Dual hypocretin receptor antagonism enhances sleep and nursing behavior in lactating rats

Dual hypocretin receptor antagonism enhances sleep and nursing behavior in lactating rats | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Dual hypocretin receptor antagonism enhances sleep and nursing behavior in lactating rats Mayda Rivas , Florencia Peña , Clara Prota , Carlos Carrera-Cañas , Miguel Garzón , Pablo Torterolo , Luciana Benedetto doi: https://doi.org/10.1101/2025.09.05.674514 Mayda Rivas 1 Unidad Académica de Fisiología, Facultad de Medicina, Universidad de la República , Montevideo, Uruguay Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: mrivas{at}fmed.edu.uy Florencia Peña 1 Unidad Académica de Fisiología, Facultad de Medicina, Universidad de la República , Montevideo, Uruguay Find this author on Google Scholar Find this author on PubMed Search for this author on this site Clara Prota 1 Unidad Académica de Fisiología, Facultad de Medicina, Universidad de la República , Montevideo, Uruguay Find this author on Google Scholar Find this author on PubMed Search for this author on this site Carlos Carrera-Cañas 2 Departamento de Anatomía , Histología y Neurociencia, Facultad de Medicina, Universidad Autónoma de Madrid , Spain Find this author on Google Scholar Find this author on PubMed Search for this author on this site Miguel Garzón 2 Departamento de Anatomía , Histología y Neurociencia, Facultad de Medicina, Universidad Autónoma de Madrid , Spain Find this author on Google Scholar Find this author on PubMed Search for this author on this site Pablo Torterolo 1 Unidad Académica de Fisiología, Facultad de Medicina, Universidad de la República , Montevideo, Uruguay Find this author on Google Scholar Find this author on PubMed Search for this author on this site Luciana Benedetto 1 Unidad Académica de Fisiología, Facultad de Medicina, Universidad de la República , Montevideo, Uruguay Find this author on Google Scholar Find this author on PubMed Search for this author on this site Abstract Full Text Info/History Metrics Preview PDF Abstract Hypocretins (also known as orexins) are neuropeptides that regulate the sleep-wake cycle and modulate various behaviors, including maternal behavior. They act through two receptor subtypes: hypocretin receptor 1 (HcrtR1) and hypocretin receptor 2 (HcrtR2). Although Dual Orexin Receptor Antagonists (DORAs) are clinically used as hypnotics, most preclinical studies with these drugs have been conducted in males, with limited research in females, leaving the postpartum period largely unexplored. Here, we examined the impact of the DORA Suvorexant on sleep and maternal behavior in lactating rats. Lactating and virgin female rats were implanted with electrodes for polysomnographic recording. Using a counterbalanced design, Suvorexant was orally administered at doses of 0, 10 (SUV10), and 30 mg/kg (SUV30) to virgin rats in diestrus and to lactating rats between postpartum days 4 and 8. Sleep recordings and maternal behaviors were assessed during the light phase for six hours following the administration of the drug. Suvorexant reduced wakefulness and increased slow wave sleep, intermediate state, and REM sleep in both groups, with a stronger effect in virgin females. In lactating rats, Suvorexant increased nursing time and milk ejections, while reducing active maternal behavior such as pup-licking. These findings demonstrate that dual hypocretin receptor antagonism produces hypnotic effects and selectively modulates maternal behavior, promoting nursing while reducing active maternal behavior. Introduction Hypocretins (HCRT), also known as orexins, are two peptides derived from a common pre-pro-HCRT precursor, HCRT-1 (orexin A) and HCRT-2 (orexin B), that exert their biological effects acting via two receptors, HCRT-R1 and HCRT-R2 1 , 2 . Initially associated with feeding regulation, HCRT are now recognized as key modulators of several functions, such as the promotion of wakefulness and motivated behaviors 3 , 4 . In addition, it is now well established that a deficiency in HCRT signaling is the underlying cause of narcolepsy 5 – 7 . The postpartum periods induce anatomical and functional changes in the HCRT system. 8 reported increased expression of pre-pro-HCRT in rats on postpartum day 1 (PPD1) compared to gestation and PPD14. Similarly, lactating rats show more HCRT-immunoreactive neurons than non-lactating animals 9 – 11 . HCRT neuronal activity during lactation, assessed via c-Fos expression, has been shown to increase during postpartum in mice and rats, followed by a decline approaching weaning 11 , 12 . Furthermore, increased levels of hypothalamic HCRT-R1 mRNA were found on PPD1 compared to PPD14 and gestation 8 . These findings suggest that the HCRT system varies along the reproductive stages of the female, likely contributing to the physiological adjustments that occur throughout the different stages of the female’s reproductive cycle. Notably, its activity is heightened in the early postpartum period, which decreases as lactation progresses, supporting its functional relevance during this period 13 . Accordingly, previous studies suggest that the HCRT system regulates maternal care. Intermediate intracerebroventricular doses of HCRT-1 enhance grooming of pups, while high doses diminish nursing 14 . Our prior research demonstrated that microinjection of HCRT-1 into the medial preoptic area (mPOA) promotes active behaviors. In contrast, administration of either a selective HCRT-R1 antagonist or a dual orexin receptor antagonist (DORA) in this area enhances passive behaviors, such as nursing, and increases litter weight gain 15 , 16 . In line with the wake-promoting role of HCRT and its increased activity during the postpartum period, mothers of various species display heightened wakefulness and fragmented sleep during this time 17 – 21 .e Indeed, elevated HCRT levels have been associated with poor sleep during pregnancy 22 , suggesting that this neuropeptide may contribute to the sleep alterations observed after parturition. Consistent with this idea, we previously found that HCRT-1 microinjected into the mPOA increased wakefulness and active maternal behaviors in lactating rats. In contrast, administration of a DORA into the same area improved sleep and nursing, highlighting the role of HCRT in coordinating both processes 16 . The wake-promoting function of HCRT has driven the development of DORAs as alternatives to traditional hypnotics 23 , 24 . Suvorexant (SUV), the first DORA approved by the U.S. Food and Drug Administration (FDA), fosters a balance between non-REM and REM sleep while preserving overall sleep architecture in individuals with insomnia 25 – 27 . Postpartum sleep disturbances, which are often linked to psychological issues, may benefit from pharmacological interventions 28 ; however, SUV is classified as a category C drug by the FDA for use during pregnancy and lactation due to a lack of comprehensive studies. This underscores the need for further research to clarify the role of HCRT during this critical period of life. Although we have examined HCRT and DORA actions in the mPOA concerning sleep and maternal behavior, their systemic effects in nursing mothers remain poorly understood. Notably, previous work has shown that postpartum rats exhibit distinct sleep patterns compared to virgins, characterized by increased wakefulness and reduced sleep 18 , 19 , 21 , a pattern considered an adaptive mechanism to support offspring care. Hence, in the present study, we first compared the basal waking and sleep patterns between lactating and virgin females. We then evaluated the systemic effects of SUV on sleep, maternal behavior, and lactation in postpartum rats, and compared the sleep-related outcomes with those observed in virgin females. Materials and methods Animals and housing Experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (8th ed., National Academy Press, 2008) and the procedures were approved by the Institutional Animal Care Committee (N° 07151-000022-23). The conditions mirrored those of our previous studies, e.g., 16 , 20 ; virgin and primiparous Sprague-Dawley female rats (250-300 g) and their pups were used. Pregnant females were housed individually from 2 to 3 days before parturition and remained in this condition until the experiments ended. On PPD1, litters were culled to four female and four male pups per dam. Similarly, virgin females were housed individually throughout the entire experiment. Animals were maintained in a temperature-controlled room (22 ± 1 °C) with a 12-hour light/dark cycle (lights turn on at 6 am) and had unrestricted access to food and water. Stereotaxic Surgery Animals were anesthetized with ketamine, xylazine, and acepromazine (80/2.8/2.0 mg/kg, i.p.), and implanted with nichrome electrodes to record electroencephalogram (EEG) activity from the frontal, parietal, and occipital cortices. A reference electrode was placed in the cerebellum, and a bipolar electrode was positioned in the neck muscles for electromyogram (EMG) recording. The electrodes and connectors were secured to the skull using acrylic cement. After surgery, the animals received sterile saline (0.9%; 10 ml/kg, s.c.), a single dose of ketoprofen (5 mg/kg, s.c.), and a topical antibiotic was applied to the surgical wound. Surgery for lactating females occurred on PPD1, after which they were returned to their home cages with their pups and housed in a soundproof chamber. Pup growth and development were monitored to confirm that maternal care and lactation were not adversely affected. Similar to lactating rats, virgin females underwent surgery three days before the experiments. Experimental Design Two experimental groups comprising eight animals were used: lactating females (PPD4-8) and virgin females ( Figure 1A ). Each animal received SUV at doses of 0, 10 (SUV10), or 30 (SUV30) mg/kg, administered in 5 ml/kg of 5% methylcellulose via oral gavage. These doses were based on prior studies demonstrating a standard hypnotic effect in male rats 25 , 29 , 30 . All administrations followed a counterbalanced design with at least one day of rest between experiments ( Figure 1A ). Download figure Open in new tab Figure 1. Schematic representation of experimental procedure involving oral administration of Suvorexant (SUV) via gavage in a virgin female (top) and a lactating rat with pups (bottom), both chronically implanted for EEG recordings (a). Representative EEG traces from frontal (FCx), parietal (PCx), and occipital (OCx) cortices, together with EMG recordings, illustrate the standard criteria for scoring wakefulness and sleep states in rats (b). Representative hypnograms depict baseline sleep architecture in a virgin (left) and a lactating (right) rat following administration of vehicle (top) and SUV30 (bottom) (c). W, wakefulness; LS, light sleep; SWS, slow wave sleep; IS, intermediate state; REM, rapid eye movement sleep. To assess nursing behavior, pups were separated from their mothers for three hours before the initiation of the drug administration 31 ( Figure 1A ). Five minutes before the end of separation, SUV or vehicle was administered, and pups were weighed. The pups were then returned to the maternal cage, and rats were connected to the polysomnographic system. Polysomnographic and behavioral recordings continued for six hours following the reunion with the pups, from 12:00 to 18:00 (ZT+3 to ZT+12). At the end of the experiments, pups were weighed for a second time to calculate litter weight gain (LWG) ( Figure 1A ). In virgin females, SUV was administered, and polysomnographic recordings were conducted similarly to those in lactating females during the same hours of the light phase. The estrous cycle phase correlates with HCRT levels and sleep patterns 8 , 32 – 34 ; thus, in virgin rats, vaginal smear samples were collected daily at 9:00 a.m. Samples were dried on glass slides, and estrous stages were identified using standard histological criteria. Experiments were conducted only during the diestrous phase. At the end of the experiments, the animals were euthanized using an overdose of ketamine and xylazine anesthesia. Recording and Analysis of Sleep and Wake Patterns For polysomnographic recordings, rats were placed in a sound-attenuated, ventilated chamber equipped with independent control of the light/dark cycle and a video camera. They were connected to a slip-ring rotor, allowing continuous recording under freely moving conditions. The bioelectrical signals obtained were amplified (x1000), filtered (low-pass: 100 Hz; high-pass: 10 Hz for EMG and 0.5 Hz for EEG), digitized (512 Hz, 16 bits), and stored for later analysis using Spike2 software. Sleep-wake states were staged in five-second epochs based on standard electrophysiological parameters, as shown in Figure 1B . Wakefulness (W) was characterized by low-amplitude, high-frequency EEG activity and high EMG tone; light sleep (LS) showed intermittent slow waves mixed with desynchronized EEG activity and a moderate reduction in EMG tone; slow-wave sleep (SWS) was identified by the presence of continuous high-amplitude low-frequency EEG waves accompanied by sleep spindles, and low EMG tone; REM sleep was defined by low-amplitude, high-frequency EEG activity and regular theta rhythm in posterior cortical areas, accompanied by muscle atonia. Additionally, the transition state between NREM sleep (LS + SWS) and REM sleep, known as the intermediate state (IS) 35 – 37 , was identified by the presence of sleep spindles in the frontal cortex and simultaneous theta activity in posterior cortical areas, along with minimal EMG activity. For this state, three variants were distinguished: entrance-IS, which precedes REM; abortive-IS, which does not precede REM; and exit-IS, which follows the end of REM. The total time spent in each behavioral state was analyzed across the entire recording period and at hourly intervals. Sleep latencies were measured, defined as the time from recording onset to the first episode of a given state lasting 20 seconds or longer. The number and duration of episodes for each state were also assessed. Recording and Analysis of Maternal Behavior Maternal activity was recorded using a webcam integrated with the polysomnographic acquisition software. The interaction of mothers with their pups was monitored and continuously recorded for six hours. The behaviors were classified into three major categories: hovering over the pups (the dam over the pups while actively engaged in any activity), nursing (being mostly immobile over the pups, characterized by low and high kyphosis and supine postures), and being away from the pups. The duration spent in each behavior was categorized into 5-second epochs and subsequently analyzed in 2-hour windows. Furthermore, the number of milk ejections was quantified based on the stretching behavior of the pups 38 – 40 , and the percentage of LWG was measured as an indirect indicator of milk ejection 38 , 41 – 43 . Additionally, we analyzed specific active maternal behaviors, including mouthing (rearranging pups within the nest), licking (both anogenital and body licking), and nest building. The analysis was focused on the first two hours following SUV administration, as maternal behavior changes were most pronounced during this period. Due to its low frequency, mouthing was excluded from the analysis. Statistics All values are presented as mean ± S.E.M. (standard error). Data normality was assessed using the Kolmogorov-Smirnov test. Sleep and wake states comparisons were analyzed using a two-way repeated measures ANOVA, with SUV dose as the within-subject factor and physiological condition (virgin or lactating) as the between-subject factor, followed by Tukey’s multiple comparison post hoc test when appropriate. Moreover, to assess whether the hypnotic effects of SUV differed between virgin and lactating rats, we calculated the percentage change from vehicle administration for each sleep state following administration of SUV10 and SUV30. These percentage change values were compared between groups using independent-samples t-tests. In addition, effect sizes were estimated using Cohen’s d to quantify the magnitude of the difference between groups. Maternal behavior states (time in minutes) were analyzed using a one-way repeated measures ANOVA. For non-parametric data, such as time spent in nest-building, the Friedman test was used, followed by Dunn’s post hoc test. The criterion used to reject the null hypothesis was p < 0.05. Results Sleep and wake patterns in lactating and virgin rats in baseline conditions As expected, under baseline conditions, lactating rats spent significantly more time awake and less time in most sleep states (SWS, IS, and REM sleep) compared to virgin females ( Table 1 ). The increased W time in lactating rats was associated with a longer duration of its episodes and occurred in parallel with a reduction in SWS episode duration. Regarding the number of episodes, lactating females exhibited fewer IS episodes as well as fewer REM episodes compared to virgins. Moreover, no differences were observed between lactating and virgin rats in the latency to initiate NREM or REM sleep under baseline conditions ( Table 1 ). View this table: View inline View popup Download powerpoint Table 1. Baseline sleep-wake parameters under vehicle condition in virgin and lactating rats. Effect of Suvorexant on sleep and wakefulness In both virgin and lactating females, SUV administration decreased total W time and increased time spent in SWS, IS, and REM sleep, whereas no changes were observed in LS ( Figure 2A , D, G, J, M). As shown in Figure 2B and E , the number of W and LS episodes remained constant following SUV administration in both virgin and lactating females compared to the vehicle. SUV30 increased the number of SWS episodes exclusively in lactating females ( Figure 2H ), while it enhanced the frequency of IS episodes in both groups ( Figure 2K ). Significant increases in REM episode frequency were just detected in virgin rats ( Figure 2N ). Regarding episode duration, only a reduction in W episode length was observed following SUV30 administration in virgin females, whereas no such change occurred in lactating ones ( Figure 2C ). On the other hand, the latencies to NREM and REM sleep were not significantly affected by SUV administration in either virgin or lactating rats ( Figure 2P , Q). Download figure Open in new tab Figure 2. Time spent in wake and sleep states in virgin and lactating rats (A, D, G, J, M, P); number of episodes (B, E, H, K, N); episode duration (C, F, I, L, O); and latency to NREM (P) and REM sleep (Q) following administration of vehicle, SUV10, and SUV30. Data are presented as mean ± SEM and were analyzed using two-way ANOVA followed by Tukey’s post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001 indicate significant differences between groups. Interestingly, the SUV effect was significantly more substantial in virgin females compared to lactating females. A significant interaction between treatment and reproductive condition indicated that SUV produced a greater reduction in total W time in virgin rats compared to lactating rats (SUV × condition F(2, 28) = 3.41, p = 0.047; Figure 2A ). Similar interaction effects were observed for the increase in the number of IS episodes (SUV × condition F(2, 28) = 5.05, p = 0.013) and REM episodes (SUV × condition F(2, 28) = 4.95, p = 0.014; Figure 2K and N ), suggesting that the impact of SUV varied according to reproductive state, with a stronger effect on virgins. Moreover, we calculated the percentage change from vehicle for total time in each sleep state, and Cohen’s d for the change induced by the administration of SUV10 and SUV30 within each reproductive condition. A significant effect was observed in W time following SUV10, where virgin females exhibited a significantly greater reduction compared to lactating females (virgin: –28.0% ± 6.0, lactating: –6.4% ± 2.7, t(14) = 3.27, p = 0.006; Cohen’s d = 1.63), indicating a larger effect size. Although SUV30 did not reach statistical significance (t(14) = 1.87, p = 0.083), the effect size was large (Cohen’s d = 0.93), suggesting that SUV30 may also reduce W more in virgin than in lactating rats. Time-course analysis at each 2-hour interval ( Figure 3 ) suggests that the effects of SUV on sleep varied over time. In lactating rats, most changes occurred during the first 2 hours after administration, particularly in the W, SWS, and REM sleep stages ( Figures 3B , 3E, and 3I, respectively). In contrast, virgin rats exhibited delayed effects, with significant changes occurring between hours 5 and 6, particularly in W and REM sleep, when both doses had an effect ( Figure 3A , 3I). Interestingly, SUV increased IS across the entire recording period in virgin females, whereas in lactating rats, this effect was limited to the 3–4-hour time block. Download figure Open in new tab Figure 3. Time spent in wake and sleep states across consecutive two-hour intervals during the 6-h recording period (2: 1–2 h; 4: 3–4 h; 6: 5–6 h) in virgin (A, C, E, G, I) and lactating (B, D, F, H, J) female rats following administration of vehicle, SUV10, and SUV30. Data are presented as mean ± SEM and were analyzed using one-way ANOVA followed by Tukey’s post hoc test . * p < 0.05, ** p < 0.01, *** p < 0.001 indicate significant differences between SUV doses. The variations in the different IS subtypes following SUV administration were examined over the total recording time ( Figure 4 ). The administration of both doses of SUV increased the number of Entrance-IS episodes in virgin females, but not in lactating females ( Figure 4A ). Abortive-IS episodes and exit-IS episodes remained unchanged after SUV administration in both virgin and lactating females ( Figure 4B-C ). Download figure Open in new tab Figure 4. Number of episodes of three IS subtypes: Entrance-IS (A), Exit-IS (B), and abortive-IS (C) in virgin and lactating rats after administration of vehicle, SUV10, and SUV30. Data are presented as mean ± SEM and were analyzed using two-way ANOVA followed by Tukey’s post hoc test, * p < 0.05, ** p < 0.01, *** p < 0.001 indicate significant differences between groups. Maternal behavior Compared to vehicle administration, both doses of SUV resulted in prolonged nursing time for lactating females ( Figure 5A and 5C ). This increase was evident in both the total recording time and during the first two hours ( Figure 5C and 5F ). In addition, SUV30 also increased the number of milk ejections, although it did not affect LWG ( Figure 5H and 5I ). Additionally, the time spent hovering over the pups decreased following SUV30 administration, while the time spent away from them remained unchanged ( Figure 5B and 5D ). Download figure Open in new tab Figure 5. Maternal parameters following administration of vehicle, SUV10, or SUV30. Representative maternograms (A) based on behavioral staging in 5-s epochs classified into nursing (N), hover over (HO), and away from pups (AP). The Time spent in maternal behaviors during the 6-h recording (B–D) and across consecutive 2-h intervals (2: 1–2 h; 4: 3–4 h; 6: 5–6 h) (E–G). Number of milk ejections (H), percentage of litter weight gain (I), and time spent in active maternal behaviors, including licking of pups (J) and nest building (K). Data are presented as mean ± SEM and were analyzed by one-way ANOVA followed by Tukey’s post hoc test, except nest-building, which was analyzed using the Friedman test. * p < 0.05, ** p < 0.01, *** p < 0.001 indicate significant differences between SUV doses. Concerning active maternal behavior during the first two hours of recording, SUV30 reduced the time spent licking the pups and tended to reduce nest-building time, although the latter did not reach statistical significance (χ²(2) = 5.44, p = 0.070; Figure 5J and 5K ). Discussion This study provides novel insights into the regulation of sleep and maternal behavior during the postpartum period by assessing, for the first time, the effects of the DORA SUV in lactating rats and comparing them with those in virgin rats. Our results indicate that SUV reduced wakefulness and increased sleep in both lactating and virgin rats, exhibiting a more potent wake-inhibitory effect in virgin compared to lactating dams. Furthermore, SUV enhanced nursing behavior and milk ejections without affecting litter weight gain, while diminishing certain active maternal behaviors. The present findings suggest that SUV could potentially serve as an effective hypnotic in mothers without markedly impairing essential maternal behaviors. However, its wake-promoting inhibition appears to be attenuated during lactation. Although further research is still needed to confirm and expand these results, this attenuation may indicate that higher doses could be required to achieve similar hypnotic effects in lactating mothers, a factor that warrants careful evaluation to balance maternal sleep improvement with the preservation of maternal behavior. Sleep and wake patterns in lactating and virgin rats In coherence with previous findings, our data revealed that under control conditions, mother rats spent significantly more time awake and less time in all sleep states, exhibiting longer W episodes, shorter SWS episodes, and fewer IS and REM episodes compared to virgin rats. It is important to note that lactating rats are typically recorded together with their pups (as would be expected in the wild), whereas virgin females are recorded in isolation. In this regard, 18 demonstrated that, from postpartum day 2 (PPD2) to PPD20, mother rats exhibited increased W and reduced REM sleep compared to virgin females, based on seven-hour recordings conducted during the light phase, conditions similar to those used in our study. Furthermore, 19 reported that postpartum rats showed increased active W during the light phase, with a significant decrease in NREM sleep. However, REM sleep and the frequency of episodes in any state were unaffected. More recently, 21 found that postpartum rats at PPD2 display increased W and a reduction in SWS and REM sleep, along with fewer REM episodes during the light phase compared to virgin rats. These earlier studies did not identify IS as a separate sleep state. Here, we demonstrate that IS shows a similar pattern to REM sleep in lactating rats, with both total time and number of episodes reduced in this condition. Despite methodological differences across studies on sleep during lactation, a consistent finding is that W duration increases during the light phase in the early postpartum days, likely serving as an adaptive response to facilitate offspring care. In this sense, previous research in different species has highlighted that sleep patterns during the lactation period are mainly driven by the constant demands of their offspring 44 . Effect of Suvorexant on sleep and wake patterns Our results showed that SUV reduced W while increasing SWS, IS, and REM sleep in lactating and virgin rats, with LS remaining unchanged in both conditions. This hypnotic effect is consistent with preclinical and clinical data that support the sleep-promoting effects of DORAs. Similar to our results, SUV30 administration decreased active W and enhanced NREM and REM sleep for 2 to 7 hours post-administration in male rats 45 , 46 . In diverse mammalian species, including rats, dogs, and humans, DORA administered orally during the active period of the circadian cycle decreased alertness and increased NREM and REM sleep 47 . Recent clinical studies suggest that SUV is clinically safe and effective in improving sleep onset and maintenance, with effects ranging from several weeks to months, in patients with insomnia 48 – 51 . Interestingly, pharmacokinetic studies have reported sex-related differences in response to SUV. Female patients showed higher plasma concentrations of SUV than males after the same dose. Women reported somnolence and other adverse effects, and only female participants discontinued driving tests due to excessive drowsiness in safety trials 49 . However, there is no previous literature about SUV in mother rats. In this regard, we found that SUV effects were more pronounced in virgin rats than in mother rats. The IS state is the natural transition into REM sleep and is particularly impaired by the absence of HCRT, as observed in narcolepsy, where animals and patients can initiate REM episodes directly from wakefulness, bypassing NREM and IS stages 52 , 53 . However, the potential role of HCRT activity in modulating the IS state has not been extensively studied. We thoroughly analyzed IS episodes, categorizing them into three subtypes. The influence of SUV on the number of entrance-IS episodes varied between lactating and virgin females. SUV increased entrance-IS episodes only in virgin females, but did not affect them in lactating females. This increase in the number of entrance-IS in virgin females parallels findings reported in males 54 . Moreover, these results align with recent findings indicating a pivotal role of HCRT neurons in modulating sleep transitions, since optogenetic inhibition of HCRT neurons in mice has been shown to facilitate the transition from NREM to REM sleep, by increasing the probability and frequency of REM initiation 55 , and we hypothesize that if measured, it would probably increase IS entrance too. Collectively, these findings support the view that IS is a pivotal transitional state in the sleep–wake cycle, sensitive to modulation by HCRT activity. To the best of our knowledge, this study is the first to evaluate the hypnotic effects of a drug on postpartum females compared to virgin counterparts. Our results show that SUV, particularly at low doses, reduced total W time more effectively in virgin rats than in lactating ones. Moreover, similar effects were found for the number of IS and REM episodes, with SUV inducing greater increases in virgins compared to lactating females. The increased number, but not duration, of SWS and IS episodes in lactating rats suggests an attempt to recover or consolidate sleep under the influence of SUV. In addition, although SUV increases the number of IS episodes in lactating females, it does not promote the transition from IS to REM, as the number of REM episodes remains unchanged in this group. Besides, the REM facilitation by SUV, although present, is less significant in lactating than in virgin rats. These changes may be caused by the active demands of the pups, which may prevent them from consolidating either SWS, IS, or REM sleep episodes. Finally, abortive-IS and exit-IS episodes remained unchanged after SUV administration in both virgin and lactating females. This could indicate that the effect of HCRT in regulating entry into REM sleep is more important than controlling the intermediate state itself. Thus, our findings contribute to the understanding of how pharmacological HCRT receptor blockade may differentially impact sleep architecture in virgin versus lactating rats, particularly by enhancing REM sleep propensity through modulation of the IS phase in the former group. Further research is needed to elucidate the specific mechanisms by which HCRT modulates IS and its implications in sleep architecture. The differential response to SUV observed between virgin and lactating females may stem from changes in sleep and W regulation experienced during the postpartum phase, during which various physiological systems adapt to promote W, a critical factor for nurturing offspring. The HCRT system changes during this time could have a significant role 13 . The reduced ability to decrease W through HCRT receptor blockade in lactating females may be linked to physiological adjustments in the HCRT system that prioritize maternal care. For instance, the postpartum period is associated with increased W and heightened arousal, possibly driven by elevated HCRT tone 12 . This major activation could counteract the effects of HCRT antagonists, making lactating females less sensitive to the impact of SUV. Alternatively, changes in HCRT receptor expression or sensitivity may contribute to this attenuated response. In support of this hypothesis, 8 reported a significant upregulation of HCRT-R1 mRNA and an increased number of HCRT-R1–immunoreactive neurons in the hypothalamus of lactating rats at PPD1. Although these findings do not directly address functional receptor sensitivity, they suggest that the brains of lactating rats may exhibit altered responsiveness to HCRT signaling. Additionally, since HCRT has been shown to modulate prolactin secretion via pituitary receptors 56 , it is plausible that hormonal states, such as lactation, could influence HCRT system responsiveness. However, to date, no studies have directly examined whether lactation-associated hormonal changes, such as elevated oxytocin or prolactin, affect HCRT receptor function or alter the pharmacological response to HCRT receptor antagonists. Additionally, the temporal profile of sleep changes induced by SUV differed between virgin and lactating rats. In lactating females, the hypnotic effects were more pronounced during the early post-administration period but dissipated more rapidly than in virgin females, suggesting a shorter efficacy. In this regard, throughout lactation, dynamic alterations in plasma volume, cardiac output, maternal glomerular filtration rate, hematocrit, and serum protein levels can modify both pharmacodynamics and pharmacokinetics 57 . In male rats, SUV exhibits a terminal half-life of approximately 0.6 hours, with high systemic clearance, and an oral bioavailability of only 20%, indicating rapid elimination following administration {Cox, 2010 #1806, although the sleep effects persist for several hours 54 . Therefore, the more transient effects observed in lactating rats could reflect a faster systemic elimination of the drug or reduced pharmacodynamic sensitivity in this physiological state. These considerations highlight the significance of reproductive status in influencing both the magnitude and duration of drug effects on sleep–wake regulation. Maternal behavior Our data indicate that SUV increased nursing behavior and the number of milk ejections, while in parallel, it reduced the time the mother spends in active behaviors over the pups, such as hovering over them. In contrast, the time spent away from them remained unchanged. These results are consistent with our previous studies, demonstrating that the administration of DORAs in the mPOA enhances nursing time 16 . However, in our previous study, nursing increased at the expense of a reduction in the time dams spent away from the pups, without affecting the time spent hovering over them. This difference could imply that the time mothers spend with their pups is less affected by systemic DORA administration than by intra-mPOA, a key area in controlling maternal behavior 58 . The blockade of both HCRT receptors enhances nursing duration and the frequency of milk ejections, regardless of whether the drug is administered systemically or directly into the mPOA. As mothers need a resting state to nurse, this effect may be increased indirectly through increased sleep duration, since mother rats often nurse while asleep 20 . Alternatively, it may suggest the simultaneous facilitation of both nursing and sleep. However, as litter weight gain was not increased by SUV administration, the former hypothesis is more probable. Additionally, the timing of the drug’s effects varies between oral and local delivery methods. Systemic administration’s peak effect on nursing occurs at two hours, whereas intra-mPOA administration reaches its peak at four hours post-administration. This suggests that blocking the HCRT receptor affects maternal behavior more directly at the systemic level than through the mPOA; it is likely that other regions involved in maternal circuits, such as the prefrontal cortex and the ventral tegmental area, are being impacted by systemic SUV, which may account for the direct effect 59 , 60 , 14 . Finally, oral administration of SUV reduces active maternal behavior, such as licking the pups. These findings parallel previous results regarding the administration of an antagonist of HCRT-R1 in the mPOA, suggesting that SUV could decrease active maternal care independently of sleep-wake cycle regulation, affecting maternal motivation to care for their offspring. In this context, future studies investigating the effects of DORAs on maternal motivation are critical from a translational perspective, given that DORAs are approved for the treatment of insomnia in several countries 61 . Technical consideration The method used for drug administration—oral gavage—ensures accurate dosing but is not without limitations. This technique can induce significant stress in experimental animals, potentially affecting physiological and behavioral outcomes, including sleep architecture and maternal behaviors. Moreover, the absorption of orally administered compounds can be influenced by the presence and composition of food in the gastrointestinal tract, introducing inter-individual variability in drug bioavailability and pharmacokinetics. In our study, the drug was prepared as a suspension, a formulation that may result in incomplete dilution and inconsistent homogenization. However, all animals underwent the same procedures, making any significant differences among groups not attributable to this concern. SUV was administered at mid-light phase to allow evaluation of maternal behavior and comparison with previous findings. However, had it been administered closer to the beginning of the resting phase, its hypnotic effects might have been different— presumably more pronounced—given that HCRT levels are typically elevated during the active phase 25 , 62 . Conclusions This study demonstrates that a dual orexin receptor antagonist exerts differential effects on sleep in lactating versus virgin rats, with its hypnotic effects significantly attenuated in lactating females. Notably, in mother rats, HCRT receptor blockade promoted nursing behavior and increased the frequency of milk ejection, without affecting litter weight gain, while reducing active maternal behaviors such as pup licking. Altogether, our results reveal a previously underappreciated interaction between pharmacological sleep promotion and maternal caregiving, highlighting the importance of physiological state in evaluating sleep-inducing treatments. This work contributes to a better understanding of how sleep pharmacotherapies may differentially affect postpartum individuals and highlights potential implications for clinical contexts involving disrupted maternal sleep. 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Share Dual hypocretin receptor antagonism enhances sleep and nursing behavior in lactating rats Mayda Rivas , Florencia Peña , Clara Prota , Carlos Carrera-Cañas , Miguel Garzón , Pablo Torterolo , Luciana Benedetto bioRxiv 2025.09.05.674514; doi: https://doi.org/10.1101/2025.09.05.674514 Share This Article: Copy Citation Tools Dual hypocretin receptor antagonism enhances sleep and nursing behavior in lactating rats Mayda Rivas , Florencia Peña , Clara Prota , Carlos Carrera-Cañas , Miguel Garzón , Pablo Torterolo , Luciana Benedetto bioRxiv 2025.09.05.674514; doi: https://doi.org/10.1101/2025.09.05.674514 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Neuroscience Subject Areas All Articles Animal Behavior and Cognition (7633) Biochemistry (17681) Bioengineering (13890) Bioinformatics (41930) Biophysics (21446) Cancer Biology (18586) Cell Biology (25493) Clinical Trials (138) Developmental Biology (13374) Ecology (19897) Epidemiology (2067) Evolutionary Biology (24308) Genetics (15607) Genomics (22498) Immunology (17736) Microbiology (40385) Molecular Biology (17175) Neuroscience (88584) Paleontology (666) Pathology (2831) Pharmacology and Toxicology (4823) Physiology (7641) Plant Biology (15149) Scientific Communication and Education (2045) Synthetic Biology (4293) Systems Biology (9823) Zoology (2271)