neurons project to several wake-promoting brain populations, including the locus coeruleus (LC; Peyron et al., 1998; Chemelli et al., 1999; Horvath et al., 1999). LC activation desynchronizes cortical activity and precedes transitions to waking, exhibiting a strongly wake-active, REM-silent firing profile (Aston-Jones and Bloom, 1981; Berridge and Foote, 1991; Takahashi et al., 2010). Optogenetic inhibition or activation of LC norepinephrine (NE) neurons increases or decreases the likelihood of sleep, respectively (Carter et al., 2010). Disruption of NE signaling via cell-type-specific LC lesions or knockout (KO) is reported to increase NREM sleep (González et al., 1998; Blanco-Centurion et al., 2004; Ouyang et al., 2004) or block wakefulness following arousing stimuli (Hunsley and Palmiter, 2004; Gompf et al., 2010), consistent with a role in maintenance of wakefulness. The LC expresses Hcrt receptor 1 (HcrtR1; Marcus et al., 2001) and Hcrt-1/orexin-A infusion into the LC increases LC neuron firing and promotes wakefulness (Hagan et al., 1999; Bourgin et al., 2000) in a HcrtR1-dependent manner (Soffin et al., 2002; Choudhary et al., 2014). Conversely, optogenetic LC inactivation blocks transitions to wakefulness following Hcrt neuron activation (Carter et al., 2012), indicating that the LC is important for Hcrt-induced wakefulness.

Hcrt neurons also strongly innervate histaminergic (HA) cells in the tuberomammillary nuclei (TMN) of the posterior hypothalamus (Peyron et al., 1998; Chemelli et al., 1999). TMN HA neurons express HcrtR2 (Marcus et al., 2001) and are excited by Hcrt peptides (Eriksson et al., 2001). HA is wake-promoting (Chu et al., 2004; Ramesh et al., 2004) and TMN HA neurons, like LC NE neurons, exhibit a wake-active, REM-off firing pattern (Takahashi et al., 2006). TMN HA lesions have relatively mild effects on sleep–wake states (Gerashchenko et al., 2004). However, mice unable to synthesize HA exhibit decreased wakefulness at lights-off, increased REM sleep time during the light phase, and short sleep latency in a novel environment (Parmentier et al., 2002; Anaclet et al., 2009). Wake promotion by Hcrt-1/orexin A is mediated in part through histaminergic neurotransmission (Huang et al., 2001). Thus, Hcrt inputs to the LC NE system and the TMN HA system are important pathways for Hcrt-induced wakefulness.

The dual Hcrt/orexin receptor antagonist (DORA) almorexant (ALM) blocks the excitatory effects of the Hcrt peptides at HcrtR1 and HcrtR2, decreasing wakefulness and increasing NREM and REM sleep time (Brisbare-Roch et al., 2007; Morairty et al., 2012). In contrast, zolpidem (ZOL; trade name Ambien) induces somnolence by activating GABAA receptors, thereby causing widespread neuronal inhibition (Dang et al., 2011). ALM, but not ZOL, requires an intact basal forebrain (BF) for maximal hypnotic efficacy and induces neurochemical events associated with the transition to normal sleep (Vazquez-DeRose et al., 2014). These findings support the hypothesis that ALM induces sleep by selectively disfacilitating subcortical wake-promoting populations whereas ZOL acts via generalized inhibition throughout the brain. Here, we tested this hypothesis by selectively ablating the LC NE neurons or the TMN HA neurons using cell-type-specific saporin conjugates, and subsequently evaluating the efficacy of ALM and ZOL in lesioned and intact rats. We find that eliminating either HcrtR1 signaling in the LC or HcrtR2 signaling in the TMN yields similar effects on ALM-induced REM sleep without affecting NREM sleep time. Because a previous study (Vazquez-DeRose et al., 2014) found the converse effects after ablation of basal forebrain cholinergic neurons, these results support the concept that Hcrt neurotransmission influences distinct aspects of NREM and REM sleep at different locations in the sleep—wake regulatory network.

Materials and Methods

Animals

Male Sprague-Dawley rats (n = 25; 200–250 g; Harlan Laboratories) were housed in light-tight, sound-attenuated environmental chambers under constant temperature (22 ± 2°C, 50 ± 25% relative humidity) on a 12 h dark/light cycle with food and water ad libitum. All dosing procedures were performed under dim red light (<2 lux). All studies were conducted in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at SRI International.

Chemicals

ALM was synthesized by the Medicinal Chemistry Laboratory at SRI International according to previously published methods (Koberstein et al., 2003, 2005). ZOL was purchased from IS Chemical. All drugs that were delivered orally were suspended and sonicated for 1 h in 1.25% hydroxypropyl methyl cellulose with 0.1% dioctyl sodium sulfosuccinate and 0.25% methylcellulose in sterile water [hereafter referred to as vehicle (VEH)]. All drug solutions were made on the day of the experiment and serially diluted to their final concentrations.

Saporin lesions

Under isoflurane anesthesia, rats were placed into a stereotaxic apparatus (Kopf Instruments) and the skull was exposed. For LC lesions, rats were injected intracerebroventricularly with 10 μl of anti-dopamine beta hydroxylase-conjugated saporin (n = 8; DBH-SAP; 0.3 μg/μl; Advanced Targeting Systems; Wrenn et al., 1996; Wiley and Kline, 2000; Brightwell and Taylor, 2009) or sterile saline (n = 7; hereafter referred to as “Sham” rats) via a 26 gauge stainless steel injection cannula connected to a 10 μl Nanofil Hamilton syringe and a digitally controlled microinjector (World Precision Instruments) at −0.8 mm AP and +1.5 mm ML relative to bregma, and 3.3 mm below dura. The infusion volume and concentration were selected based on previously published methods and were verified in pilot studies. Injections lasted ∼10 min; the cannula was left in place for 5 min after the injection. For TMN lesions, rats were injected bilaterally with 250–350 nl of Hcrt2-saporin (n = 13; Hcrt2-SAP; 0.228 μg/μl; Advanced Targeting Systems; Gerashchenko et al., 2001, 2004) or sterile saline (n = 7) via glass micropipettes (inner tip diameter ∼30–50 μm) using a Picospritzer (Parker Hannifin) at −4.2 or −4.35 mm AP and ±0.8 mm ML relative to bregma, and 9.3 mm below dura. Injectate volume was measured via precalibrated marks on the barrel of the pipette. Injections lasted 5 min/side; the pipette was left in place for 5 min after the injection. Following SAP injections, rats were instrumented for EEG/EMG telemetry.

Telemetry surgery

All rats were surgically implanted with a sterile abdominal transmitter (F40-EET, DSI) for continuous telemetric recordings of electroencephalograph (EEG), electromyograph (EMG), core body temperature (Tb), and locomotor activity as described previously (Morairty et al., 2008, 2012). Briefly, the wires from the transmitter were subcutaneously channeled rostrally to the head. Two biopotential leads (EEG electrodes) were inserted into drilled holes over the dura (lead 1: +2.0 mm AP, +1.5 mm LM; lead 2: −7.0 mm AP, −2.0 mm LM; all coordinates relative to bregma) and affixed with dental acrylic. Two additional biopotential leads (EMG electrodes) were sutured into the neck musculature and closed with non-absorbable suture. Both DBH-SAP (Wrenn et al., 1996; Blanco-Centurion et al., 2004; Gompf et al., 2010) and Hcrt2-SAP (Gerashchenko et al., 2004) induce maximal degeneration by 12–14 d postinjection. Accordingly, animals were singly housed after surgery and allowed to recover undisturbed in their home cage for 3 weeks to allow sufficient time for SAP-induced neurodegeneration and sleep–wake behavior to stabilize prior to any recordings.

Assessment of hypnotic efficacy in saporin-lesioned rats

Rats were kept in their home cages for the duration of the study in ventilated, light-tight, and sound-attenuated chambers in 12 h light/dark cycles. Prior to initiation of sleep recordings, animals were acclimated to handling for ∼1 week, and were dosed with VEH once per day for the last 3 d of the acclimation period. Animals were then left undisturbed for 2 d after acclimation was complete. Rats were administered ALM (30, 100, and 300 mg/kg), ZOL (10, 30, and 100 mg/kg), or VEH orally starting at lights-out (ZT 12) with at least 3 d between treatments to allow sufficient time for washout between doses. Drug treatments were balanced across lesion condition and treatment day, such that every dose was administered on each treatment day, and an approximately equal number of lesioned and sham rats received each dose on each treatment day. EEG was recorded for 24 h following dosing; the first 6 h following dosing was scored and analyzed (from ZT12 to ZT18).

To confirm the extent of lesions, rats were deeply anesthetized and transcardially perfused with heparinized 0.1 m phosphate-buffered saline followed by 4% paraformaldehyde. Brains were removed, postfixed in 4% paraformaldehyde, and then transferred to 30% sucrose until sectioning. Brains were sectioned at 40 µm on a freezing microtome. Free-floating sections containing the LC (bregma −9.16 mm to −10.30 mm) were incubated with 1% H2O2 for 15 min to quench endogenous peroxidase activity, followed by: (1) 1 h in blocking buffer containing 3% normal donkey serum, (2) overnight in mouse anti-DBH (1:100,000, MAB308, EMD Millipore), (3) 2 h in biotinylated donkey anti-mouse IgG (1:500; Jackson ImmunoResearch), and (4) 2 h in avidin–biotin complex (Vector Laboratories). DBH was visualized by reacting sections in 0.05% diaminobenzidine tetrahydrochloride and 0.01% H2O2 to form a brown reaction product. Sections were then mounted, dehydrated and coverslipped. To visualize HA neurons, sections containing the TMN (bregma −3.80 to −4.80 mm) were processed using a similar protocol that was modified as follows: (1) sections were incubated overnight in rabbit anti-adenosine deaminase (ADA; 1:20,000, ab176, EMD Millipore), followed by (2) 2 h in biotinylated donkey anti-rabbit IgG (1:500; Jackson ImmunoResearch).

The extent of the LC was delineated by the fourth ventricle and other landmarks. The exact number of DBH-positive LC neurons could not be accurately counted in Sham rats because of the high density of these cells (Fig. 1B); accordingly, Sham rats were only scored for the presence of DBH-positive cells. In DBH-SAP injected rats, all residual DBH-positive cells in the LC region were counted. To evaluate the extent of TMN HA neuronal loss, ADA-positive neurons were counted in the dorsal (dTMN), ventral (vTMN), and caudal TMN (cTMN) subregions as identified previously (Ko et al., 2003; Parks et al., 2016). All neurons expressing ADA in each subregion were scored.

Figure 1.
Figure 1.

Characterization of DBH-SAP lesions. A, Schematic showing location of LC NE neurons targeted by DBH-SAP infusions (red) and the more ventrally located A5 noradrenergic neurons (yellow). B, DBH immunostaining of the LC in a Sham-injected rat shows densely-packed NE neurons, which were destroyed following DBH-SAP injections (C). By contrast, A5 neurons were intact in both Sham (D) and DBH-SAP-injected rats (E). 4v, Forth ventricle; A5, A5 NE group; PRN, pontine reticular nucleus; scp, superior cerebellar peduncle, SOC, superior olivary complex; SubC, subcoeruleus; VIIn, facial nerve. Scale bar, 200 μm. Adapted from Swanson (2004).

EEG and EMG analyses and sleep–wake determinations

EEG and EMG were recorded via telemetry on a PC running Dataquest ART 3.1 (DSI). All recordings were manually scored off-line by a trained expert in 10 s epochs as Wake, NREM, or REM sleep using NeuroScore 2.1 (DSI). Any epochs that contained recording artifacts were tagged and excluded from spectral analyses. Individual state data were quantified as time spent in each state per 6 h. Latency to NREM and REM onset for each animal was calculated from the time of drug injection. Bouts were defined as a minimum of three consecutive epochs of wake or NREM, and two consecutive epochs of REM sleep.

Statistical analyses

Latency to NREM and REM sleep, total time in each state (wake, NREM, REM), REM–NREM ratio, average bout duration, and total number of bouts for the 6 h following dosing were analyzed by a two-way ANOVA comparing lesion condition (between-subjects) and drug treatment (within-subjects). Bout architecture was further analyzed using a three-way mixed model ANOVA comparing the effects of lesion condition (between-subjects), drug treatment (within-subjects) and bout duration (within-subjects) on bout number. To assess fragmentation of arousal states, we included all sleep–wake bouts without a minimum bout length requirement. Significant main effects and interactions (p < 0.05) were subsequently analyzed with Bonferroni post hoc tests. In some cases, near-significant trends in the omnibus ANOVA were followed up with planned comparisons (F test), examining the effects of lesion at each drug dose; these planned comparisons are specified in the results. TMN lesion efficacy was assessed via a Student’s t-test. All statistical analyses were run using Statistica (Statsoft), except the t test and accompanying power analysis (Table 1, row n), which were run using R and G*Power, respectively.

View this table:
Table 1.

Statistical table

Results

LC lesion evaluation

Figure 1A shows the LC area targeted by the DBH-SAP lesions (red), as well as the approximate location of the nearby A5 NE neurons (yellow). In Sham-injected rats, the LC was clearly delineated by densely packed DBH-positive cells and fibers (Fig. 1B). The darkly stained neuropil and proximity of DBH-positive cells to each other made it difficult to accurately count individual cells. In DBH-SAP-injected rats, only a few scattered DBH-positive neurons were visible in the LC (Fig. 1C); bilateral counts in the LC revealed 15.75 ± 4.2 DBH-positive cells in SAP-treated rats, ranging from 2 to 38 neurons remaining in individual animals. In contrast, the more ventral A5 neurons were largely or entirely spared following DBH-SAP injections (Sham, Fig. 1D; DBH-SAP, Fig. 1E) as previously reported for a similar DBH-SAP dose (Wrenn et al., 1996; Brightwell and Taylor, 2009). All DBH-SAP-injected rats were thus considered to have complete LC lesions.

LCx attenuates sleep induction by ALM

Both ALM and ZOL (all doses) shortened the latency to NREM sleep compared with VEH in Sham rats, whereas only ZOL (10 and 100 mg/kg) was effective in LCx rats (interaction: F(6,78) = 2.553, p = 0.026; Fig. 2A,B)a. Thus, LCx attenuated the ALM-induced but not the ZOL-induced decrease in NREM latency. Although LC lesions shortened NREM sleep latency following VEH from ∼60 to ∼40 min, the decrease was not significant compared to VEH-treated Sham rats likely because of the large variance in NREM latency among the Sham rats. Importantly, ZOL further decreased NREM latency from this baseline in LCx rats, whereas ALM only decreased NREM latency in Shams (Fig. 2B), indicating that lesioned rats could still respond to hypnotics. REM sleep latency was significantly increased by ZOL at 100 mg/kg independently of lesion status (main effect of drug: F(6,78) = 12.58, p < 0.001; Fig. 2C,D)b.

Figure 2.
Figure 2.

Latency to NREM (A, B) and REM sleep (C, D) following ALM (A, C), and ZOL (B, D) in LCx and Sham lesioned rats. Doses are mg/kg. *p < 0.05 versus vehicle; *'p < 0.06 versus vehicle.

LCx attenuates REM sleep increases following ALM

Both ALM (all doses) and ZOL (30, 100 mg/kg) decreased wake time for the 6 h period following dosing (ZT 12 to ZT 18) independent of lesion status (main effect of drug: F(6,78) = 21.532, p < 0.001; Fig. 3A,B)c. NREM sleep time was increased by LC lesions (main effect of lesion: F(1,13) = 5.722, p = 0.033)d and by all doses of ALM and ZOL (main effect of drug: Fig. 3C,D; F(6,78) = 18.821, p < 0.001)d, with no drug–lesion interaction (Fig. 3C,D). By contrast, ALM (100, 300 mg/kg) increased REM sleep time compared to VEH in both Sham and LCx rats, but this increase was attenuated in LCx compared with Sham rats at the ALM 300 mg/kg dose (interaction: F(6,78 = 4.439, p < 0.001; Fig. 3E,F)e. Similarly, ALM (100, 300 mg/kg) increased the ratio of REM to NREM sleep (REM–NREM) compared to VEH in Shams, but not LCx rats (interaction: F(6,78) = 5.010, p < 0.001; Fig. 3G)f, such that REM–NREM was significantly attenuated in LCx rats compared to Shams following ALM (100 and 300 mg/kg). Although ZOL did not significantly affect REM sleep time (Fig. 3F), ZOL decreased REM–NREM in both Sham (30 and 100 mg/kg) and LCx rats (100 mg/kg; Fig. 3H). Thus, LCx blocked ALM-induced, but not ZOL-induced shortening of NREM sleep latency, attenuated the ALM-mediated increase of REM sleep time, and increased NREM sleep time independent of drug effects.

Figure 3.
Figure 3.

Total Wake (A, B), NREM (C, D) and REM (E, F) sleep time, and the ratio of REM to NREM sleep (G, H) following ALM (A, C, E, G) and ZOL (B, D, F, H) for 6 h following dosing in LCx and Sham lesioned rats. Doses are mg/kg. *p<0.05 versus vehicle; #p<0.05 (LCx vs Sham).

LCx attenuates NREM and REM bout increases following ALM

LC lesions decreased the total number of wake bouts compared with Shams (main effect: F(1,13) = 5.891, p = 0.030; Fig. 4A,B)g. ALM (all doses) increased the total number of wake bouts, whereas ZOL did not (main effect of drug: F(6,78) = 29.152, p < 0.001; Fig. 4A,B)g. There was a borderline drug x lesion interaction (F(6,78) = 2.136; p = 0.058)g; planned comparisons revealed that LCx rats had fewer wake bouts than Shams following VEH and ALM (100 and 300 mg/kg) but not after ZOL (Fig. 4A,B).

Figure 4.
Figure 4.

Total number of wake (A, B), NREM (C, D) and REM (E, F) bouts for 6 h following ALM (A, C, E) and ZOL (B, D, F) LCx and Sham lesioned rats. Doses are mg/kg. *p<0.05 versus vehicle; #p<0.05 (LCx vs Sham); #p<0.06 (LCx vs Sham); +p<0.05, paired comparison F test (LCx vs Sham).

A similar effect was observed for the number of bouts of NREM sleep (interaction: F(6,78) = 2.514, p = 0.028, Fig. 4C,D)h; ALM (all doses) significantly increased NREM bout number compared with VEH in all rats, but bout numbers were consistently lower in LCx compared with Sham rats at all doses (Fig. 4C). By contrast, ZOL tended to equalize NREM bout number between lesion conditions, especially at the highest dose (100 mg/kg; Fig. 4D).

ANOVA for the number of REM bouts revealed a significant drug x lesion interaction (F(6,78) = 4.519; p < 0.001; Fig. 4E,F)i. ALM increased the number of REM bouts compared with VEH in both Sham (all doses) and LCx rats (100 and 300 mg/kg); however, LCx attenuated the ALM-induced increase at 300 mg/kg, with a borderline effect at the 100 mg/kg dose (Bonferroni, p=0.055). ZOL did not affect REM bout number in either Sham or LCx rats.

The mean duration of NREM bouts was independently increased by LC lesions (main effect of lesion: F(1,13) = 8.848; p = 0.011)j and by ZOL (100 mg/kg; main effect of drug: F(6,78) = 6.734; p < 0.001)j, with no interaction between the factors (data not shown). There were no other effects on the mean duration of NREM, REM, or wake bouts.

LCx preferentially attenuates short wake and NREM bouts following ALM

We next asked whether changes in bout number were associated with changes in bout duration (Fig. 5). ALM preferentially increased the number of short (<0.5 min) wake bouts in both LCx and Sham rats (F(24,312) = 3.814, p < 0.001; Fig. 5A,D)k, but LCx rats had fewer short wake bouts compared to Shams following VEH and ALM. Similarly, ALM also increased the number of short NREM bouts in both LCx and Sham rats (F(24,312) = 4.739, p < 0.001; Fig. 5B,E)l, but LCx rats had fewer NREM bouts <1 min than Shams following VEH and ALM. In other words, ALM increased the number of short sleep–wake bouts, whereas LC lesions decreased the number of short bouts. By contrast, LC lesions had very little effect on wake and NREM bout architecture following ZOL. ZOL appeared to increase the number of long NREM bouts (>4 min) in both Sham (Fig. 5H) and LCx (Fig. 5K) rats. Although the post hoc comparisons were not statistically significant, the additional long bouts likely account for the significant increase in mean NREM bout duration under ZOL treatment.

Figure 5.
Figure 5.

Number of wake (A, D, G, J), NREM (B, E, H, K), and REM (C, F, I, L) bouts as a function of bout duration in Sham (A–C, G–I) and LCx rats (D–F, J–L) following ALM (A–F) or ZOL (G–L). *p<0.05 vs vehicle; #p<0.05 (LCx vs Sham).

There were significant drug × lesion (F(6,78) = 4.473; p < 0.001)m and bout x lesion interactions (F(4,52) = 6.421; p < 0.001)m that affected REM bout composition, but there was no three-way interaction between drug, bout, and lesion (Fig. 5C,F,I,L). As described above for total bout number, LCx attenuated the increase in REM bout number following ALM (100 and 300 mg/kg); Figure 5, F and L, show that the changes in REM bout number were distributed across short and long REM bouts and were observed following both ALM and ZOL.

TMN lesion evaluation

Figure 6A–C shows the posterior hypothalamic region targeted by the Hcrt2-SAP lesions, with the dorsal, ventral, and caudal TMN subgroups highlighted. ADA-immunostaining clearly visualized the HA neurons in Sham-injected rats (Fig. 6D). There were 1375 ± 78 ADA-positive cells in the TMN of Sham rats (combined count of dorsal, ventral, and caudal TMN). Hcrt2-SAP injections decreased ADA-immunostaining in the TMN (Fig. 6E,G). Inspection of Nissl-stained sections revealed that Hcrt2-SAP also destroyed some cells surrounding the TMN (Fig. 6H), as described in previous studies (Gerashchenko et al., 2004; Blanco-Centurion et al., 2007).

Figure 6.
Figure 6.

Characterization of Hcrt2-SAP lesions. A–C, Schematic showing location of TMN HA neurons targeted by Hcrt2-SAP infusions. Panels adapted from Swanson (2004). D, E, HA-positive neurons in the dTMN and vTMN in a Sham-injected rat (D) and a Hcrt2-SAP injected rat (E). Photomicrographs depict the TMN at approximately the same rostrocaudal level as B. F, The TMNx group (n = 6) exhibited 445 ± 73 ADA-positive TMN cells, with individual lesions ranging from 216to 612 ADA-positive cells. G, H, Composite photomicrographs depicting ADA-immunostaining (G) and Nissl (H) in adjacent brain sections of a rat that was unilaterally injected with Hcrt2-SAP. Dotted lines in each panel indicate location of the vTMN in the injected hemisphere. 3v, Third ventricle; cpd, cerebral peduncle; f, fornix; LHA, lateral hypothalamic area; MM, medial mammillary nuclei; mt, mammillary tract; PH, posterior hypothalamic nucleus. Scale bars: D, E, 200 μm; G, H, 500 μm. *p < 0.05 (LCx vs Sham).

Of 13 Hcrt2-SAP injected rats, six exhibited substantial bilateral reductions in ADA-positive cell number (ranging from 16% to 45% of the Sham group mean). These six rats were used as the TMNx group. This TMNx group exhibited 445 ± 73 ADA-positive TMN cells, with individual lesions ranging from 216 to 612 ADA-positive cells, significantly fewer than in the Sham group (t(11) = 8.731; p < 0.001n; Fig. 6F, individual counts from each TMNx rat superimposed on the group mean). The remaining rats exhibited little to no ADA cell loss (>75% of Sham group mean), and were excluded from further analysis on the basis of being essentially unlesioned.

TMNx attenuates REM sleep promotion by ALM

As in the LCx study described above, there was a significant main effect of drug treatment on NREM sleep latency (F(6,66) = 11.243; p < 0.001)o such that ZOL but not ALM significantly shortened the latency to NREM sleep (data not shown). There was also a significant main effect of drug treatment on REM sleep latency (F(6,66) = 5.390; p < 0.001)p; whereas post hoc tests showed no significant changes compared with VEH, ALM tended to decrease REM sleep latency, whereas ZOL tended to increase it. Neither NREM nor REM sleep latency was affected by TMN lesion.

Consistent with the LCx study, both ALM and ZOL decreased wake time (main effect of drug: F(6,66) = 29.346, p < 0.001; Fig. 7A,B)q and increased NREM sleep time (main effect of drug: F(6,66) = 27.612, p < 0.001; Fig. 7C,D)r, but with no main or interaction effect of TMN lesion. By contrast, ALM (100 and 300 mg/kg) increased REM sleep time compared with VEH in both Sham and TMNx rats (drug × lesion interaction: F(6,66) = 2.436, p = 0.035; Fig. 7E,F)s. Pairwise comparisons of TMNx and Sham rats in each drug treatment condition revealed that TMNx rats had less total REM sleep time following ALM (30 and 300 mg/kg) compared to Shams, whereas there were no differences in REM sleep time between Sham and TMNx rats following ZOL (Fig. 7E,F). REM–NREM was significantly increased by ALM (100 and 300 mg/kg) and decreased by ZOL (100 mg/kg; main effect of drug: F(6,66) = 28.419, p < 0.001; Fig. 7G,H)t. However, these drug effects were qualified by a borderline interaction effect (F(6,66) = 2.131; p = 0.061)t, such that TMNx decreased REM–NREM compared to Shams following ALM (300 mg/kg; pairwise comparison, p< 0.05). Thus, TMNx affected ALM-induced REM sleep increases in a similar manner to that seen following LCx.

Figure 7.
Figure 7.

Total wake (A, B), NREM (C, D), and REM (E, F) sleep time, and the ratio of REM to NREM sleep (G, H) for 6 h following ALM (A, C, E, G) and ZOL (B, D, F, H) in TMNx and Sham lesioned rats. Doses are mg/kg. *p < 0.05 vs vehicle; +p < 0.05, paired comparison F test (TMNx vs Sham).

TMNx attenuates increases in REM bout number following ALM

ALM (all doses) increased the total number of wake and NREM bouts compared to VEH (main effect of drug: wake, F(6,66) = 48.670, p < 0.001u; NREM, F(6,66) = 46.346, p < 0.001v) without an effect of TMN lesion (Fig. 8A,C). ZOL did not affect the total number of wake or NREM bouts (Fig. 8B,D). Further analysis of bout duration histograms showed that TMNx preferentially increased the number of short (<0.5 min) NREM bouts (bout × lesion interaction: F(5,55) = 3.401; p = 0.010)w with no additional influence of drug treatment (data not shown).

Figure 8.
Figure 8.

Total number of wake (A, B), NREM (C, D) and REM (E, F) bouts for 6 h following ALM (A, C, E) and ZOL (B, D, F) in TMNx and Sham lesioned rats. Doses are mg/kg. *p < 0.05 versus vehicle; #p < 0.05 (TMNx vs Sham).

Whereas ALM (100 and 300 mg/kg) increased REM bout number in TMNx and Sham rats, TMNx significantly attenuated this increase at the highest dose of ALM (interaction: F(6,66) = 2.860, p = 0.015; Fig. 8E)x. ZOL did not affect the total number of REM bouts in either lesion group (Fig. 8F). There were no additional effects of lesion or drug treatment on total REM, wake bout numbers, or on the distribution of bout numbers as a function of their duration (data not shown). Thus, TMNx attenuated the promotion of REM sleep by ALM primarily by decreasing the number of REM episodes, while increasing the number of short NREM sleep bouts independently of drug treatment.

Discussion

The Hcrt system promotes wakefulness in part through excitation of wake-active monoaminergic populations, including the noradrenergic LC and histaminergic TMN. In this study, neurotoxic lesions of the LC NE neurons or TMN HA neurons selectively attenuated ALM-mediated increases in REM sleep but not NREM sleep. Furthermore, neither lesion altered efficacy of the GABAA receptor agonist ZOL. These findings support the hypothesis that ALM promotes sleep via selective disfacilitation of subcortical arousal systems. In addition, these results highlight the important role of Hcrt input to monoaminergic populations in regulating REM sleep.

Lesion efficacy

DBH-SAP infusion caused a near-complete loss of LC DBH-immunoreactive cells, with no signs of collateral or nonspecific damage. DBH-SAP is highly selective for LC NE neurons when delivered intracerebroventricularly or intraparenchymally (Wrenn et al., 1996; Blanco-Centurion et al., 2004). Medullary and pontine NE populations receive Hcrt projections (Baldo et al., 2003) and are suggested to play a role in the inhibition of REM sleep (Fenik et al., 2002; Rukhadze et al., 2008; Léger et al., 2009). Although we cannot rule out the possibility of collateral damage to these non-LC NE groups, the nearby A5 noradrenergic neurons appeared intact in our LCx rats following 3µg DBH-SAP, consistent with previous work showing that higher doses are required to lesion these populations (Wrenn et al., 1996). Furthermore, LC lesions increased NREM sleep but not REM sleep following VEH. We therefore conclude that the observed effects on ALM and ZOL efficacy are attributable to NE cell loss in the LC, and not to damage of neighboring noradrenergic or other cell groups.

In contrast to our LC lesions, our TMN-HA lesions were less extensive (55–84% ADA-positive cell loss in TMNx rats). However, more complete TMN lesions still leave basal sleep–wake parameters largely intact (Gerashchenko et al., 2004; Blanco-Centurion et al., 2007). Furthermore, significant deficits in motivated behavior were reported at a threshold of 45% ADA-positive cell loss (Valdés et al., 2010), whereas lesions comparable to ours (∼70% ADA-positive cell loss) impaired hippocampal LTP during walking (Luo and Leung, 2010) and enhanced isoflurane anesthesia induction (Luo and Leung, 2011), suggesting that our lesions, although not complete ablations, were sufficient to assess TMN function. On the other hand, we cannot rule out the possibility that loss of non-HA neurons expressing Hcrt receptors may contribute to our observed results. Inspection of Nissl-stained sections revealed that Hcrt2-SAP lesions destroyed (presumably non-HA) cells surrounding the TMN, as described in previous studies (Gerashchenko et al., 2004; Blanco-Centurion et al., 2007). Both wake-active and wake/REM-active neurons have been observed in this region (Steininger et al., 1999; Takahashi et al., 2006). The posterior hypothalamus also contains GABAergic neurons active in REM recovery sleep (Steininger et al., 1999; Sapin et al., 2010); destruction of such neurons could contribute to attenuated REM sleep in ALM-treated TMNx rats. However, such REM-active neurons appear to be relatively sparse in the immediate vicinity of the TMN (Sapin et al., 2010). These populations present great potential for future inquiry.

LC and TMN influences on basal sleep and waking

LCx, but not TMNx, increased NREM sleep time overall, independently of drug treatment. Although some studies found no effect of LC NE ablation on basal NREM sleep time (Hunsley and Palmiter, 2003; Gompf et al., 2010), others observed increased NREM sleep in the dark phase or around lights-off (González et al., 1998; Blanco-Centurion et al., 2004; Ouyang et al., 2004), similar to our results obtained between ZT12 and ZT18, and consistent with the idea that the loss of LC NE signaling impairs the ability to maintain wakefulness. However, LC lesions did not impair the capacity of either drug to increase NREM sleep, as both ALM and ZOL increased NREM sleep time compared to VEH in both lesioned and unlesioned rats.

Neither LC nor TMN ablation affected basal REM sleep in the dark phase, as previously reported (González et al., 1998; Blanco-Centurion et al., 2004; Gerashchenko et al., 2004; Gompf et al., 2010), suggesting that multiple REM-suppressing or wake-promoting systems exist which compensate when one is lost (Blanco-Centurion et al., 2007). It is unlikely that such compensatory responses masked the response to ALM, as the response to another sleep-promoting agent (ZOL) was similar in LCx, TMNx, and Sham rats in both studies (eg, increased NREM sleep coupled with decreased REM sleep). Furthermore, neither lesion impacted NREM sleep increases following ALM, and LC lesions actually increased NREM sleep time (see previous paragraph). These targeted LC and TMN lesions therefore attenuated specific aspects of ALM-mediated sleep while leaving responses to ZOL intact, supporting the hypothesis that Hcrt antagonism acts at subcortical sleep–wake regulatory targets to promote sleep.

The LC is a critical site of action for sleep induction by ALM

LC lesions blocked reductions in NREM sleep latency following ALM, whereas ZOL reduced NREM latency to a comparable extent in both lesioned and Sham rats, demonstrating that sleep latency changes following hypnotics are readily observed in LCx rats. LC lesions are thus unlikely to have obscured the sleep latency response to ALM. Although the persistence of rapid sleep onset following ZOL may be related to pharmacokinetics as well as site of action, the LC is clearly important for induction of NREM sleep by ALM. Increases in spontaneous LC firing rate anticipate transitions to wakefulness (Aston-Jones and Bloom, 1981; Takahashi et al., 2010); LC stimulation activates the cortex and hippocampus (Berridge and Foote, 1991) and promotes transitions from sleep to wakefulness (Carter et al., 2010). Ablation of NE neurons causes deficits in arousal following stress or novelty (Hunsley and Palmiter, 2004; Ouyang et al., 2004; Gompf et al., 2010). LC NE signaling is thus a key component of arousal, particularly in association with attention; these observations suggest that the LC is a critical site of action for ALM-mediated sleep induction.

LCx increased NREM sleep time and NREM bout duration while decreasing the number of short wake and NREM bouts, suggesting increased NREM consolidation. ALM-induced sleep is typically fragmented compared with ZOL-induced sleep in normal, healthy rodents (Morairty et al., 2012; Black et al., 2013). Although ALM increased the number of wake and NREM bouts in LCx as well as Sham rats, increased NREM bout numbers in LCx rats were still evident at the highest dose of ALM. By contrast, ZOL increased NREM bout duration while equalizing NREM bout numbers between LCx and Sham rats (Figs. 4B,D, 5A,B,D,E). LC ablation thus attenuated ALM- but not ZOL-induced changes in sleep architecture. Stimulating Hcrt neurons (Adamantidis et al., 2007; Choudhary et al., 2014) or infusing Hcrt-1 centrally (Piper et al., 2000; Morairty et al., 2011) or directly into the LC (Bourgin et al., 2000) promotes waking, whereas local application of HcrtR1 antagonists or optogenetic LC inhibition blocks transitions to wakefulness (Carter et al., 2012), suggesting that Hcrt-mediated wakefulness is highly dependent on the LC NE system. Our results support this concept by demonstrating that LC ablation impacts both the sleep-induction profile of ALM and its effects on sleep bout architecture.

By contrast, TMNx increased the number of short NREM bouts but had no effects on initiation or duration of NREM sleep in any drug condition. Like the LC NE neurons, HA neurons exhibit a wake-active, REM-off firing pattern (Takahashi et al., 2006), elevated HA levels are correlated with wakefulness (Chu et al., 2004; Ramesh et al., 2004), and mice lacking HA exhibit deficits in wakefulness at lights-off and in a novel environment (Parmentier et al., 2002)—all of which suggest that HA is important for arousal. Hcrt directly and indirectly excites TMN HA neurons (Eriksson et al., 2001; 2004; Schöne et al., 2014) and Hcrt infusion into the TMN promotes waking and induces cortical HA release (Huang et al., 2001). Although it was surprising that TMN lesions did not affect NREM latency or NREM sleep time, Hcrt2-SAP TMN lesions exhibited few effects on basal sleep in a previous study (Gerashchenko et al., 2004).

Lesioning either LC or TMN attenuates ALM-induced REM sleep

Blockade of Hcrt signaling with ALM increased REM sleep in Sham rats, as previously reported in intact animals (Brisbare-Roch et al., 2007); lesioning either the LC NE or TMN HA neurons selectively attenuated the promotion of REM sleep by ALM. LC lesions significantly decreased both REM–NREM and REM bout number at 100 and 300 mg/kg, whereas these measures were only affected at the 300 mg/kg dose in TMNx rats (Figs. 7E,G, 8E), suggesting a less robust influence of TMN HA neurons on REM sleep compared to the LC NE neurons. Alternatively, surviving HA neurons in lesioned rats may have been sufficient to maintain normal histaminergic regulation of REM sleep. However, the common influence of either lesion on ALM-induced REM sleep suggests a specialized role for Hcrt signaling to both of these nuclei in regulating REM sleep.

The LC NE neurons exhibit a wake-active, “REM-off” firing profile (Takahashi et al., 2010). The LC inhibits cholinergic brainstem “REM-on” neurons (Hobson et al., 1975; McCarley and Hobson, 1975). The LC NE neurons are a major target of the Hcrt neurons (Peyron et al., 1998) and express HcrtR1 almost exclusively (Marcus et al., 2001). Local Hcrt-1/orexin-A infusion activates the LC and suppresses REM sleep (Bourgin et al., 2000), whereas HcrtR1 knockout (Mieda et al., 2011), systemic HcrtR1 antagonism (Smith et al., 2003; Morairty et al., 2012), or intra-LC HcrtR1 blockade blocks Hcrt-mediated REM suppression (Bourgin et al., 2000; Choudhary et al., 2014), and siRNA downregulation of HcrtR1 in the LC increases spontaneous REM sleep in the dark phase (Chen et al., 2010). HcrtR1-mediated Hcrt signaling thus powerfully regulates the REM-suppressing role of the LC NE neurons, although this effect may be more clearly observed following LC-specific manipulations rather than systemic treatments.

TMN HA neurons, which express HcrtR2 (Marcus et al., 2001), also exhibit a wake-active, REM-off firing profile (Takahashi et al., 2006). Mice unable to synthesize HA exhibit increased REM sleep time (Parmentier et al., 2002; Anaclet et al., 2009). HA inhibits hypothalamic melanin-concentrating hormone neurons (Parks et al., 2014) that have been implicated in REM sleep (Verret et al., 2003; Clément et al., 2012; Jego et al., 2013), and administration of either ALM or a HcrtR2 antagonist decreases extracellular HA levels in the LH (Dugovic et al., 2009), consistent with a role for Hcrt-HA signaling in REM sleep regulation. Thus, the acute blockade of excitatory Hcrt input by ALM would “disfacilitate” REM-off activity in the LC and the TMN, resulting in the downstream disinhibition of REM-active populations such as brainstem cholinergic neurons targeted by the LC or the hypothalamic MCH neurons targeted by HA, respectively.

Lesioning either the LC, which expresses only HcrtR1, or the TMN, which expresses only HcrtR2 (Marcus et al., 2001), produced similar effects on ALM efficacy. Recent studies differ regarding whether blockade of Hcrt signaling at one or both Hcrt receptors is critical for promoting sleep (Smith et al., 2003; Dugovic et al., 2009; Mang et al., 2012; Morairty et al., 2012; Steiner et al., 2013). However, deletion of either Hcrt receptor modulates REM sleep response following Hcrt1 infusion (Mieda et al., 2011), while coadministration of HcrtR1 and R2 antagonists elicited synergistic effects distinct from those of either drug administered alone (Dugovic et al., 2009). The sleep-promoting effects of Hcrt receptor antagonism may thus depend on the resulting balance between HcrtR1 and HcrtR2 activity (Hoyer et al., 2013). In this light, localized manipulations such as lesions could also significantly alter drug efficacy by eliminating critical points in the Hcrt signaling pathway. Such manipulations would depend as much on the anatomical site of action as the receptor type(s) expressed there. For example, REM sleep is increased by siRNA knockdown of either HcrtR1 in the LC (Chen et al., 2010) or HcrtR2 in the lateral pontomesencephalic tegmentum (Chen et al., 2013), whereas lesioning the basal forebrain, which expresses both Hcrt receptors (Marcus et al., 2001), attenuates ALM-induced NREM, but not REM sleep (Vazquez-DeRose et al., 2014). In the present study, eliminating either HcrtR1 signaling in the LC or HcrtR2 signaling in the TMN yielded similar effects on ALM-induced REM sleep, independently of the Hcrt receptor type expressed in each region. Indeed, the effects of ablating the LC, which is thought to respond to Hcrt exclusively via HcrtR1, suggest strongly that HcrtR1-mediated LC NE activity represents a critical pathway for ALM-mediated sleep induction and REM promotion. Together, these findings suggest that the site of action, not just the specific receptor or receptors targeted, is a key determinant of how Hcrt receptor antagonism facilitates sleep.

Conclusions

DORAs, including ALM, promote sleep by blocking Hcrt signaling. In this study, lesions of the wake-promoting LC NE or TMN HA populations compromised the REM sleep-promoting efficacy of ALM, whereas previous work has shown that ALM, but not ZOL, requires an intact BF for maximum NREM-promoting efficacy (Vazquez-DeRose et al., 2014). Together, these studies indicate that Hcrt neurotransmission influences distinct aspects of sleep at different locations in the sleep–wake regulatory network. Furthermore, our results, particularly our finding that LC lesions attenuate ALM efficacy, suggest that site of action is at least as important a consideration for Hcrt antagonist efficacy as the receptor targeted. By selectively disfacilitating these subcortical wake-promoting populations, ALM effectively promotes sleep by eliciting neurochemical events consistent with the transition to normal sleep (Vazquez-DeRose et al., 2014). These properties are likely to underlie the findings that ALM also promotes sleep without negatively impacting cognitive performance (Morairty et al., 2014) and without globally blocking the capability for arousal (Parks et al., 2016).

Footnotes

  • 1 The authors report no conflict of interest.

  • 3 This work was supported by the U.S. Army Medical Research Acquisition Activity award number W81XWH-09-2-0081 and NIH R01 NS077408 to T.S.K. We thank Drs Priyattam J. Shiromani and Carlos Blanco-Centurion for providing Hcrt2-SAP; Tsui-Ming Chen, Alan Wilk, and Dr Simon Fisher for technical assistance; Dr Ling Jong for synthesis of almorexant; and Drs Sarah Black and Gregory Parks for helpful comments on data analysis and the paper.

This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

References

  1. ↵
    Adamantidis AR, Zhang F, Aravanis AM, Deisseroth K, de Lecea L (2007) Neural substrates of awakening probed with optogenetic control of hypocretin neurons. Nature 450:420–424. doi:10.1038/nature06310 pmid:17943086
    OpenUrlCrossRefPubMed
  2. ↵
    Anaclet C, Parmentier R, Ouk K, Guidon G, Buda C, Sastre JP, Akaoka H, Sergeeva OA, Yanagisawa M, Ohtsu H, Franco P, Haas HL, Lin JS (2009) Orexin/hypocretin and histamine: distinct roles in the control of wakefulness demonstrated using knock-out mouse models. J Neurosci 29:14423–14438. doi:10.1523/JNEUROSCI.2604-09.2009 pmid:19923277
    OpenUrlAbstract/FREE Full Text
  3. ↵
    Aston-Jones G, Bloom FE (1981) Activity of norepinephrine-containing locus coeruleus neurons in behaving rats anticipates fluctuations in the sleep-waking cycle. J Neurosci 1:876–886. pmid:7346592
    OpenUrlAbstract
  4. ↵
    Baldo BA, Daniel RA, Berridge CW, Kelley AE (2003) Overlapping distributions of orexin/hypocretin- and dopamine-beta-hydroxylase immunoreactive fibers in rat brain regions mediating arousal, motivation, and stress. J Comp Neurol 464:220–237. doi:10.1002/cne.10783 pmid:12898614
    OpenUrlCrossRefPubMed
  5. ↵
    Berridge CW, Foote SL (1991) Effects of locus coeruleus activation on electroencephalographic activity in neocortex and hippocampus. J Neurosci 11:3135–3145. pmid:1682425
    OpenUrlAbstract
  6. ↵
    Black SW, Morairty SR, Fisher SP, Chen T-M, Warrier DR, Kilduff TS (2013) Almorexant promotes sleep and exacerbates cataplexy in a murine model of narcolepsy. Sleep 36:325–336. doi:10.5665/sleep.2442 pmid:23449602
    OpenUrlCrossRefPubMed
  7. ↵
    Blanco-Centurion C, Gerashchenko D, Salin-Pascual RJ, Shiromani PJ (2004) Effects of hypocretin2-saporin and antidopamine-beta-hydroxylase-saporin neurotoxic lesions of the dorsolateral pons on sleep and muscle tone. Eur J Neurosci 19:2741–2752. doi:10.1111/j.0953-816X.2004.03366.x
    OpenUrlCrossRefPubMed
  8. ↵
    Blanco-Centurion C, Gerashchenko D, Shiromani PJ (2007) Effects of saporin-induced lesions of three arousal populations on daily levels of sleep and wake. J Neurosci 27:14041–14048. doi:10.1523/JNEUROSCI.3217-07.2007 pmid:18094243
    OpenUrlAbstract/FREE Full Text
  9. ↵
    Bourgin P, Huitron-Huitrón-Résendiz S, Spier AD, Fabre V, Morte B, Criado JR, Sutcliffe JG, Henriksen SJ, de Lecea L (2000) Hypocretin-1 modulates rapid eye movement sleep through activation of locus coeruleus neurons. J Neurosci 20:7760–7765. pmid:11027239
    OpenUrlAbstract/FREE Full Text
  10. ↵
    Brightwell JJ, Taylor BK (2009) Noradrenergic neurons in the locus coeruleus contribute to neuropathic pain. Neuroscience 160:174–185. doi:10.1016/j.neuroscience.2009.02.023 pmid:19223010
    OpenUrlCrossRefPubMed
  11. ↵
    Brisbare-Roch C, Dingemanse J, Koberstein R, Hoever P, Aissaoui H, Flores S, Mueller C, Nayler O, van Gerven J, de Haas SL, Hess P, Qiu C, Buchmann S, Scherz M, Weller T, Fischli W, Clozel M, Jenck F (2007) Promotion of sleep by targeting the orexin system in rats, dogs and humans. Nat Med 13:150–155. doi:10.1038/nm1544 pmid:17259994
    OpenUrlCrossRefPubMed
  12. ↵
    Carter ME, Brill J, Bonnavion P, Huguenard JR, Huerta R, de Lecea L (2012) Mechanism for Hypocretin-mediated sleep-to-wake transitions. Proc Natl Acad Sci U S A 109:E2635–E2644. doi:10.1073/pnas.1202526109 pmid:22955882
    OpenUrlAbstract/FREE Full Text
  13. ↵
    Carter ME, Yizhar O, Chikahisa S, Nguyen H, Adamantidis A, Nishino S, Deisseroth K, de Lecea L (2010) Tuning arousal with optogenetic modulation of locus coeruleus neurons. Nat Neurosci 13:1526–1533. doi:10.1038/nn.2682 pmid:21037585
    OpenUrlCrossRefPubMed
  14. ↵
    Chemelli RM, Willie JT, Sinton CM, Elmquist JK, Scammell T, Lee C, Richardson JA, Williams SC, Xiong Y, Kisanuki Y, Fitch TE, Nakazato M, Hammer RE, Saper CB, Yanagisawa M (1999) Narcolepsy in orexin knockout mice: molecular genetics of sleep regulation. Cell 98:437–451. pmid:10481909
    OpenUrlCrossRefPubMed
  15. ↵
    Chen L, McKenna JT, Bolortuya Y, Brown RE, McCarley RW (2013) Knockdown of orexin type 2 receptor in the lateral pontomesencephalic tegmentum of rats increases REM sleep. Eur J Neurosci 37:957–963. doi:10.1111/ejn.12101 pmid:23282008
    OpenUrlCrossRefPubMed
  16. ↵
    Chen L, McKenna JT, Bolortuya Y, Winston S, Thakkar MM, Basheer R, Brown RE, McCarley RW (2010) Knockdown of orexin type 1 receptor in rat locus coeruleus increases REM sleep during the dark period. Eur J Neurosci 32:1528–1536. pmid:21089218
    OpenUrlCrossRefPubMed
  17. ↵
    Choudhary RC, Khanday MA, Mitra A, Mallick BN (2014) Perifornical orexinergic neurons modulate REM sleep by influencing locus coeruleus neurons in rats. Neuroscience 279:33–43. doi:10.1016/j.neuroscience.2014.08.017 pmid:25168734
    OpenUrlCrossRefPubMed
  18. ↵
    Chu M, Huang ZL, Qu WM, Eguchi N, Yao MH, Urade Y (2004) Extracellular histamine level in the frontal cortex is positively correlated with the amount of wakefulness in rats. Neurosci Res 49:417–420. doi:10.1016/j.neures.2004.05.001 pmid:15236867
    OpenUrlCrossRefPubMed
  19. ↵
    Clément O, Sapin E, Libourel PA, Arthaud S, Brischoux F, Fort P, Luppi PH (2012) The lateral hypothalamic area controls paradoxical (REM) sleep by means of descending projections to brainstem GABAergic neurons. J Neurosci 32:16763–16774. doi:10.1523/JNEUROSCI.1885-12.2012 pmid:23175830
    OpenUrlAbstract/FREE Full Text
  20. ↵
    Dang A, Garg A, Rataboli PV (2011) Role of zolpidem in the management of insomnia. CNS Neurosci Ther 17:387–397. doi:10.1111/j.1755-5949.2010.00158.x pmid:20553305
    OpenUrlCrossRefPubMed
  21. ↵
    Dugovic C, Shelton JE, Aluisio LE, Fraser IC, Jiang X, Sutton SW, Bonaventure P, Yun S, Li X, Lord B, Dvorak CA, Carruthers NI, Lovenberg TW (2009) Blockade of orexin-1 receptors attenuates orexin-2 receptor antagonism-induced sleep promotion in the rat. J Pharmacol Exp Therapeut 330:142–151. doi:10.1124/jpet.109.152009 pmid:19363060
    OpenUrlAbstract/FREE Full Text
  22. ↵
    Eriksson KS, Sergeeva O, Brown RE, Haas HL (2001) Orexin/hypocretin excites the histaminergic neurons of the tuberomammillary nucleus. J Neurosci 21:9273–9279. pmid:11717361
    OpenUrlAbstract/FREE Full Text
  23. ↵
    Eriksson KS, Sergeeva OA, Selbach O, Haas HL (2004) Orexin (hypocretin)/dynorphin neurons control GABAergic inputs to tuberomammillary neurons. Eur J Neurosci 19:1278–1284. doi:10.1111/j.1460-9568.2004.03243.x pmid:15016085
    OpenUrlCrossRefPubMed
  24. ↵
    Estabrooke I, McCarthy M, Ko E, Chou T, Chemelli R, Yanagisawa M, Saper C, Scammell TE (2001) Fos expression in orexin neurons varies with behavioral state. J Neurosci 21:1656–62.
    OpenUrlAbstract/FREE Full Text
  25. ↵
    Fenik V, Marchenko V, Janssen P, Davies RO, Kubin L (2002) A5 cells are silenced when REM sleep-like signs are elicited by pontine carbachol. J Appl Physiol 93:1448–1456. doi:10.1152/japplphysiol.00225.2002 pmid:12235046
    OpenUrlAbstract/FREE Full Text
  26. ↵
    Gerashchenko D, Chou TC, Blanco-Centurion CA, Saper CB, Shiromani PJ (2004) Effects of lesions of the histaminergic tuberomammillary nucleus on spontaneous sleep in rats. Sleep 27:1275–1281. pmid:15586780
    OpenUrlPubMed
  27. ↵
    Gerashchenko D, Kohls MD, Greco M, Waleh NS, Salin-Pascual R, Kilduff TS, Lappi DA, Shiromani PJ (2001) Hypocretin-2-saporin lesions of the lateral hypothalamus produce narcoleptic-like sleep behavior in the rat. J Neurosci 21:7273–7283. pmid:11549737
    OpenUrlAbstract/FREE Full Text
  28. ↵
    Gompf HS, Mathai C, Fuller PM, Wood DA, Pedersen NP, Saper CB, Lu J (2010) Locus ceruleus and anterior cingulate cortex sustain wakefulness in a novel environment. J Neurosci 30:14543–14551. doi:10.1523/JNEUROSCI.3037-10.2010 pmid:20980612
    OpenUrlAbstract/FREE Full Text
  29. ↵
    González MM, Debilly G, Valatx JL (1998) Noradrenaline neurotoxin DSP-4 effects on sleep and brain temperature in the rat. Neurosci Lett 248:93–96. pmid:9654350
    OpenUrlCrossRefPubMed
  30. Hagan J, Leslie RA, Patel S, Evans ML, Wattam TA, Holmes S, Benham CD, Taylor SG, Routledge C, Hemmati P, Munton RP, Ashmeade TE, Shah AS, Hatcher JP, Hatcher PD, Jones DN, Smith MI, Piper DC, Hunter AJ, Porter RA. (1999) Orexin A activates locus coeruleus cell firing and increases arousal in the rat. Proc Natl Acad Sci U S A 96:10911–6. pmid:10485925
    OpenUrlAbstract/FREE Full Text
  31. ↵
    Hobson JA, McCarley RW, Wyzinski PW (1975) Sleep cycle oscillation: reciprocal discharge by two brainstem neuronal groups. Science 189:55–58. pmid:1094539
    OpenUrlAbstract/FREE Full Text
  32. ↵
    Horvath TL, Peyron C, Diano S, Ivanov A, Aston-Jones G, Kilduff TS, van den Pol AN (1999) Hypocretin (orexin) activation and synaptic innervation of the locus coeruleus noradrenergic system. J Comp Neurol 415:145–159. pmid:10545156
    OpenUrlCrossRefPubMed
  33. ↵
    Hoyer D, Dürst T, Fendt M, Jacobson LH, Betschart C, Hintermann S, Behnke D, Cotesta S, Laue G, Ofner S, Legangneux E, Gee CE (2013) Distinct effects of IPSU and suvorexant on mouse sleep architecture. Front Neurosci 7:235. doi:10.3389/fnins.2013.00235 pmid:24368893
    OpenUrlCrossRefPubMed
  34. ↵
    Huang ZL, Qu WM, Li WD, Mochizuki T, Eguchi N, Watanabe T, Urade Y, Hayaishi O (2001) Arousal effect of orexin A depends on activation of the histaminergic system. Proc Natl Acad Sci U S A 98:9965–9970. doi:10.1073/pnas.181330998 pmid:11493714
    OpenUrlAbstract/FREE Full Text
  35. ↵
    Hunsley MS, Palmiter RD (2003) Norepinephrine-deficient mice exhibit normal sleep-wake states but have shorter sleep latency after mild stress and low doses of amphetamine. Sleep 26:521–526. pmid:12938804
    OpenUrlPubMed
  36. ↵
    Hunsley MS, Palmiter RD (2004) Altered sleep latency and arousal regulation in mice lacking norepinephrine. Pharmacol Biochem Behav 78:765–773. doi:10.1016/j.pbb.2004.05.008 pmid:15301933
    OpenUrlCrossRefPubMed
  37. ↵
    Jego S, Glasgow SD, Herrera CG, Ekstrand M, Reed SJ, Boyce R, Friedman J, Burdakov D, Adamantidis AR (2013) Optogenetic identification of a rapid eye movement sleep modulatory circuit in the hypothalamus. Nat Neurosci 16:1637–1643. doi:10.1038/nn.3522 pmid:24056699
    OpenUrlCrossRefPubMed
  38. ↵
    Ko EM, Estabrooke IV, McCarthy M, Scammell TE (2003) Wake-related activity of tuberomammillary neurons in rats. Brain Res 992:220–226. pmid:14625060
    OpenUrlCrossRefPubMed
  39. ↵
    Koberstein R, Aissaoui H, Bur D, Clozel M, Fischli W, Jenck F, Mueller C, Nayler O, Sifferlen T, Treiber A, Weller T (2003) Tetrahydroisoquinolines as orexin receptor antagonists: strategies for lead optimization by solution-phase chemistry. CHIMIA 57:270–275. doi:10.2533/000942903777679361
    OpenUrlCrossRef
  40. ↵
    Koberstein R, Fischli W, Clozel M, Aissaoui H, Weller T (2005) Substituted 1,2,3,4-tetrahydroisoquinoline derivatives. World patent: WO 2005118548.
  41. ↵
    Lee MG, Hassani OK, Jones BE (2005) Discharge of identified orexin/hypocretin neurons across the sleep-waking cycle. J Neurosci 25:6716–6720. doi:10.1523/JNEUROSCI.1887-05.2005 pmid:16014733
    OpenUrlAbstract/FREE Full Text
  42. ↵
    Léger L, Goutagny R, Sapin E, Salvert D, Fort P, Luppi P-H (2009) Noradrenergic neurons expressing Fos during waking and paradoxical sleep deprivation in the rat. J Chem Neuroanat 37:149–157. doi:10.1016/j.jchemneu.2008.12.008 pmid:19152834
    OpenUrlCrossRefPubMed
  43. ↵
    Lin L, Faraco J, Li R, Kadotani H, Rogers W, Lin X, Qiu X, de Jong PJ, Nishino S, Mignot E (1999) The sleep disorder canine narcolepsy is caused by a mutation in the hypocretin (orexin) receptor 2 gene. Cell 98:365–376. pmid:10458611
    OpenUrlCrossRefPubMed
  44. ↵
    Luo T, Leung LS (2010) Endogenous histamine facilitates long-term potentiation in the hippocampus during walking. J Neurosci 30:7845–7852. doi:10.1523/JNEUROSCI.1127-10.2010 pmid:20534833
    OpenUrlAbstract/FREE Full Text
  45. ↵
    Luo T, Leung LS (2011) Involvement of tuberomamillary histaminergic neurons in isoflurane anesthesia. Anesthesiology 115:36–43. doi:10.1097/ALN.0b013e3182207655 pmid:21562401
    OpenUrlCrossRefPubMed
  46. ↵
    Mang GM, Dürst T, Bürki H, Imobersteg S, Abramowski D, Schuepbach E, Hoyer D, Fendt M, Gee CE (2012) The dual orexin receptor antagonist almorexant induces sleep and decreases orexin-induced locomotion by blocking orexin 2 receptors. Sleep 35:1625–1635. doi:10.5665/sleep.2232 pmid:23204605
    OpenUrlCrossRefPubMed
  47. ↵
    Marcus JN, Aschkenasi CJ, Lee CE, Chemelli RM, Saper CB, Yanagisawa M, Elmquist JK (2001) Differential expression of orexin receptors 1 and 2 in the rat brain. J Comp Neurol 435:6–25. pmid:11370008
    OpenUrlCrossRefPubMed
  48. ↵
    McCarley RW, Hobson JA (1975) Neuronal excitability modulation over the sleep cycle: a structural and mathematical model. Science 189:58–60. pmid:1135627
    OpenUrlAbstract/FREE Full Text
  49. ↵
    Mieda M, Hasegawa E, Kisanuki YY, Sinton CM, Yanagisawa M, Sakurai T (2011) Differential roles of orexin receptor-1 and -2 in the regulation of non-REM and REM sleep. J Neurosci 31:6518–6526. doi:10.1523/JNEUROSCI.6506-10.2011 pmid:21525292
    OpenUrlAbstract/FREE Full Text
  50. ↵
    Morairty SR, Hedley L, Flores J, Martin R, Kilduff TS (2008) Selective 5HT(2A) and 5HT(6) receptor antagonists promote sleep in rats. Sleep 31:34–44. pmid:18220076
    OpenUrlPubMed
  51. ↵
    Morairty SR, Revel FG, Malherbe P, Moreau J-L, Valladao D, Wettstein JG, Kilduff TS, Borroni E (2012) Dual hypocretin receptor antagonism is more effective for sleep promotion than antagonism of either receptor alone. PLoS One 7:e39131. doi:10.1371/journal.pone.0039131 pmid:22768296
    OpenUrlCrossRefPubMed
  52. ↵
    Morairty SR, Wilk AJ, Lincoln WU, Neylan TC, Kilduff TS (2014) The hypocretin/orexin antagonist almorexant promotes sleep without impairment of performance in rats. Front Neurosci 8:3. doi:10.3389/fnins.2014.00003 pmid:24550767
    OpenUrlCrossRefPubMed
  53. ↵
    Morairty SR, Wisor J, Silveira K, Sinko W, Kilduff TS (2011) The wake-promoting effects of hypocretin-1 are attenuated in old rats. Neurobiol Aging 32:1514–1527. doi:10.1016/j.neurobiolaging.2009.07.017 pmid:19781813
    OpenUrlCrossRefPubMed
  54. ↵
    Ohno K, Sakurai T (2008) Orexin neuronal circuitry: role in the regulation of sleep and wakefulness. Front Neuroendocrinol 29:70–87. doi:10.1016/j.yfrne.2007.08.001 pmid:17910982
    OpenUrlCrossRefPubMed
  55. ↵
    Ouyang M, Hellman K, Abel T, Thomas SA (2004) Adrenergic signaling plays a critical role in the maintenance of waking and in the regulation of REM sleep. J Neurophysiol 92:2071–2082. doi:10.1152/jn.00226.2004 pmid:15190089
    OpenUrlAbstract/FREE Full Text
  56. ↵
    Parks GS, Olivas ND, Ikrar T, Sanathara NM, Wang L, Wang Z, Civelli O, Xu X (2014) Histamine inhibits the melanin-concentrating hormone system: implications for sleep and arousal. J Physiol (Lond) 592:2183–2196. doi:10.1113/jphysiol.2013.268771 pmid:24639485
    OpenUrlCrossRefPubMed
  57. ↵
    Parks GS, Warrier DR, Dittrich L, Schwartz MD, Palmerston JB, Neylan TC, Morairty SR, Kilduff TS (2016) The dual hypocretin receptor antagonist almorexant is permissive for activation of wake-promoting systems. Neuropsychopharmacology 41:1144-1155.
    OpenUrlPubMed
  58. ↵
    Parmentier R, Ohtsu H, Djebbara-Hannas Z, Valatx JL, Watanabe T, Lin JS (2002) Anatomical, physiological, and pharmacological characteristics of histidine decarboxylase knock-out mice: evidence for the role of brain histamine in behavioral and sleep-wake control. J Neurosci 22:7695–7711. pmid:12196593
    OpenUrlAbstract/FREE Full Text
  59. ↵
    Peyron C, Tighe DK, van den Pol AN, de Lecea L, Heller HC, Sutcliffe JG, Kilduff TS (1998) Neurons containing hypocretin (orexin) project to multiple neuronal systems. J Neurosci 18:9996–10015. pmid:9822755
    OpenUrlAbstract/FREE Full Text
  60. ↵
    Piper DC, Upton N, Smith MI, Hunter AJ (2000) The novel brain neuropeptide, orexin-A, modulates the sleep-wake cycle of rats. Eur J Neurosci 12:726–730. pmid:10712652
    OpenUrlCrossRefPubMed
  61. ↵
    Ramesh V, Thakkar MM, Strecker RE, Basheer R, McCarley RW (2004) Wakefulness-inducing effects of histamine in the basal forebrain of freely moving rats. Behav Brain Res 152:271–278. doi:10.1016/j.bbr.2003.10.031 pmid:15196795
    OpenUrlCrossRefPubMed
  62. ↵
    Rukhadze I, Fenik VB, Branconi JL, Kubin L (2008) Fos expression in pontomedullary catecholaminergic cells following rapid eye movement sleep-like episodes elicited by pontine carbachol in urethane-anesthetized rats. Neuroscience 152:208–222. doi:10.1016/j.neuroscience.2007.11.013 pmid:18155849
    OpenUrlCrossRefPubMed
  63. ↵
    Sapin E, Bérod A, Léger L, Herman PA, Luppi P-H, Peyron C (2010) A very large number of GABAergic neurons are activated in the tuberal hypothalamus during paradoxical (REM) sleep hypersomnia. PLoS One 5:e11766. doi:10.1371/journal.pone.0011766
    OpenUrlCrossRefPubMed
  64. ↵
    Schöne C, Apergis-Schoute J, Sakurai T, Adamantidis A, Burdakov D (2014) Coreleased orexin and glutamate evoke nonredundant spike outputs and computations in histamine neurons. Cell Rep 7:697–704. doi:10.1016/j.celrep.2014.03.055 pmid:24767990
    OpenUrlCrossRefPubMed
  65. ↵
    Smith MI, Piper DC, Duxon MS, Upton N (2003) Evidence implicating a role for orexin-1 receptor modulation of paradoxical sleep in the rat. Neurosci Lett 341:256–258. pmid:12697296
    OpenUrlCrossRefPubMed
  66. ↵
    Soffin EM, Evans ML, Gill CH, Harries MH, Benham CD, Davies CH (2002) SB-334867-A antagonises orexin mediated excitation in the locus coeruleus. Neuropharmacology 42:127–133. pmid:11750922
    OpenUrlCrossRefPubMed
  67. ↵
    Steiner MA, Gatfield J, Brisbare-Roch C, Dietrich H, Treiber A, Jenck F, Boss C (2013) Discovery and characterization of ACT-335827, an orally available, brain penetrant orexin receptor type 1 selective antagonist. Chem Med Chem 8:898–903. doi:10.1002/cmdc.201300003 pmid:23589487
    OpenUrlCrossRefPubMed
  68. ↵
    Steininger TL, Alam MN, Gong H, Szymusiak R, McGinty D (1999) Sleep-waking discharge of neurons in the posterior lateral hypothalamus of the albino rat. Brain Res 840:138–147. pmid:10517961
    OpenUrlCrossRefPubMed
  69. ↵
    Swanson LW (2004) Brain maps: structure of the rat brain, 3rd edition.
  70. ↵
    Takahashi K, Kayama Y, Lin JS, Sakai K (2010) Locus coeruleus neuronal activity during the sleep-waking cycle in mice. Neuroscience 169:1115–1126. doi:10.1016/j.neuroscience.2010.06.009 pmid:20542093
    OpenUrlCrossRefPubMed
  71. ↵
    Takahashi K, Lin JS, Sakai K (2006) Neuronal activity of histaminergic tuberomammillary neurons during wake-sleep states in the mouse. J Neurosci 26:10292–10298. doi:10.1523/JNEUROSCI.2341-06.2006 pmid:17021184
    OpenUrlAbstract/FREE Full Text
  72. ↵
    Thannickal TC, Moore RY, Nienhuis R, Ramanathan L, Gulyani S, Aldrich M, Cornford M, Siegel JM (2000) Reduced number of hypocretin neurons in human narcolepsy. Neuron 27:469–474. pmid:11055430
    OpenUrlCrossRefPubMed
  73. ↵
    Valdés JL, Sánchez C, Riveros ME, Blandina P, Contreras M, Farías P, Torrealba F (2010) The histaminergic tuberomammillary nucleus is critical for motivated arousal. Eur J Neurosci 31:2073–2085. doi:10.1111/j.1460-9568.2010.07241.x pmid:20529118
    OpenUrlCrossRefPubMed
  74. ↵
    Vazquez-DeRose J, Schwartz MD, Nguyen AT, Warrier DR, Gulati S, Mathew TK, Neylan TC, Kilduff TS (2014) Hypocretin/orexin antagonism enhances sleep-related adenosine and GABA neurotransmission in rat basal forebrain. Brain Struct Funct 221:923–940.
    OpenUrlPubMed
  75. ↵
    Verret L, Goutagny R, Fort P, Cagnon L, Salvert D, Léger L, Boissard R, Salin P, Peyron C, Luppi PH (2003) A role of melanin-concentrating hormone producing neurons in the central regulation of paradoxical sleep. BMC Neurosci 4:19. doi:10.1186/1471-2202-4-19
    OpenUrlCrossRefPubMed
  76. Wiley RG, Kline RH IV. (2000) Neuronal lesioning with axonally transported toxins. J Neurosci Methods 103:73–82. pmid:11074097
    OpenUrlCrossRefPubMed
  77. ↵
    Wrenn CC, Picklo MJ, Lappi DA, Robertson D, Wiley RG (1996) Central noradrenergic lesioning using anti-DBH-saporin: anatomical findings. Brain Res 740:175–184. pmid:8973812
    OpenUrlCrossRefPubMed

Synthesis

Reviewing Editor: Rae Silver, Columbia University

The decision was a result of the Reviewing Editor Rae Silver and the peer reviewers coming together and discussing their recommendations until a consensus was reached. A fact-based synthesis statement explaining their decision and outlining what is needed to prepare a revision is listed below. The following reviewer(s) agreed to reveal their identity: Thomas Scammell

With regard to the minor comments, the following amplifications/clarifications are requested.

1) They authors acknowledge that saporin likely kills more than just the TMN neurons, and it would be helpful to examine Nissl-stained sections to estimate the size of the affected region so one can assess the likelihood of whether some of the behavioral change is caused by injury to non-histamine neurons.

2) On average, about 32% of the TMN neurons remained in the "TMN lesioned" group. Is this enough to really assess the TMN? How does this compare to prior lesion papers?

3) The photomicrographs convey the key message but could still be improved (lighting is uneven, parts are dark).

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Locus Coeruleus and Tuberomammillary Nuclei Ablations Attenuate Hypocretin/Orexin Antagonist-Mediated REM Sleep
Michael D. Schwartz, Alexander T. Nguyen, Deepti R. Warrier, Jeremiah B. Palmerston, Alexia M. Thomas, Stephen R. Morairty, Thomas C. Neylan, Thomas S. Kilduff
eNeuro 28 February 2016, 3 (1) ENEURO.0018-16.2016; DOI: 10.1523/ENEURO.0018-16.2016
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