Bidirectional modulation of sleep and wakefulness via prefrontal cortical chemogenetics

Bidirectional modulation of sleep and wakefulness via prefrontal cortical chemogenetics | 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 Bidirectional modulation of sleep and wakefulness via prefrontal cortical chemogenetics View ORCID Profile Lukas B. Krone , View ORCID Profile Jack D. Hamilton , View ORCID Profile Anna Hoerder-Suabedissen , View ORCID Profile Clara Marnitz , View ORCID Profile Anna B. Szabo , View ORCID Profile Colin J. Akerman , View ORCID Profile Zoltán Molnár , View ORCID Profile Vladyslav V. Vyazovskiy doi: https://doi.org/10.1101/2025.09.07.673922 Lukas B. Krone 1 Centre for Neural Circuits and Behaviour, University of Oxford , Oxford, UK 2 University Hospital of Psychiatry and Psychotherapy, University of Bern , Bern, Switzerland 3 Department of Physiology, Anatomy and Genetics, University of Oxford , Oxford, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Lukas B. Krone For correspondence: lukas.krone{at}dpag.ox.ac.uk Jack D. Hamilton 1 Centre for Neural Circuits and Behaviour, University of Oxford , Oxford, UK 3 Department of Physiology, Anatomy and Genetics, University of Oxford , Oxford, UK 4 Sir Jules Thorn Sleep and Circadian Neuroscience Institute, Nuffield Department of Clinical Neuroscience, University of Oxford , Oxford, UK 5 Kavli Institute for Nanoscience Discovery, University of Oxford , Oxford, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jack D. Hamilton Anna Hoerder-Suabedissen 3 Department of Physiology, Anatomy and Genetics, University of Oxford , Oxford, UK 5 Kavli Institute for Nanoscience Discovery, University of Oxford , Oxford, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Anna Hoerder-Suabedissen Clara Marnitz 1 Centre for Neural Circuits and Behaviour, University of Oxford , Oxford, UK 3 Department of Physiology, Anatomy and Genetics, University of Oxford , Oxford, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Clara Marnitz Anna B. Szabo 4 Sir Jules Thorn Sleep and Circadian Neuroscience Institute, Nuffield Department of Clinical Neuroscience, University of Oxford , Oxford, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Anna B. Szabo Colin J. Akerman 6 Department of Pharmacology, University of Oxford , Oxford, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Colin J. Akerman Zoltán Molnár 3 Department of Physiology, Anatomy and Genetics, University of Oxford , Oxford, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Zoltán Molnár Vladyslav V. Vyazovskiy 3 Department of Physiology, Anatomy and Genetics, University of Oxford , Oxford, UK 4 Sir Jules Thorn Sleep and Circadian Neuroscience Institute, Nuffield Department of Clinical Neuroscience, University of Oxford , Oxford, UK 5 Kavli Institute for Nanoscience Discovery, University of Oxford , Oxford, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Vladyslav V. Vyazovskiy Abstract Full Text Info/History Metrics Supplementary material Data/Code Preview PDF Abstract The drive to sleep is inescapable but its biological substrate remains largely elusive. Until recently, the search for sleep centres in the mammalian brain focussed on subcortical neurons that promote sleep or wakefulness, or induce state transitions. However, the regulatory elements of mammalian sleep circuitry, which govern the timing, amount and intensity of sleep, have proven difficult to elucidate. Growing evidence suggests an essential role for the cerebral cortex. Here we used a chemogenetic approach to test whether prefrontal cortex (PFC) layer 5 pyramidal neurons partake in the regulation of sleep. We found that inhibiting or exciting Rbp4-Cre neurons in the PFC with hM4Di/hM3Dq Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) increases and decreases sleep, respectively. Targeted PFC DREADD inhibition of Rbp4-Cre neurons induces sleep that electrophysiologically and architecturally resembles spontaneous sleep. In contrast, widespread cortical DREADD inhibition induces sleep that is punctuated by frequent brief awakenings, has suppressed rapid-eye-movement (REM) sleep, and is followed by a rebound in electroencephalogram (EEG) slow wave activity (SWA), typically observed after disrupted or lost sleep. Our work demonstrates that local modulation of cortical activity can bidirectionally change the amount of global sleep. This represents an important step in the dissection of sleep circuitry, aids the identification of molecular counterparts of sleep pressure, and accelerates the delineation of top-down sleep-regulatory pathways. Together, these lay the foundation for neurobiologically-informed sleep treatments, such as targeted non-invasive brain stimulation and cell-type specific pharmaceuticals, to better modulate sleep in humans. Introduction Sleep is an essential behaviour for which the neurobiological drivers in mammals remain largely enigmatic ( Cirelli and Tononi, 2008 ). Consequently, long-term treatments for poor sleep, particularly insomnia disorder, are limited to psychotherapeutic and other non-pharmacological interventions ( Riemann et al ., 2023 ), as most currently available hypnotics do not restore the physiological characteristics and benefits of sleep, and their chronic use has detrimental consequences ( De Crescenzo et al ., 2022 ). The challenge of understanding and manipulating the drive to sleep in mammals arises from three peculiarities in its regulation. First, the brain is thought to be the central organ regulating sleep and benefitting from it ( Hobson, 2005 ); therefore, cellular and molecular mechanisms of sleep drive are more complex to study than other essential drives such as thirst and appetite, for which key signals are generated by peripheral organs. Second, sleep need primarily occurs on a ‘local’ level of individual brain cells and cortical columns ( Krueger et al ., 2008 ); several brain regions interact in generating the ‘global’ response which leads to the behavioural manifestation of sleep ( Franks and Wisden, 2021 ). This redundancy in sleep circuitry highlights the vital importance of sleep but makes it difficult to pinpoint key centers. Third, the timescale of the build-up and dissipation of sleep pressure (hours) ( Borbély, 1982 ) is considerably longer than that of many other homeostatically-controlled processes such as respiration (seconds), and studying it requires chronic in vivo experiments with subacute manipulations of the brain. Additionally, the homeostatic drive to sleep is modulated by internal circadian rhythmicity and external ‘zeitgebers’ such as light ( Borbély, 2022 ; Franken and Dijk, 2024 ), and animals can (choose to) temporarily forfeit sleep if other opportunities or demands prevail. The momentary drive to sleep has been quantified for almost a century using parameters such as the readiness of an animal to engage in sleep-characteristic behaviours ( Hess, 1929 ), the amplitude of slow waves in the electroencephalogram (EEG) ( Blake and Gerard, 1937 ), and the level of sleep slow wave activity (SWA, 0.5-4 Hz) during NREM sleep in EEG spectral analysis ( Borbély et al ., 1981 ; Borbély, Tobler and Hanagasioglu, 1984 ). However, the identification and manipulation of the centres generating this drive in the mammalian brain is only at its beginning. Intriguingly, only few sleep-regulatory centres overlap with the well-studied subcortical sleep-control circuitry ( Franks and Wisden, 2021 ). The cerebral cortex generates most of the established EEG readouts for the level of sleep drive ( Adamantidis, Gutierrez Herrera and Gent, 2019 ), is strongly influenced by sleep regulatory substances ( Krueger et al ., 2019 ), and harbours neuronal populations that are sleep active and reflect the level of homeostatic sleep drive ( Gerashchenko et al ., 2008 ). However, the possibility that the cerebral cortex may actively contribute to the regulation of sleep was long overlooked despite some evidence of a putative involvement ( Kilduff, Cauli and Gerashchenko, 2011 ; Krone et al ., 2017 ). We demonstrated an active role of the cerebral cortex in sleep regulation by observing a three hour reduction in daily sleep time and a diminished response to extended wakefulness in Rbp4 Cre :Snap25 fl/fl mice that lack activity-dependent neurotransmitter release from a subpopulation of neocortical layer 5 pyramidal neurons and archicortical dentate gyrus granule cells ( Krone et al ., 2021 ). However, this initial work in a conditional knockout mouse model could not exclude the possibility that developmental or neurodegenerative effects may have contributed to the sleep phenotype, nor could it resolve which cortical regions contribute to the regulation of sleep ( Hoerder-Suabedissen et al ., 2019 ). This changed with the identification of somatostatin-expressing, γ-aminobutyric acid (GABA) neurons in the prefrontal cortex (PFC Sst -GABA ) that respond to sleep deprivation and can trigger sleep preparation and initiation via specific projections to the lateral preoptic hypothalamus (LPO) and lateral hypothalamus (LH) ( Tossell et al ., 2023 ). Among cortical areas, the prefrontal cortex (PFC) appears to have a particular relevance for sleep. It has long been postulated that the PFC ‘gets tired first’ based on the frontocentral pattern of EEG synchronisation during extended wakefulness and the onset of sleep ( Marzano et al ., 2013 ), the fronto-posterior gradient in the level of SWA during non-rapid eye movement (NREM) sleep following extended wakefulness ( Werth, Achermann and Borbély, 1996 ; Cajochen, Foy and Dijk, 1999 ; Huber, Deboer and Tobler, 2000 ), and the typical frontocentral origin of NREM sleep slow waves ( Massimini et al ., 2004 ; Vyazovskiy, Faraguna, et al ., 2009 ). The work on PFC Sst -GABA neurons also suggests that the PFC has a particular ability to regulate sleep, as manipulations of equivalent neurons in the occipital cortex did not elicit sleep preparation or initiation ( Tossell et al ., 2023 ). Additional evidence for the special role of the PFC in sleep regulation was recently provided by the finding that chemogenetically increasing synaptic spine size of pyramidal neurons of the PFC, but not the occipital cortex, can increase the amount of sleep ( Sawada et al ., 2024 ), and that the PFC directly regulates sleep amount in rats ( Groenhout et al ., 2025 ). Together, these recent studies suggest that the PFC can respond to plasticity-dependent sleep need with sleep preparation and initiation, raising three important questions: (1) Are the sleep-inducing properties of PFC pyramidal neurons solely plasticity-dependent or also activity-dependent? (2) Is the PFC a hub for sleep-induction or would manipulations of wider cortical areas yield stronger sleep-modulatory effects? (3) Can cortical pyramidal neurons bidirectionally alter the drive to sleep? In this study, we address these three questions using cortical chemogenetic manipulations with Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) during chronic EEG and electromyogram (EMG) recordings in Rbp4 Cre mice. We created an Rbp4 hM4Di mouse line to assess sleep during widespread chemogenetic inhibition of layer 5 pyramidal neurons across the cortical mantle and in the archicortex. Subsequently, we use viral vectors to perform local hM4Di and hM3Dq DREADD-manipulations of PFC layer 5 pyramidal neurons. Results Chemogenetic inhibition of a subset of cortical excitatory neurons increases the amount but alters the characteristics of sleep To address the question of whether acute manipulations of activity in layer 5 pyramidal neurons can modulate sleep, we crossed the pan-layer 5 driver line Rbp4 Cre to the inhibitory DREADD-reporter line R26-LSL-Gi-DREADD (abbreviated R26 LSL-hM4Di ) and performed chemogenetic manipulations combined with chronic EEG/EMG recordings in young adult male Rbp4 hM4Di mice ( Fig. 1a,b ). The choice of this driver line was based on our previous finding that Rbp4 Cre :SNAP25 fl/fl mice sleep approximately three hours less per day compared to Cre-negative controls and have a diminished homeostatic response to extended wakefulness ( Krone et al ., 2021 ). Download figure Open in new tab Figure 1: Chemogenetic inhibition of a subset of cortical excitatory neurons increases sleep time but alters sleep architecture and electrophysiology. (a) Schematic of electrophysiological recordings in Rbp4 hM4Di mice. Scale bars represent 500 µV on the y-axis and 1 s on the x-axis, respectively. (b) Experimental paradigm: counterbalanced intraperitoneal injections of saline and 0.5 mg/kg CNO were administered at light onset (ZT 0). (c) Representative hypnograms and EEG SWA (0.5-4 Hz, 4-s epochs) of a mouse following saline (top panel) and CNO (bottom panel). (d) Time courses (left column) of Wake, NREM, and REM sleep in the first 6 hr post-i.p. injection of saline and CNO, with the corresponding percentages of time spent in each vigilance state during hours 1 (DREADD-on column) and 6 (DREADD-off column) post-injection. Note that REM sleep is presented as a percentage of total sleep time. (e) Representative EEG (F, frontal; O, occipital) and EMG traces after saline and CNO injections. (f) Time course (left) of brief awakenings (BA; <16 s movement during sleep; n/h TST) during the first 6 hr after saline and CNO, with the corresponding number of BA in hours 1+2 (DREADD-on) and 5+6 (DREADD-off) post-i.p. injection. (g) Time course (left) of EEG NREM SWA for the first 6 hr after saline and CNO, with the corresponding % of NREM SWA in hours 1+2 (DREADD-on) and 5+6 (DREADD-off) post-i.p. injection. (h) NREM (left column) and REM (right column) sleep architecture in hours 1+2 (DREADD-on) post-injection of saline and CNO. Note that all time analyses were aligned from 15 min post-injection (as T=0) to ensure full DREADD activation. Asterisks indicate comparisons with significant differences (*p<0.05; **p<0.01; ***p<0.001) for analyses with significant main effects. n = 9 for panels d, g, h; n = 8 for panel f. BA: Brief awakenings; CNO: Clozapine- N -oxide; DREADD: Designer Receptors Exclusively Activated by Designer Drugs; EEG: Electroencephalogram; NREM: Non-rapid eye movement sleep; REM: Rapid eye movement sleep; SWA: Slow wave activity; TST: Total sleep time; ZT: Zeitgeber time. In pilot experiments, we observed that intraperitoneal (i.p.) injections of 0.5, 1, and 5 mg/kg clozapine- N -oxide dihydrochloride (CNO) can induce sleep in Rbp4 hM4Di mice (Suppl. Fig. 1a). Based on effect-size calculations, the strength of this effect appeared dose-dependent (Suppl. Fig. 1b). The 5 mg/kg CNO dose caused a profound response such that within a few minutes after CNO injections, all mice started to engage in sleep preparatory behaviours and then fell asleep, even when exposed to a novel object ( Suppl. Video 1 ). Since even the lowest CNO dose of 0.5 mg/kg reliably increased the amount of sleep ( Fig 1c,d ), we conducted all subsequent experiments with counterbalanced injections of 0.5 mg/kg CNO and saline. Rbp4 hM4Di mice showed an increase in total sleep time from a median of 36.8 min (saline) to 52.8 min (CNO) in the first hour of the DREADD manipulation (0:15 - 1:15h after i.p. injections, (W = 9, p = 0.008)). This increase was due to a larger amount of NREM sleep (from 30.33±8.33 min after saline to 45.82±7.57 min after CNO, t(8) = 4.301, p = 0.003), while the proportion of REM sleep relative to total sleep time (TST) was reduced (t(8) = 2.471, p = 0.039; Fig. 1d ). The amount of sleep remained unchanged in DREADD-free R26 LSL-hM4Di controls injected with the same CNO dose (Suppl Fig. 2a). To further investigate potential changes in the characteristics of sleep, we analysed sleep architecture and EEG spectra. For these analyses, we chose a two-hour time window (0:15 - 2:15h after i.p. injections) to ensure that all mice in all conditions had a sufficient amount of NREM and REM sleep episodes for statistical analysis. Brief awakenings – short bouts (<16 s) of wake-like EEG/EMG activity during NREM or REM episodes, or between sleep-state transitions – were substantially increased in the CNO condition (t(7) = 5.821, p = 0.0006; Fig. 1e,f ). Despite the increase in NREM sleep during the DREADD manipulation (Med = 81.89) compared to saline (Med = 54.89, p = 0.004), the level of SWA during NREM sleep – an established marker of sleep intensity ( Borbély et al ., 1981 ; Borbély, Tobler and Hanagasioglu, 1984 ; Huber, Deboer and Tobler, 2000 ) – remained unchanged between the CNO and saline condition ( Fig. 1g ), but the frequency range of 3.75 Hz and above had reduced spectral power during NREM sleep (Suppl. Fig. 3a). REM sleep spectra showed a leftward shift in the theta peak frequency (Suppl. Fig. 3b) similar to mouse models with chronic ablation of SNAP25 (synaptosomal-associated protein 25 kDa) from cortical excitatory neurons ( Krone et al ., 2021 ; Meijer et al ., 2025 ). Consistent with the observed NREM-promoting and REM-suppressing effects observed in the analysis of sleep amount, all animals fell asleep within 30 minutes after CNO injections and had longer NREM episodes (t(8) = 2.527, p = 0.035), while REM onset was delayed (W = 45, p = 0.004) and fewer REM episodes occurred (t(8) = 2.484, p = 0.038; Fig. 1h ). At the end of our six-hour observation time window – when the DREADD effects are expected to have ceased – sleep time, frequency of brief awakenings, and characteristics of the EEG power spectra of REM normalised (4:15 - 6:15h after i.p. injections, Fig. 1d-f ). However, the NREM sleep EEG power spectra showed a significant rebound in SWA ( Fig. 1g ), based on a selective increase in power in the low frequency range (Suppl. Fig. 3a), which typically characterises the response to sleep deprivation. The R26 LSL-hM4Di control group showed no differences in any of the assessed sleep architectural or spectral parameters between saline and 0.5 mg/kg CNO conditions (Suppl Fig. 2b-e). Together, these findings demonstrate that sleep can be induced by acute chemogenetic inhibition of Rbp4-expressing neurons across the entire cortex with hM4Di DREADDs, but that this sleep has different characteristics to spontaneous sleep. To narrow down the neocortical region(s) and cell population(s) responsible for the sleep-inducing chemogenetic effect in the Rbp4 hM4Di mice, we next performed targeted manipulations of layer 5 pyramidal neurons via focal injections of viral vectors in the PFC. Targeted, chemogenetic inhibition/excitation of prefrontal cortical layer 5 pyramidal neurons increases/decreases sleep, respectively We focused the local, viral vector-based, chemogenetic manipulations of layer 5 pyramidal neurons on the PFC, due to previous work indicating a particular role of the PFC in sensing and responding to sleep pressure ( Marzano et al ., 2013 ; Tossell et al ., 2023 ; Sawada et al ., 2024 ). For bidirectional manipulations, we injected adeno-associated viruses for Cre-dependent expression of inhibitory ( pAAV8-hSyn-DIO-hM4D(Gi)-mCherry ) or excitatory ( pAAV8-hSyn-DIO-hM3D(Gq)-mCherry) DREADDs ( Fig. 2a ) in Rbp4 Cre mice, resulting in mice with a subpopulation of layer 5 pyramidal neurons that can be inhibited (PFC-Rbp4 hM4Di ) or excited (PFC-Rbp4 hM3Dq ), respectively, with CNO ( Fig. 2b ). Download figure Open in new tab Figure 2: Targeted, chemogenetic inhibition/excitation of prefrontal cortical layer 5 pyramidal neurons bidirectionally change sleep amount. (a) Schematic of viral vector injections (inhibitory hM4Di, blue; excitatory hM3Dq, red) for localised DREADD expression in the PFC of Rbp4 Cre mice. (b) Coronal brain section (top) and dissection microscope image (bottom) showing PFC DREADD expression. Scale bars, 1000 µm. (c) Representative hypnograms and EEG SWA (0.5-4 Hz, 4-s epochs) of PFC-Rbp4 hM4Di (left) and PFC-Rbp4 hM3Dq (right) mice following i.p. injections of saline (top) and CNO (bottom) at light onset (ZT 0). (d and h) Time courses (left column) of Wake, NREM, and REM sleep in the first 6 hr post-injection of saline and CNO in PFC-Rbp4 hM4Di (d) and PFC-Rbp4 hM3Dq (h) mice, with the corresponding percentages of time spent in each vigilance state during hour 1 (DREADD-on column) post-injection. Note that REM sleep is presented as a percentage of total sleep time. (e and i) Time courses (left) of brief awakenings (BA; <16 s movement during sleep; n/h TST) during the first 6 hr after saline and CNO in PFC-Rbp4 hM4Di (e) and PFC-Rbp4 hM3Dq (i) mice, with the corresponding number of BA in hours 1+2 (DREADD-on) post-injection. (f and j) Time courses (left) of EEG SWA for the first 6 hr after saline and CNO in PFC-Rbp4 hM4Di (f) and PFC-Rbp4 hM3Dq (j) mice, with the corresponding % of NREM SWA in hours 1+2 (DREADD-on) post-injection. (g and k) Sleep architecture in hours 1+2 (DREADD-on) post-injection of saline and CNO in PFC-Rbp4 hM4Di (g) and PFC-Rbp4 hM3Dq (k) mice. Note that all time analyses were aligned from 15 min post-injection (as T=0) to ensure full DREADD activation. Asterisks indicate post-hoc comparisons with significant differences (*p<0.05; **p<0.01; ***p<0.001) for analyses with significant main effects. n = 11 PFC-Rbp4 hM4Di and n=10 PFC-Rbp4 hM3Dq for panels d, f, g, h, j, k; n = 9 PFC-Rbp4 hM4Di and n=10 PFC-Rbp4 hM3Dq for panels e and i. BA: Brief awakenings; CNO: Clozapine- N -oxide; DREADD: Designer Receptors Exclusively Activated by Designer Drugs; EEG: Electroencephalogram; i.p.: Intraperitoneal; NREM: Non-rapid eye movement sleep; PFC: Prefrontal cortex; REM: Rapid eye movement sleep; SWA: Slow wave activity; TST: Total sleep time; ZT: Zeitgeber time. Administration of 0.5 mg/kg CNO i.p. at light onset significantly increased the amount of sleep in PFC-Rbp4 hM4Di mice (W = 64, p = 0.002; Fig. 2c,d ) and decreased it in PFC-Rbp4 hM3Dq mice (W = - 45, p = 0.02; Fig. 2c,h ; time window for both analyses 0:15 - 1:15h after i.p. injections). Specifically, the proportion of NREM sleep was significantly increased in PFC-Rbp4 hM4Di mice (M Sal = 60.98%, SD Sal = 15.32; M CNO = 79.52, SD CNO = 9.034; t(10)=5.113, p = 0.0005) and decreased in PFC-Rbp4 hM3Dq mice (Med Sal = 58.89, Med CNO = 31; W =-47, p = 0.014) during the first hour of the DREADD manipulations, while the percentage of REM sleep remained unchanged in both groups following injection of CNO compared to saline ( Fig. 2d,h ; t(10) = 0.578, p = 0.576 and Z = 3, P = 0.91, respectively). The frequency of brief awakenings was unaltered during the DREADD manipulation in PFC-Rbp4 hM4Di mice (W =-24, p = 0.32; Fig. 2e ) and slightly reduced in PFC-Rbp4 hM3Dq mice (t(10) = 2.454, p = 0.037; Fig. 2i ) once they entered sleep. NREM sleep episodes were longer in PFC-Rbp4 hM4Di mice (t(10) = 2.45, p = 0.034; Fig. 2g ) following CNO injections compared to saline, whilst latencies to NREM and REM sleep did not change in either of the two groups ( Fig. 2g,k ). For both DREADD types, NREM spectra showed a reduction of power in higher frequencies, but no systematic change in REM spectra during the DREADD manipulations, nor in NREM or REM spectra during the time window in which rebound effects occurred in Rbp4 hM4Di mice (Suppl. Fig. 4a-d). Consistently, for both DREADD types, the time course of SWA in saline and CNO conditions showed the usual decay in power during the early light period without any indication of rebound effects. However, an exploratory analysis of the exponential decay constant indicated a potentially faster reduction of SWA in PFC-Rbp4 hM4Di than in PFC-Rbp4 hM3Dq mice ( Fig. 2f,j ). Overall, PFC-targeted bidirectional DREADD manipulations of layer 5 pyramidal neurons up-or down-regulated the amount of sleep with largely normal physiological characteristics. Discussion The cerebral cortex is the evolutionarily youngest and most complex region of the mammalian brain ( Geschwind and Rakic, 2013 ). Cortical network oscillations with neuronal UP/DOWN states, population ON/OFF states, and slow waves in local field potentials (LFP) and the EEG, electrophysiologically define sleep in mammals ( Vyazovskiy and Harris, 2013 ; Adamantidis, Gutierrez Herrera and Gent, 2019 ). Moreover, the amplitude of slow waves and level of SWA in LFP or EEG power spectra indicates sleep intensity ( Blake and Gerard, 1937 ; Borbély et al ., 1981 ; Vyazovskiy et al ., 2007 ), and cortical activity patterns reflect the homeostatic drive to sleep ( Vyazovskiy, Olcese, et al ., 2009 ; Thomas et al ., 2020 ). The cortex also recalibrates its synaptic structure particularly during sleep ( Tononi and Cirelli, 2006 ; Vyazovskiy et al ., 2008 ; de Vivo et al ., 2017 ), and cortical pyramidal neurons adapt their intracellular chloride levels during sleep for subsequent optimal task performance ( Alfonsa et al ., 2023 ). Hence, the cortex tracks sleep need and itself benefits from sleep. Since the cerebral cortex allows mammals to perform many functions in a more refined way than subcortical areas alone ( Preuss and Wise, 2022 ), it is surprising that its important role in regulating sleep was only recently discovered ( Krone et al ., 2021 ). Among cortical areas, the prefrontal cortex develops particularly late and has an unparalleled complexity ( Kolk and Rakic, 2022 ). Many functions of the PFC remain to be explored, including its newly-ascribed role in the top-down regulation of sleep ( Sawada et al ., 2024 ; Groenhout et al ., 2025 ), specifically through different projections to subcortical centres for sleep preparation and initiation ( Tossell et al ., 2023 ) and REM sleep control ( Hong et al ., 2023 ). Here, we build on our recent work in the conditional knockout mouse model Rbp4 Cre :SNAP25 fl/fl which indicated, through a reduction in sleep time and altered sleep homeostasis dynamics, that layer 5 pyramidal neurons regulate sleep amount ( Krone et al ., 2021 ). Our cortex-wide chemogenetic inhibition experiments in Rbp4 hM4Di mice now demonstrate that acute manipulations of their activity can strongly induce sleep. Our targeted, bidirectional chemogenetic manipulations of Rbp4-expressing layer 5 pyramidal neurons in the PFC further reveal that even local modulations of neocortical activity can change the amount and architecture of sleep. This provides evidence for the hypothesis that ‘global’ sleep – that is, sleep as a brain state and behaviour – can emerge from ‘local’ sleep, i.e. local sleep-like activity patterns in the neocortex ( Krueger et al ., 2008 ; Andrillon, 2023 ). Importantly, the sleep induced by local chemogenetic inhibition of layer 5 pyramidal neurons in the PFC preserves the characteristics of sleep electrophysiology and architecture, suggesting that chemogenetically induced sleep in PFC-Rbp4 hM4Di mice resembles spontaneous sleep, and may convey the same restorative properties. This is in stark contrast to chemogenetically-induced sleep through a cortex-wide manipulation in Rbp4 hM4Di mice, which is characterised by frequent brief awakenings and suppression of REM sleep, and is followed by rebound effects. Notably, classical hypnotics such as ‘Z-drugs’ (benzodiazepine-receptor agonists) and benzodiazepines also increase sleep time but disrupt sleep electrophysiology ( McKillop et al ., 2021 ) and impair some functions of sleep ( Hauglund et al ., 2025 ). This may represent a shared disadvantage of sleep-promoting approaches that alter cortex-or even brain-wide activity. Such pharmacological strategies come at the cost of detrimental health consequences ( De Crescenzo et al ., 2022 ), highlighting how increasing the amount of sleep alone is likely insufficient to harvest sleep’s benefits. More targeted manipulations of sleep-regulatory areas are needed to induce sleep with physiological features and functions. The reduction of sleep observed following excitatory DREADD manipulations in PFC-Rbp4 hM3Dq mice is reminiscent of the reduction of total sleep time in our previous experiments with an excitatory protocol of bifrontal transcranial direct current stimulation (tDCS) applied to human volunteers in an overnight sleep study ( Frase et al ., 2016 ). However, the mechanisms underlying sleep-modulatory effects of local cortical activity modulations remain to be explored, both in mice and humans. There are several possible, non-mutually-exclusive explanations. First, PFC layer 5 pyramidal cells could operate the ‘sleep switch’ in the hypothalamus and brainstem via projections similar to those of PFC Sst -GABA neurons, which trigger sleep preparatory behaviours and induce sleep through projections to the LPO and LH ( Tossell et al ., 2023 ). Second, layer 5 pyramidal neurons could initiate slow oscillations that propagate through the neocortex ( Sanchez-Vives and McCormick, 2000 ), and layer 5 but not layer 2/3 pyramidal may be sufficient and necessary for recurrent low-frequency dynamics ( Beltramo et al ., 2013 ). Considering that NREM sleep is characterised by slow waves and their underlying ON-and OFF-periods in large populations of cortical neurons ( Vyazovskiy, Olcese, et al ., 2009 ; Thomas et al ., 2020 ) – and that burst activation of the centromedial thalamus (CMT) induces brain-wide synchrony of cortical slow waves during sleep and accelerates the decay to slow wave activity ( Gent et al ., 2018 ) –, the hM4Di DREADD might induce bistability in a subset of intrinsically-bursting layer 5 pyramidal cells ( Lőrincz et al ., 2015 ) through hyperpolarisation of their membrane potentials (i.e., the main effect of inhibitory DREADDs ( Roth, 2016 ). This would resolve the seemingly contradictory finding that ‘inhibition’ of layer 5 pyramidal neurons in Rbp4 hM4Di and PFC-Rbp4 hM4Di increases the amount of sleep whilst ‘silencing’ the axonal terminals of layer 5 pyramidal neurons in the Rbp4 Cre :SNAP25 fl/fl mice reduced the amount of sleep. Finally, intracortically-projecting layer 5 pyramidal neurons may engage in crosstalk with PFC Sst -GABA neurons or other cortical sleep-regulatory cells, such as the transcriptomically re-defined population of SST/Chodl/Nos1/Tacr1 (SCNT) neurons ( Tasic et al ., 2016 ), which comprise the previously discovered sleep-active neural nitric oxide synthase (nNOS) expressing GABA neurons ( Gerashchenko et al ., 2008 ; Morairty et al ., 2013 ) and which promote cortical synchrony and sleep ( Ratliff et al ., 2024 ). Since the Rbp4-Cre mouse line is a pan-layer 5 driver line with pyramidal neurons projecting intracortically and subcortically from sublayers 5a and 5b, manipulations of individual subpopulations using established Cre-driver lines ( Gerfen, Paletzki and Heintz, 2013 ) or novel viral vectors ( Ben-Simon et al ., 2025 ) is an important next step in the dissection of the sleep-regulatory circuitry that involves layer 5 pyramidal neurons. Chemogenetic experiments of such subpopulations and selective optogenetic stimulation of their axonal terminals in target regions, will allow the sleep-modulating effects of subacute activity modulations, as well as a potential neural code for low or high sleep need, to be assessed. For example, it is possible that tonic vs. burst firing may represent a corticothalamic neural code for wakefulness vs. sleep. This hypothesis is based on previous findings that thalamic CMT neurons promote wakefulness in tonic firing mode but sleep recovery in bust firing mode ( Gent et al ., 2018 ). Hints regarding potential intracellular mechanisms may arise from recent findings in other model organisms. Universally, a drop or increase in neurochemical processes during wakefulness reliably evokes sleep through complex neuronal mechanisms ( Anafi, Kayser and Raizen, 2019 ). In Drosophila , a detailed neurobiological interpretation of sleep homeostasis is rapidly emerging, which defines neurochemical substrates ( Rorsman et al ., 2025 ), changes in ion channel properties ( Pimentel et al ., 2016 ; Kempf et al ., 2019 ), adaptations in intracellular organelles ( Sarnataro et al ., 2025 ), neuronal structure ( Ho et al ., 2022 ), neurophysiological patterns ( Hasenhuetl et al ., 2025 ; Raccuglia et al ., 2025 ), neuro-glial crosstalk ( Haynes et al ., 2024 ), and a dedicated neuroanatomical circuitry ( Donlea, Pimentel and Miesenböck, 2014 ; Liu et al ., 2016 ; Donlea et al ., 2018 ). Future work in mammals could benefit from considering similar fundamental, evolutionarily-conserved neuronal and biochemical processes. As it stands, our current finding has implications that go beyond the delineation of another cell population with sleep-inducing properties in the mammalian brain. The approach presented here can be used to elucidate the intracellular molecular and structural counterparts of sleep need in the cortex, which is currently emerging as a key regulator of sleep ( Pickup and Weber, 2025 ). Vice versa, the identification of a circumscribed PFC cell population that can exert bidirectional control of sleep drive, can be used to map the emerging cortical network for sleep regulation as well as its inputs and outputs, which likely include well-established centres of the subcortical ‘sleep-wake switch’, but may also include circuits previously not attributed to sleep regulation such as cortico-thalamic loops. The PFC now also provides an anatomical target to further test through which cell type(s) neurochemical modulators such as noradrenaline or adenosine ‘wake the cortex up’ or ‘put the cortex to sleep’. Such investigations could confirm the PFC as a novel neocortical hub for regulating the drive to sleep. From a clinical perspective, neocortical activity and related networks can be effectively modulated with non-invasive brain stimulation approaches. Subregions of the prefrontal cortex are already evidence-based targets for neuromodulation in depression ( Cole et al ., 2022 ) and can now be precisely stimulated with individualised parameters to enhance SWA during NREM sleep ( Schaeffer et al ., 2025 ). However, evidence for the induction of physiological sleep with non-invasive brain stimulation methods is still lacking ( Krone et al ., 2023 ; Luff and de Lecea, 2024 ). This is partially due to the low precision of conventional non-invasive brain stimulation tools and to the lack of neurobiologically-informed reachable targets for sleep neuromodulation. Applying refined methods of stimulation such as temporal interference stimulation (TIS) and transcranial focussed ultrasound (FUS) to the newly discovered sleep-regulatory centres in the neocortex may just open the door for the effective neuromodulation of sleep in humans ( Krone et al ., 2025 ). Methods Mice The following types of mice were used: Rbp4 Cre (Tg(Rbp4-Cre)KL100Gsat/Mmucd) expressing Cre-recombinase in layer 5 pyramidal neurons of the neocortex and dentate gyrus granule cells of the archicortex ( Gerfen, Paletzki and Heintz, 2013 ; Grant, Hoerder-Suabedissen and Molnár, 2016 ), kindly provided by R.M. Bruno (University of Oxford, UK); B6.129-Gt(ROSA)26Sortm1(CAG-CHRM4*,-mCitrine)Ute/J (Jackson Laboratory stock #026219, abbreviated here as R26 LSL-hM4Di mice) for Cre recombinase-inducible expression of the HA-tagged DREADD receptors hM4Di and the yellow fluorescent protein mCitrine; and a cross of the first two strains (abbreviated here as Rbp4 hM4Di mice). The Rbp4 Cre mouse line was bred to homozygosity and congenic on the C57BL/6N background, and the R26 LSL-hM4Di mouse line was supplied by the Jackson Laboratory on a C57BL/6N background and maintained homozygously in our local colony. A total of 42 young adult male mice were used: 11 Rbp4 hM4Di (2 for pilot experiments, 9 for main experiment); 9 R26 LSL-hM4Di ; and 22 Rbp4 Cre (12 PFC-Rbp4 hM4Di and 10 PFC-Rbp4 hM3Dq ). All mice were housed on a 12 h light/12 h dark cycle, at constant temperature and humidity and with ad libitum access to food and water. Stereotaxic surgery EEG/EMG implants All surgeries were aseptic and performed under isoflurane anesthesia as previously described ( McKillop et al ., 2018 ). Two electroencephalogram (EEG) screw electrodes were placed at mediolateral (ML) +2 mm/anteroposterior (AP) +2 mm (frontal derivation) and ML +2.5/AP-3.5 mm (occipital derivation), relative to bregma. Where viral injections were done (see below), the frontal screw was placed slightly more posteriorly (AP +1.8 mm). A reference screw electrode was placed over the cerebellum at ∼ML 0/-1.5 mm. Two electromyography (EMG) wire electrodes were inserted bilaterally into the neck extensor muscles. Viral injections Local expression of inhibitory and excitatory DREADD receptors in the prefrontal cortex (PFC) of Rbp4-Cre mice was achieved by injecting the viruses pAAV8-hSyn-DIO-hM4D(Gi)-mCherry (Addgene plasmid #292479) and pAAV8-hSyn-DIO-hM3D(Gq)-mCherry (Addgene plasmid #148598), respectively. 100 nL of virus was bilaterally injected at 1-4 sites per hemisphere (ML ± 0.65 mm/AP ± 2.6; ML ± 1.5 mm/AP ± 2.6; ML ± 0.65 mm/AP ± 1.9; ML ± 1.5 mm/AP ± 1.9; all relative to bregma), and always at 2 different depths (dorsoventral (DV)-1.5 and-0.8 mm, relative to bregma). Medial injections (that is, injections done at ML ± 0.65 mm) were angled six degrees inwards. The virus infusion rate was 40 nL/min using a Nanoject II (Drummond Scientific). We waited at least 3 weeks from viral injections to the first recordings to ensure adequate viral transgene expression. Chronic electrophysiological recordings Mice were acclimatised to custom-built Plexiglas recording chambers (20.3 x 32 x 35 cm) and tethered recording conditions for at least 3 days prior to the start of experiments. The chambers were placed in sound-attenuated and light-controlled Faraday cages (Campden Instruments Ltd., London, UK). EEG/EMG signals were recorded with the 128-channel Neurophysiology Recording System (Tucker-Davis Technologies Inc., Alachua, FL, USA) and the electrophysiological recording software Synapse. Raw electrophysiological signals were filtered (0.1-100 Hz) and amplified with a PZ5 NeuroDigitizer (Tucker-Davis Technologies Inc., Alachua, FL, USA), and stored at a sampling rate of 256 Hz on a local computer. Chemogenetic manipulations To chemogenetically modulate cortical neurons, mice were intraperitoneally injected with clozapine -N -oxide dihydrochloride (CNO) and saline (control). The CNO solution used was prepared from clozapine- N -oxide dihydrochloride powder (Tocris, Bio-Techne LTD, Abingdon, UK, catalog no.: 6329) with sterile 0.9% saline and passed through a Millipore filter. The main experiments were done with counterbalanced conditions of 0.5 mg/kg CNO and saline. To assess a potential dose-dependency, a subset of mice subsequently also received injections of 1 mg/kg CNO and 5 mg/kg CNO as in previous work ( Traut et al ., 2023 ). For all mice, the first condition was 0.5 mg/kg CNO vs. saline at light onset, zeitgeber time (ZT) 0, when homeostatic sleep pressure is typically at a moderate level. CNO considerations While generally considered biologically inert, CNO in fact undergoes partial back-conversion to clozapine ( Gomez et al ., 2017 ) and has off-target binding at endogenous neurotransmitter receptors ( Jendryka et al ., 2019 ). We previously tested the effects of different CNO doses (1, 5 and 10 mg/kg) on sleep systematically in wild-type (C57BL/6) mice that do not express hM3Dq or hM4Di DREADD receptors ( Traut et al ., 2023 ). We reported that CNO elicits a dose-dependent effect on several sleep parameters at medium and high doses (5 and 10 mg/kg) of this water-soluble CNO product. More specifically, it causes a mild REM suppression, alters individual NREM and REM bout number and duration, and increases sleep state stability and sleep continuity ( Traut et al ., 2023 ). For low-dose CNO (1 mg/kg) we found no significant effects on sleep amount, sleep electrophysiology, and nearly all other assessed sleep architectural parameters reported in this current study, but observed some potential indications of small DREADD-independent effects. Consequently, here we used an even lower dose of CNO (0.5 mg/kg), in line with other recent DREADD-based sleep studies in mice which show no DREADD-independent effects in their control groups but a sufficient activation of DREADDs to test sleep-wake regulation ( Lee et al ., 2025 ). In addition, we provide data from inhibitory/excitatory DREADDs or use a DREADD-free control group injected with the same CNO dose and product to exclude off-target effects of CNO. Histology Mice were deeply anaesthetised and transcardially perfused with 0.01 M PBS followed by 4% paraformaldehyde (PFA) made up from 37% stock solution (product code F8775, Sigma-Aldrich). Brains were removed and fixed in PFA overnight then stored in PBS. Whole brains were imaged on a fluorescence dissection microscope to assess the regional spread of viral expression before being sliced for histological assessment into 50 µm coronal sections using a Leica VT1000S vibrating microtome. Sections were stained with DAPI before being mounted onto glass slides and coverslipped. Fluorescence images were taken with a Zeiss LSM710 microscope. Images were analyzed and merged and scale bars were added using FIJI ImageJ (v2.9.0). All figures were created using Inkscape (v1.0.2, Inkscape Project 2020; https://inkscape.org ). The results across all animals were similar to those presented in the representative images. One mouse injected with the hM4Di construct showed unilateral expression only and was thus excluded from all analyses. Electrophysiological data processing and analysis Filtered and amplified EEG/EMG signals were resampled at 256 Hz offline using custom MATLAB (The MathWorks Inc., Natick, MA, USA, v. R2023b) scripts and converted into the European Data Format (EDF) via the open-source software Neurotraces. All files were sleep scored in a blinded, semi-automated fashion with SleepSign for Animals (v3.3.6.1602, SleepSign Kissei Comtec). EEG/EMG recordings were partitioned into 4-second epochs and automatically pre-annotated with a vigilance state assignment (wake, NREM, or REM). Annotations were manually checked to ensure mainly that artefacts, movements, and vigilance state transitions were correctly scored, based on visual inspection of the frontal and occipital EEG derivations and EMG traces. Epochs with recording artifacts arising from gross movements, chewing or external electrostatic noise were still assigned to the respective vigilance state but not included in the spectral analysis. EEG power spectra were computed using a fast Fourier transform routine (Hanning window) with a 0.25-Hz resolution and exported in the frequency range between 0 and 30 Hz for spectral analysis. NREM SWA (%) was calculated as a percentage of the average NREM SWA (the spectral power in the frequency bins between 0.5 and 4 Hz) in the 6-hour observation time window following saline injections. Sleep architecture analyses were based on EEG/EMG recordings scored according to criteria detailed in ( Traut et al ., 2023 ). Statistics Data were analysed using MATLAB (version R2023b; The MathWorks Inc, Natick, MA, USA) and GraphPad Prism (version 9.1.1 for Windows; GraphPad Software, San Diego, CA, USA, https://www.graphpad.com/ ). Reported averages represent the mean ± SD and are rounded to three decimals, unless stated otherwise. A significance level of p=0.05 was used for all analyses. For time-course analyses of time spent awake, in NREM, or in REM sleep, we compared the first and sixth post-injection hours between saline and CNO conditions using paired t-tests. These windows were chosen to capture the period of putative maximal DREADD activity (first hour) versus minimal residual activity (sixth hour) ( Jendryka et al ., 2019 ). For spectral analyses and sleep-architecture variables (e.g., average duration and number of REM and NREM episodes or average spectral power in REM and NREM), longer windows were required to ensure sufficient time in each vigilance state; therefore, we averaged across the first two and last two post-injection hours here. Comparisons of sleep architecture-related variables between the 0.5 mg/kg CNO and saline conditions were performed using paired t-tests or nonparametric analogues when appropriate. Note that all time window details above were defined relative to 15 min after intraperitoneal (i.p.) injection, informed by previous pharmacokinetic work showing that after this time CNO has passed the blood-brain barrier and reached peak CSF levels ( Jendryka et al ., 2019 ), and by examples from similar chemogenetic studies which allow up to 20 min for the onset of DREADD-mediated effects ( Lee et al ., 2025 ). Ethical approval All experiments were performed in accordance with the UK Home Office Animal Scientific Procedures Act (1986) under personal and project licenses granted by the United Kingdom Home Office. Ethical approval was provided by the Local Ethical Review Panel at the University of Oxford. Animal holding and experimentation was located at the Biomedical Sciences Building, University of Oxford. Author contributions L.B.K.: conceptualisation, funding, electrophysiology, behavioural experiments, histology, sleep scoring, data analysis, supervision, manuscript writing and editing. J.D.H.: conceptualisation, electrophysiology, behavioural experiments, histology, sleep scoring, data analysis, manuscript writing and editing. A.H.S.: histology, supervision, manuscript editing. C.M.: electrophysiology, behavioural experiments, histology, sleep scoring, manuscript editing. A.B.S.: data analysis, manuscript writing and editing. C.J.A.: conceptualisation, supervision, manuscript writing and editing. Z.M.: conceptualisation, funding, supervision, manuscript editing. V.V.V.: conceptualisation, funding, supervision, manuscript editing. Download figure Open in new tab Suppl. Fig. 1: The strength of chemogenetic sleep modulation in Rbp4 hM4Di mice is CNO-dose-dependent. (a) Representative hypnograms and EEG SWA (0.5-4 Hz, 4-s epochs) of an Rbp4 hM4Di mouse during the first 6 hr post-i.p. injection of saline and 0.5, 1, and 5 mg/kg CNO at light onset (ZT 0). (b) Time courses (left column) of Wake, NREM, and REM sleep in the first 6 hr post-injection of saline and 1 and 5 mg/kg CNO, with the corresponding percentages of time spent in each vigilance state during hour 1 (DREADD-on) post-injection. Note that REM sleep is presented as a percentage of total sleep time. Note that all time analyses were aligned from 15 min post-injection (as T=0) to ensure full DREADD activation. Asterisks indicate comparisons with significant differences (*p<0.05; **p<0.01; ***p<0.001) for analyses with significant main effects. n = 6 for 1 mg/kg CNO condition; n = 4 for 5 mg/kg CNO condition. CNO: Clozapine- N -oxide; DREADD: Designer Receptors Exclusively Activated by Designer Drugs; EEG: Electroencephalogram; i.p.: Intraperitoneal; NREM: Non-rapid eye movement sleep; REM: Rapid eye movement sleep; SWA: Slow wave activity; TST: Total sleep time; ZT: Zeitgeber time. Download figure Open in new tab Suppl. Fig. 2: CNO administration does not alter sleep amount, architecture or slow wave activity in a DREADD-free control group (R26 LSL-hM4Di ). (a) Time courses (left column) of Wake, NREM, and REM sleep over the first 6 hr post-i.p. injection of saline and CNO in DREADD-free control mice (R26 LSL-hM4Di ) at light onset (ZT 0), with the corresponding percentages of time spent in each vigilance state during hours 1+2 and 5+6 post-injection. Note that REM sleep is presented as a percentage of total sleep time. (b) Representative EEG (F, frontal; O, occipital) and EMG traces after saline and CNO injections. (c) Time course (left) of brief awakenings (BA; <16 s movement during sleep; n/h TST) during the first 6 hr after saline and CNO, with the corresponding number of BA in hours 1+2 and 5+6 post-injection. (d) Time course (left) of EEG NREM SWA for the first 6 hr after saline and CNO, with the corresponding % of NREM SWA in hours 1+2 (DREADD-on) and 5+6 (DREADD-off) post-injection. (e) NREM (left column) and REM (right column) sleep architecture in hours 1+2 post-injection of saline and CNO. Note that all time analyses were aligned from 15 min post-injection (as T=0) as in the main figs. n = 9 for panels a and e, and n = 8 for panels c and d. BA: Brief awakenings; CNO: Clozapine- N -oxide; DREADD: Designer Receptors Exclusively Activated by Designer Drugs; EEG: Electroencephalogram; EMG: Electromyogram; i.p.: Intraperitoneal; NREM: Non-rapid eye movement sleep; REM: Rapid eye movement sleep; SWA: Slow wave activity; TST: Total sleep time; ZT: Zeitgeber time. Download figure Open in new tab Suppl. Fig. 3: CNO administration has acute and rebound effects on EEG spectra in Rbp4 hM4Di mice but not in R26 LSL-hM4Di mice. (a and b) Frontal EEG spectra during NREM (a) and REM (b) sleep in Rbp4 hM4Di mice in hours 1 (DREADD-on) and 6 (DREADD-off) after i.p. injection of saline and 0.5 mg/kg CNO at light onset (ZT 0). (c and d) Frontal EEG spectra during NREM (c) and REM (d) sleep in R26 LSL-hM4Di mice in hours 1+2 and 5+6 after i.p. injection of saline and 0.5 mg/kg CNO. Note all inlays represent respective spectral power following CNO as a percentage of the saline condition. n = 9 Rbp4 hM4Di and n = 8 R26 LSL-hM4Di mice. Asterisks indicate post-hoc contrasts with significant differences (grey and blue (inlay) *p<0.05; black *p<0.01). CNO: Clozapine- N -oxide; DREADD: Designer Receptors Exclusively Activated by Designer Drugs; EEG: Electroencephalogram; i.p.: Intraperitoneal; NREM: Non-rapid eye movement sleep; REM: Rapid eye movement sleep; SWA: Slow wave activity; ZT: Zeitgeber time. Download figure Open in new tab Suppl. Fig. 4: CNO administration has acute but no rebound effects on NREM EEG spectra in mice expressing hM4Di and hM3Dq receptors in the PFC. (a-d) Frontal EEG spectra during NREM (a,c) and REM (b,d) sleep in PFC-Rbp4 hM4Di (a-b) and PFC-Rbp4 hM3Dq (b-d) mice in hours 1+2 (DREADD-on) and 5+6 (DREADD-off) after i.p. injection of saline and 0.5 mg/kg CNO at light onset (ZT 0). Note all inlays represent respective spectral power following CNO as a percentage of the saline condition. n = 9 PFC-Rbp4 hM4Di and n = 10 PFC-Rbp4 hM3Dq mice. Asterisks indicate post-hoc contrasts with significant differences (grey and blue (inlay) *p<0.05; black *p<0.01). CNO: Clozapine- N -oxide; DREADD: Designer Receptors Exclusively Activated by Designer Drugs; EEG: Electroencephalogram; i.p.: Intraperitoneal; NREM: Non-rapid eye movement sleep; PFC: Prefrontal cortex; REM: Rapid eye movement sleep; SWA: Slow wave activity; ZT: Zeitgeber time. Suppl. Video 1: Reduced exploratory behaviour and earlier sleep onset following CNO administration in Rbp4 hM4Di mice. One-hour time-lapsed video (60x speed) of two representative Rbp4 hM4Di mice for 1 hr post-i.p. injection of saline (left mouse) and 5 mg/kg CNO (right mouse) at light onset (ZT 0). Immediately following the i.p. injection, the mice’s own nesting material was scattered evenly across the cage and a novel object (taped toilet roll) was introduced. Camera light sensors were deactivated to generate black and white footage. CNO: Clozapine- N -oxide; i.p.: Intraperitoneal; ZT: Zeitgeber time. Video available at: https://doi.org/10.6084/m9.figshare.30069778 Acknowledgements We thank the members of the Vyazovskiy and Miesenböck labs for discussions and feedback on the project. In particular we thank Dr. Linus Milinski for detailed feedback on the figures and text, and for helping supervise students. We thank Liliana Mendes da Paz, James Metcalf, and Laura Thomas of the Oxford Mouse Sleep Core (OMSC) for their technical support, and the Oxford Biomedical Sciences Level 1 and 2 teams for taking care of the animals. We thank Madison Bartley and Prof. Randy Bruno for the provision of breeder pairs for the Rbp4 Cre mouse line. We thank Louise Aarons for assistance with surgeries, electrophysiology experiments, and sleep scoring as part of a student project. We thank Dr. Natalie Hauglund for the mouse illustration and Dr. Clifford Talbot for helping with statistical analyses. Finally, we thank L.B.K.’s Sir Henry Wellcome Fellowship sponsors Prof. Chiara Cirelli, Prof. Antoine Adamantidis, and Prof. Gero Miesenböck for their ongoing advice and support. We thank the following funders for their financial support: L.B.K. was supported by a Wellcome Trust Doctoral Studentship in Neuroscience (203971/Z/16/Z) and is currently supported by a Wellcome Trust Sir Henry Wellcome Fellowship (224083/Z/21/Z), and the Exeter College Staines Medical Research Fellowship. J.D.H. is supported by an Australian Ramsay Postgraduate Scholarship, James Fairfax Oxford-Australia Scholarship, and previously the Oxford Clarendon Fund. C.M. is supported by the German Academic Scholarship Foundation, BMEP Stipend (University Clinic Jena), and an Oxford Berlin Research Partnership (Early Career Researcher Mobility). Z.M. and V.V.V. are supported by a BBSRC grant (BB/X008711/1) and Z.M. is supported by an MRC Project Grant (G00900901) and a Research Grant from St John’s College Research Centre (21138077). No artificial intelligence was used for data analysis or writing of this manuscript. The large language model ChatGPT (version 5, OpenAI, 2025) was used to simplify and annotate Matlab scripts previously written by L.B.K and V.V.V. and to adapt the colour scheme of illustrations. Funder Information Declared Wellcome Trust , 203971/Z/16/Z , 224083/Z/21/Z Oxford Exeter College Staines Medical Research Fellowship Australian Ramsay Postgraduate Scholarship James Fairfax Oxford-Australia Scholarship Foundation Oxford Clarendon Fund German Academic Scholarship Foundation BMEP Stipend (University Clinic Jena) Oxford Berlin Research Partnership BBSRC Grant , BB/X008711/1 MRC Project Grant , G00900901 St John's Research Centre , 21138077 Footnotes https://doi.org/10.6084/m9.figshare.30069778 References ↵ Adamantidis , A.R. , Gutierrez Herrera , C. and Gent , T.C . ( 2019 ) ‘ Oscillating circuitries in the sleeping brain ’, Nature Reviews Neuroscience , 20 ( 12 ), pp. 746 – 762 . Available at : doi: 10.1038/s41583-019-0223-4 . OpenUrl CrossRef ↵ Alfonsa , H. et al. ( 2023 ) ‘ Intracellular chloride regulation mediates local sleep pressure in the cortex ’, Nature Neuroscience , 26 ( 1 ), pp. 64 – 78 . Available at : doi: 10.1038/s41593-022-01214-2 . 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