GABAergic network from AVP neurons to VIP neurons in the suprachiasmatic nucleus sets the activity/rest time of the circadian behavior rhythm

GABAergic network from AVP neurons to VIP neurons in the suprachiasmatic nucleus sets the activity/rest time of the circadian behavior rhythm | 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 GABAergic network from AVP neurons to VIP neurons in the suprachiasmatic nucleus sets the activity/rest time of the circadian behavior rhythm Yubo Peng , Yusuke Tsuno , Takashi Maejima , Mohan Wang , Ayako Matsui , View ORCID Profile Michihiro Mieda doi: https://doi.org/10.1101/2025.04.28.650944 Yubo Peng 1 Department of Integrative Neurophysiology, Graduate School of Medical Sciences, Kanazawa University , Kanazawa, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yusuke Tsuno 1 Department of Integrative Neurophysiology, Graduate School of Medical Sciences, Kanazawa University , Kanazawa, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Takashi Maejima 1 Department of Integrative Neurophysiology, Graduate School of Medical Sciences, Kanazawa University , Kanazawa, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Mohan Wang 1 Department of Integrative Neurophysiology, Graduate School of Medical Sciences, Kanazawa University , Kanazawa, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ayako Matsui 1 Department of Integrative Neurophysiology, Graduate School of Medical Sciences, Kanazawa University , Kanazawa, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Michihiro Mieda 1 Department of Integrative Neurophysiology, Graduate School of Medical Sciences, Kanazawa University , Kanazawa, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Michihiro Mieda For correspondence: mieda{at}med.kanazawa-u.ac.jp tsuno{at}med.kanazawa-u.ac.jp Abstract Full Text Info/History Metrics Preview PDF Abstract The central circadian clock of the suprachiasmatic nucleus (SCN) is a network composed of multiple types of γ-aminobutyric acid (GABA)-ergic neurons and glial cells. However, the precise role of GABAergic transmission in the SCN remains unclear. In this study, we investigated the GABAergic regulation from arginine vasopressin (AVP)-producing neurons in the SCN shell to vasoactive intestinal polypeptide (VIP)-producing neurons in the SCN core. Blocking GABA release from AVP neurons by a vesicular GABA transporter ( Vgat ) gene deletion lengthened the activity time (the interval between the onset and offset of locomotor activity) and shortened the duration of high Ca 2+ activity in VIP neurons to match the behavioral rest time. Conversely, eliminating functional GABA A receptors (GABA A R) in VIP neurons by in vivo genome editing reduced locomotor activity level and the activity time, and lengthened the high Ca 2+ duration in VIP neurons. Optogenetic activation of AVP neurons in vivo increased Ca 2+ in VIP neurons during the night. A similar Ca 2+ response of VIP neurons to AVP neuronal activation was also observed in SCN slices and was inhibited by a GABA A R antagonist, gabazine. Importantly, gabazine application alone raised the baseline Ca 2+ in VIP neurons, suggesting a tonic depression of these neurons by GABA. Moreover, AVP neuronal activation decreased Ca 2+ in non-AVP neurons located between AVP- and VIP-rich regions in the SCN. These results suggest that GABA from AVP neurons disinhibits VIP neurons indirectly by suppressing other intermediate GABA neurons to set the behavior activity/rest time precisely. Introduction The suprachiasmatic nucleus (SCN) of the hypothalamus serves as the central circadian clock in mammals, orchestrating multiple circadian biological rhythms in the body [ 1 ]. The SCN is composed of approximately 20,000 cells. Individual SCN cells can generate cellular circadian oscillations driven by the autoregulatory transcription-translation feedback loop (TTFL) of clock genes, including Bmal1 , Clock , Per1/2/3 , and Cry1/2 . Interestingly, it is not only the SCN cells that share TTFL-driven cellular clocks but also most cells throughout the body. Rather, intercellular communication between SCN cells is essential to generate a highly robust, coherent circadian rhythm [ 1 , 2 ]. The SCN is a network of neurochemically heterogeneous γ-aminobutyric acid (GABA)-ergic neurons. Several types of GABA neurons can be distinguished by the co-expressed neuropeptides [ 1 , 2 ]. Arginine vasopressin (AVP)-producing GABAergic neurons in the SCN shell, the dorsomedial part of the SCN, and vasoactive intestinal polypeptide (VIP)-producing GABAergic neurons in the SCN core, the ventrolateral part, are the two of the major neuron types. VIP has been reported as a critical factor in the maintenance and synchronization of SCN neurons [ 3 – 6 ]. In addition, these neurons also contribute to the output pathway from the SCN [ 7 – 9 ]. In contrast, AVP neurons have been suggested to be the primary pacesetter cells that determine the period length of the circadian rhythm generated by the SCN network [ 10 – 13 ]. The functional roles of GABA-mediated signaling in the SCN network remain controversial [ 14 ]. GABA was initially reported to synchronize action potential firing rhythms of dispersed SCN neurons through GABA A receptors (GABA A R) [ 15 ]. Studies using SCN slices and GABA A R antagonists showed that GABA synchronizes or desynchronizes the cellular circadian oscillations depending on the lighting conditions before the slice preparation [ 16 – 18 ]. In addition, the regulation of extracellular GABA in the SCN by astrocytes was reported to be critical for circadian timekeeping in neonatal SCN cultures [ 19 – 21 ]. The SCN-specific in vivo deletion of the vesicular GABA transporter gene ( Vgat , also called Slc32a1 ), which is necessary for filling synaptic vesicles with GABA and thus for synaptic GABA release [ 22 ], attenuated the circadian behavior rhythm but remained the TTFL oscillation more or less normal [ 23 , 24 ]. We previously demonstrated that AVP neuron-specific deletion of Vgat drastically alters the daily locomotor activity pattern without changing the free-running period [ 25 ]. Namely, these mice showed a marked lengthening of the activity time in the circadian behavior rhythm due to an extended interval between morning and evening locomotor activities. GABA released by AVP neurons was suggested not to significantly affect the synchrony of TTFLs among SCN neurons but to regulate the phase relationships between TTFLs in the SCN and circadian morning/evening locomotor activities [ 25 ]. In addition, the daily rhythm of the in vivo multiunit activity (MUA) in the SCN clearly changed to an aberrant bimodal pattern that correlated with dissociated morning/evening locomotor activities [ 25 ]. These observations indicated that GABAergic transmission from AVP neurons regulates the activities of other SCN neurons to temporally restrict circadian behavior to appropriate time windows in SCN TTFLs [ 25 ]. In this study, we show the critical role of indirect GABAergic regulation of AVP neurons on VIP neurons in the activity/rest time setting. Results In vivo Ca 2+ rhythm of SCN VIP neurons tracks the compressed rest period of the behavior rhythm in AVP neuron-specific Vgat deficiency AVP neuron-specific Vgat -deficient mice ( Avp-Vgat −/− ) showed a lengthened activity time (the interval between the locomotor activity onset and offset) of behavior rhythm with little change in the intracellular Ca 2+ rhythm of AVP neurons, resulting in a misalignment of locomotor activity and AVP neuronal Ca 2+ rhythm [ 25 ]. To elucidate how GABA released from AVP neurons regulates the activity of VIP neurons in the SCN, we first examined the temporal relationship between the circadian behavioral rhythm and the intracellular Ca 2+ in VIP neurons (VIP-Ca 2+ ) of Avp-Vgat −/− mice in vivo using fiber photometry. To specifically target the jGCaMP7s expression in VIP neurons of Avp-Vgat −/− mice, we introduced a Vip-tTA allele [ 26 ] into Avp-Cre; Vgat flox/flox ( Avp-Vgat −/− ) or Avp-Cre; Vgat wt/flox (control) mice. In Vip-tTA mice, the tetracycline transactivator (tTA) is expressed specifically in VIP neurons. We then specifically expressed the fluorescent Ca 2+ indicator jGCaMP7s [ 27 ] in SCN VIP neurons by focally injecting a tTA-dependent AAV vector (AAV- TRE-jGCaMP7s ) and implanted an optical fiber just above the SCN ( Fig 1A and 1B ) [ 12 ]. Download figure Open in new tab Figure 1. VIP-Ca 2+ rhythm matches the compressed rest time of the locomotor activity in Avp-Vgat −/− mice (A) Schematic diagram of viral vector ( AAV-TRE-jGCaMP7s ) injection and optical fiber implantation at the SCN in control ( Avp-Cre; Vgat wt/flox ; Vip-tTA ) or Avp-Vgat −/− ( Avp-Cre; Vgat flox/flox ; Vip-tTA ) mice for fiber photometry recording. (B) A representative coronal section of mice with jGCaMP7s expression in SCN VIP neurons. A white dotted square shows the estimated position of the implanted optical fiber. Green, jGCaMP7s; blue, DAPI. Scale bar, 1 mm. (C) Representative plots of the in vivo jGCaMP7s signal of SCN VIP neurons (green: VIP-Ca 2+ ) overlaid with locomotor activity (black) in actograms. Control (Left) and Avp-Vgat −/− (Right) mice were initially housed in LD (LD1 to LD5), then in DD (DD1 to DD13). Gray shading indicates the time when the lights were off. (D) Plots of locomotor activity onset (black), activity offset (gray), VIP-Ca 2+ onset (green), VIP-Ca 2+ offset (light green), and VIP-Ca 2+ midpoint (magenta) of mean ± SEM (left column) and individual (right column) in control and Avp-Vgat −/− mice. Identical marker shapes indicate data from the same animal. (E) High VIP-Ca 2+ duration in LD (LD1-5, left) or in DD (DD6-10, right). (F) Normalized VIP-Ca 2+ daily rhythm profiles in LD (LD1-5, top) or in DD (DD6-10, bottom). In DD, CT12 was determined as the onset of locomotor activity. Red, Avp-Vgat −/− (n = 8); blue, Control ( Avp-Vgat +/− , i.e., Avp-Cre; Vgat wt/flox , n = 5); Values are mean ± SEM. *P < 0.05 by two-tailed Welch’s t-test. The VIP-Ca 2+ rhythm was synchronized antiphasically with the locomotor activity rhythm in control ( Avp-Cre; Vgat wt/flox ; Vip-tTA ) mice ( Fig 1C and 1D ) and showed high levels of Ca 2+ across the entire span of the behavioral rest period, as reported previously [ 12 , 28 – 30 ]. In Avp-Vgat −/− mice, in contrast, the duration of high VIP-Ca 2+ was clearly compressed ( Avp-Vgat −/− , 10.0 ± 0.7 h; Control, 12.2 ± 0.2 h, P = 0.02) complementarily to the expansion of behavioral activity time ( Avp-Vgat −/− , 19.2 ± 0.3 h; Control, 13.6 ± 0.4 h, P < 0.001) in constant darkness (DD) condition ( Figs 1C - 1F and S1). The VIP-Ca 2+ offset almost coincided with the locomotor activity onset ( Fig 1D ). These results suggest that the temporal relationship between the VIP-Ca 2+ rhythm and the locomotor activity is essentially maintained in the Vgat -deficiency of AVP neurons and that GABA from AVP neurons regulates the VIP-Ca 2+ rhythm. Elimination of GABA A R in SCN VIP neurons reduces locomotor activity and shortens the activity time of the behavior rhythm Thus, SCN VIP neurons likely receive direct or indirect GABAergic regulation from AVP neurons, as well as GABA from other types of SCN neurons. To investigate the role of GABAergic signaling in VIP neurons in generating the behavior rhythm, we next aimed to eliminate ionotropic GABA A R specifically in these neurons. GABA A R is essentially a pentameric protein composed of α, β, and γ or δ subunits [ 31 ]. Each subunit has multiple subtype genes, but we had no information about which genes to delete. Considering that the β subunit is necessary for functional GABA A Rs and that only three subtype genes encode the β subunit ( Gabrb1 - 3 ), we introduced indel mutations in all three simultaneously and specifically in VIP neurons by in vivo genome editing. We first crossed Cre-dependent SpCas9-expressing ( Rosa-LSL-SpCas9-2A-EGFP ) mice [ 32 ] with VIP neuron-specific Cre driver ( Vip-ires-Cre ) mice [ 33 ]. Then we injected the mixture of three AAV vectors, each expressing gRNAs targeting one of the Gabrb genes (AAV- U6-gGabrb1, 2, 3-EF1α-DIO-mCherry ), into the SCN of these mice ( Vip-GABA A R −/− mice) ( Figs 2A , 2B , S2A and S2B). Indeed, GABA A R-mediated postsynaptic currents (GPSCs) disappeared almost completely in VIP neurons of SCN slices prepared from Vip-GABA A R −/− mice, confirming the effectiveness of this method (S2C and S2D Figs). Download figure Open in new tab Figure 2. Vip-GABA A R − / − mice reduce the locomotor activity and shorten the activity time (A) Schematic diagram of viral vector (AAV- U6-gGabrb1,2,3-EF1α-DIO-mCherry or AAV- U6-gControl-EF1α-DIO-mCherry ) injection at the SCN in Rosa26-CAG-LSL-SpCas9-2A-EGFP; Vip-ires-Cre mice to generate control or Vip-GABA A R −/− mice for locomotor activity recording. (B) A representative coronal section of mice with SpCas9-2A-EGFP and gRNA-DIO-mCherry expression in SCN VIP neurons. Green, SpCas9-EGFP; magenta, mCherry. (C) Representative locomotor activity of control and Vip-GABA A R −/− mice (home-cage activity). Gray shading indicates the time when the lights were off. (D) Averaged daily profile of locomotor activity in LD (left) or DD (right). (E) Average locomotor activity counts per 6-hour interval in LD (left) or DD (right). (F) Activity time of locomotor activity rhythm in LD (left) or in DD (right). Blue, Control; red, Vip-GABA A R −/− . Values are mean ± SEM. n = 8 for Control, n = 16 for Vip-GABA A R −/− mice. *P < 0.05; **P < 0.01 by two-way repeated measures ANOVA post-hoc two-tailed Student’s t-test with Bonferroni correction (E), or by two-tailed Student t tests (F). In LD, Vip-GABA A R −/− mice showed a daily locomotor activity rhythm comparable to controls. In DD, however, the nocturnal locomotor activity of Vip-GABA A R −/− mice was significantly reduced with less obvious locomotor onset and offset without the bimodal morning and evening locomotor activities ( Fig 2C - 2E ), resulting in a shortened activity time ( Vip-GABA A R −/− , 12.73 ± 0.21 h; Control, 13.79 ± 0.43 h, P = 0.0189) ( Fig 2F ). In contrast, their free-running period and amplitude in DD did not alter significantly (S2E and S2F Figs). These results suggest that the disinhibition of VIP neurons due to the absence of GABA A Rs may suppress the locomotor activity in the subjective night. The high Ca 2+ duration in VIP neurons is lengthened in Vip-GABA A R − / − mice In Avp-Vgat −/− mice, the high VIP-Ca 2+ duration of the VIP-Ca 2+ rhythm was shortened while the behavioral activity time was lengthened. Therefore, we next investigated how the VIP-Ca 2+ rhythm alters in vivo in Vip-GABA A R −/− mice ( Fig 3A and 3B ). Download figure Open in new tab Figure 3. Duration of high VIP-Ca 2+ lengthens in Vip-GABA A R −/− mice. (A) Schematic diagram of viral vector (AAV- CAG-FLEX-jGCaMP7s and AAV- U6-gGabrb1,2,3-EF1α-DIO-mCherry or AAV- U6-gControl-EF1α-DIO-mCherry ) injection and optical fiber implantation at the SCN in Rosa26-CAG-LSL-SpCas9-2A-EGFP; Vip-ires-Cre mice to generate control or Vip-GABA A R −/− mice for fiber photometry recording. (B) A representative coronal section of mice with jGCaMP7s expression and gRNA-DIO-mCherry expression in SCN VIP neurons. A white dotted square shows the estimated position of the implanted optical fiber. Green, jGCaMP7s; magenta, mCherry. (C) Representative plots of the in vivo jGCaMP7s signal of SCN VIP neurons (green) overlaid with locomotor activity (home-cage activity) (black) in actograms. Control (Left) and Vip-GABA A R −/− (Right) mice were initially housed in LD (LD1 to LD7) and then in DD (DD1 to DD15). Gray shading indicates the time when the lights were off. (D) Plots of locomotor activity onset (black), activity offset (gray), VIP-Ca 2+ onset (green), VIP-Ca 2+ offset (light green), and VIP-Ca 2+ midpoint (magenta) of mean ± SEM (left column) and individual (right column) in control and Vip-GABA A R -/- mice. (E) Normalized VIP-Ca 2+ daily rhythm profiles in LD (LD3-7, top) or in DD (DD5-14,middle and bottom). In DD, circadian time 12 was determined as the onset of locomotor activity and VIP-Ca 2+ circadian time 0 was determined as the onset of VIP-Ca 2+ . (F) High VIP-Ca 2+ duration in LD (LD3-7, left) or in DD (DD5-14, right). (G) High VIP-Ca 2+ duration in LD (LD3-7, left) or in DD (DD5-14, right) calculated with 20% amplitude of the VIP-Ca 2+ profile as the threshold to determine the Ca 2+ onset and offset. Values are mean ± SEM. n = 7 for control, n = 5 for Vip-GABA A R −/− mice. *P < 0.05; **P < 0.01 by two-tailed Student t tests. In Vip-GABA A R −/− mice, the locomotor activity onset and offset became obscure due to the reduced activity levels ( Fig 3C ), as described earlier ( Fig 2 ). Correspondingly, the high Ca 2+ duration of the VIP-Ca 2+ rhythm was significantly extended in these mice ( Vip-GABA A R −/− , 12.30 ± 0.25 h; Control, 11.62 ± 0.19 h, P = 0.0483), which is consistent with the reduced behavioral activity time observed in these mice ( Figs 3D - 3F , S3B and S3C). The free-running period of the VIP-Ca 2+ rhythm was comparable between genotypes (S3D Fig). Notably, the shape of the daily VIP-Ca 2+ profiles appeared to be qualitatively different between Vip-GABA A R −/− and control mice ( Fig 3E ). In fact, the latter showed a relatively smooth quasi-rectangular wave, while the former fluctuated more on the plateau ( Figs 3E and S3A). These observations might reflect the presumed higher basal Ca 2+ level in VIP neurons lacking GABA A R, causing a ceiling effect on the plateau. Indeed, the high VIP-Ca 2+ duration lengthening was more pronounced when the threshold for determining Ca 2+ onset and offset was set at 20% of the amplitude (max-min) rather than 50%, as in other analyses ( Fig 3E and 3G ). These data suggest that the GABA A R elimination in VIP neurons causes a significant extension of high Ca 2+ duration in vivo, which may subsequently suppress locomotor activity. Optogenetic activation of AVP neurons increases VIP-Ca 2+ in a time-dependent manner in vivo Previous studies have reported that AVP neuronal fibers make sparse contacts onto VIP neurons and that VIP neurons respond to the optogenetic activation of AVP neurons by increasing Ca 2+ at around ZT22 in vivo [ 12 , 34 ]. Therefore, to further investigate the functional connectivity between AVP and VIP neurons, we next tested the time-of-day dependency of VIP-Ca 2 response to the optogenetic activation of AVP neurons in vivo. To this end, ChrimsonR-mCherry, a red light-gated cation channel [ 35 , 36 ], and jGCaMP7s were expressed specifically in AVP and VIP neurons, respectively, by injecting AAV- CAG-FLEX-ChrimsonR-mCherry and AAV- TRE-jGCaMP7s into the SCN of Avp-Cre; Vip-tTA mice. Interestingly, optogenetic stimulation of AVP neurons increased VIP-Ca 2+ only during the night, when basal Ca 2+ is low, but not during the day, when basal Ca 2+ is high ( Fig 4A - 4E ). The most potent responses were observed at ZT14 and ZT22. These results suggest that AVP neurons can activate VIP neurons during the night in vivo. Download figure Open in new tab Figure 4. Optogenetic stimulation of AVP neurons increases VIP-Ca 2+ in vivo during the night (A, B) Top: Representative traces of the jGCaMP7s signal of SCN VIP neurons upon optogenetic stimulation of AVP neurons at various timing in vivo (A, ChrimsonR; B, mCherry-Control). Green traces indicate the fluorescence (F) value at the 470 nm light excitation (F470), Ca 2+ -dependent signal. Magenta traces indicate the fluorescence value at the 415 nm light excitation (F415), Ca 2+ -independent control signal. Red shading indicates the timing of optical stimulation (635 nm, 50 ms pulse, 5 Hz, 120 s). Bottom: Ratio (R) calculated by F470/F415 from the upper traces. The baseline ratio (R 0 ) is the mean R-value of the pre-stimulation period (-30 s - 0 s). ΔR is the difference between the mean R-value of the late phase during the stimulation period (R 1 , 90 s - 120 s, R 1 ) and R 0 . a.u., arbitrary unit. (C, D) Daily rhythms of the baseline ratio (R 0 , open circle) and the ratio during the late phase of stimulation (R 1 , closed circle). Each color represents an individual mouse. C, ChrimsonR; D, mCherry-Control. (E) Comparison of the ΔR/R 0 %. Optogenetic stimulation of SCN AVP neurons increases VIP-Ca 2+ during the night in freely moving mice. n = 6 for ChrimsonR, n = 4 for control. ***P < 0.001 by two-tailed Welch’s t-test. Four out of 6 in ChrimsonR and all control animals are from a previously used cohort [ 12 ]. GABA A R antagonist increases VIP-Ca 2+ and inhibits the response of VIP-Ca 2+ to the optogenetic activation of AVP neurons in SCN slices To further understand how AVP neurons regulate VIP neurons in the SCN network, we next recorded the VIP-Ca 2+ response to the optogenetic activation of AVP neurons in coronal slices of the middle SCN along the rostro-caudal axis. ChrimsonR-mCherry and jGCaMP7s were expressed in AVP and VIP neurons of Avp-Cre; Vip-tTA mice, respectively, by injecting AAV vectors into the SCN ( Fig 5A and 5B ). Then, SCN slices were prepared, and an optical fiber was placed close to the ChrimsonR-mCherry-positive region in the SCN for optogenetic activation ( Fig 5B ). Download figure Open in new tab Figure 5. GABA A R antagonist increases VIP-Ca 2+ and inhibits the response of VIP-Ca 2+ to the optogenetic activation of AVP neurons in slices (A) Schematic diagram of viral vector injection at the SCN in Avp-Cre; Vip-tTA mice for slice recording of VIP-Ca 2+ and optogenetic stimulation of AVP neurons. (B) A representative coronal section of mice with ChrimsonR expression in AVP neurons (left) and jGCaMP7s expression in VIP neurons (right). White dotted squares indicate the estimated position of the optical fiber. (C) Average traces of the jGCaMP7s signal of SCN VIP neurons with (ChrimsonR, blue) or without (Control, purple) optogenetic stimulation of AVP neurons at ZT22. Red shading indicates the timing of optical stimulation (617 nm, 40 ms pulse, 10 Hz, 120 s). (D) The mean ΔF/F 0 % values during the last 30 s of optogenetic stimulation (90 s – 120 s in C). Values are mean ± SEM. n=3 for control (mCherry) mice, n = 6 for ChrimsonR mice. *P < 0.05 by two-tailed Welch’ s t-test. (E) Average trace of jGCaMP7s signal in VIP neurons after administration of gabazine (GABA A R antagonist, 10 μM) via bath perfusion. The green shading indicates when Gabazine was present. (F) The mean ΔF/F 0 % value during the last 60 s of gabazine application (180 s –240 s in E). Baseline is the mean ΔF/F 0 % values 2 min prior to gabazine application (-120 – 0 s). Values are mean ± SEM. n = 6. *P < 0.05 by two-tailed paired t-test. (G) Average traces of the jGCaMP7s signal of SCN VIP neurons upon optogenetic stimulation of AVP neurons at ZT22 without (ChrimsonR, blue) or with (ChrimsonR + Gabazine, red) gabazine application. Red shading indicates the timing of optical stimulation (617 nm, 40 ms pulse, 10 Hz, 120 s). The ChrimsonR traces in (C, blue) and (G, blue) are identical. (H) The mean ΔF/F 0 % values during the last 30 s of optogenetic stimulation (90 s – 120 s in G). Values are mean ± SEM. n = 6. *P < 0.05 by two-tailed paired t-test. Slices were photo-stimulated around ZT22 when VIP neurons exhibited a strong response in vivo. Optogenetic activation of AVP neurons increased VIP-Ca 2+ , as observed in vivo ( Figs 5C , 5D , S4A and S4B). Strikingly, the application of a GABA A R antagonist, gabazine, alone raised the baseline VIP-Ca 2 ( Figs 5E , 5F , and S4A), suggesting that VIP neurons are suppressed by GABA during the night. Moreover, the VIP-Ca 2+ increase induced by the optogenetic activation of AVP neurons was largely inhibited in the presence of gabazine ( Figs 5G , 5H , S4A and S4C). These data may suggest that GABA released from AVP neurons indirectly activates VIP neurons by inhibiting intermediate GABA neurons that suppress VIP neurons. On the other hand, the fact that gabazine failed to inhibit the VIP-Ca 2+ response completely may indicate the presence of parallel pathways mediated by transmitters of AVP neurons other than GABA, such as AVP and other neuropeptides. Optogenetic activation of SCN AVP neurons decreases Ca 2+ in the adjacent non-AVP neurons If AVP neurons disinhibit VIP neurons, as suggested in the previous section, there should be some population of intermediate GABA neurons that are inhibited by AVP neurons. To test this possibility, we made a spatial map of the Ca 2+ responses of non-AVP neurons to the optogenetic activation of AVP neurons in SCN slices. To do this, we expressed ChrimsonR-mCherry and jGCaMP7s in AVP and non-AVP neurons, respectively, by injecting AAV- CAG-FLEX-ChrimsonR-mCherry and AAV- EF1-rDIO (reverse DIO)-jGCaMP7s into the SCN of Avp-Cre mice ( Fig 6A and 6B ). Then, we prepared slices of the middle SCN along the rostro-caudal axis and monitored the jGCaMP7s signal throughout the slices. Download figure Open in new tab Figure 6. Optogenetic activation of AVP neurons decreases Ca 2+ in the adjacent non-AVP cells (A) Schematic diagram of viral vector injection at the SCN in Avp-Cre mice for slice recording of SCN non-AVP cellular Ca 2+ and optogenetic stimulation of AVP neurons. (B) A representative coronal section of mice with jGCaMP7s expression in non-AVP cells (left) and ChrimsonR expression in AVP neurons (right). White dotted squares indicate the estimated position of the optical fiber. The red rectangles in (B) indicate the regions of the enlarged images (C). (C) Left: Representative coronal section of Avp-Cre mice with jGCaMP7s expression in non-AVP cells (2.6 μm/pixl). White outline indicates the regions considered as the SCN. Right: Representative pixel-level heat maps (20.8 μm/pixel) showing the jGCaMP7s signal from non-AVP cells in response to the optogenetic stimulation of AVP neurons at ZT22. Optical stimulation was applied from time 0 to 120 s. Blue and red squares on the maps indicate regions (4 x 4 pixels) considered as the middle and ventral regions, respectively, for the subsequent analyses. (D) Representative responses of the jGCaMP7s signals in the middle (left) and ventral (right) regions to the optogenetic activation of AVP neurons. Red shading indicates the timing of optical stimulation (617 nm, 40 ms pulse, 10 Hz, 120 s). M P0 and M T0 are the highest (peak) and lowest (trough) ΔF/F 0 %-value of the pre-stimulation (-60 s ∼ 0 s) in the middle region, respectively. M P and M T are the highest and lowest ΔF/F 0 %-value during the stimulation (0 s ∼ 120 s). Correspondingly, V P0 , V T0 , V P , and V T refer to the respective points in the ventral region. (E) Mean responses of the meddle and ventral SCN regions to the optogenetic activation of AVP neurons. Values are mean ± SEM. n = 5. *P < 0.05 by two-tailed Welch’ s t-test. The jGCaMP7s signals of non-AVP neurons began to decrease shortly (roughly 10 s) after the onset of optogenetic stimulation of AVP neurons in most of the SCN area ( Fig 6C ). As the stimulation continued, the signals gradually recovered from this inhibitory response and eventually shifted to an increasing response in some areas ( Fig 6D ). Cells exhibiting such an excitatory response tended to be distributed in the ventral region of SCN, where VIP neurons are located. In contrast, cells in the intermediate area tended to demonstrate larger inhibitory responses with little excitation ( Figs 6C-E , S5A and S5B). These results support the idea that AVP neurons indirectly activate VIP neurons via GABA by inhibiting another population of GABA neurons in the SCN network. Discussion In this study, we investigated the GABAergic network through which AVP neurons in the SCN shell regulate VIP neurons in the SCN core to set the timing of locomotor activity. To our knowledge, this is the first mechanistic analysis of the functional regulation from the SCN shell to the core, highlighting the critical role of the GABAergic network within the SCN. The Vgat deficiency in AVP neurons extended the activity time of locomotor activity and compressed the duration of high VIP-Ca 2+ accordingly to fit in the behavioral rest time. Conversely, the GABA A R deficiency in VIP neurons shortened the activity time by reducing morning and evening locomotor activities and lengthened the high VIP-Ca 2+ duration accordingly. Thus, GABAergic communication from AVP neurons to VIP neurons may account for the inverse correlation between the locomotor activity time and the high VIP-Ca 2+ duration. Besides, the optogenetic activation of AVP neurons increased VIP-Ca 2+ both in vivo and ex vivo during the night, which was shown to be inhibited by GABA A R antagonism in slices. Furthermore, baseline VIP-Ca 2+ rose upon GABA A R antagonism, and AVP neuronal activation reduced Ca 2+ in cells located in the SCN area between AVP and VIP neurons. Together with our previous observation that the daily AVP-Ca 2+ rhythm starts to rise slightly before the onset of VIP-Ca 2+ [ 12 ], these findings raise a model in which GABA released from AVP neurons activates VIP neurons indirectly by suppressing intermediate GABAergic neurons that inhibit VIP neurons, then VIP neurons in turn suppress the locomotor activity to set the rest time, probably indirectly via other neurons that regulate locomotion (S6 Fig). Therefore, the lack of GABA release from AVP neurons may fail to disinhibit VIP neurons in the evening and morning, leading to an earlier onset and later offset of the locomotor activity. Conversely, the lack of GABA A R signaling in VIP neurons may reduce their inhibition by the intermediate GABA neurons in the evening and morning, resulting in a later onset and earlier offset of the locomotor activity. It should be noted that not only neurons but also astrocytes may contribute as the intermediate GABA-releasing/regulating cells, as discussed later. Both the inhibitory and excitatory effects of GABA on SCN neurons have been reported, which may vary with the time of the day, area within the SCN, or photoperiod [ 14 , 37 – 40 ]. Therefore, we also considered the possibility that GABA from AVP neurons directly excited VIP neurons. However, the fact that gabazine application increased VIP-Ca 2+ in slices indicated that GABA inhibited most, if not all, VIP neurons. This observation is consistent with a previous study reporting that GABA has a predominantly inhibitory effect on the ventral SCN [ 37 ]. In addition, AVP neurons send only sparse projections directly onto VIP neurons [ 12 , 34 ]. On the other hand, there are many GABA neurons that receive abundant AVP fibers and densely project to VIP neurons between AVP-rich and VIP-rich regions in the SCN. Indeed, we confirmed the existence of cells in such intermediate regions that respond to AVP neuronal activation by reducing Ca 2+ . Collectively, the above-mentioned disinhibition pathway via the intermediate neurons is likely the primary regulatory pathway, although an accessory direct GABAergic pathway may exist. In addition, other transmitters, such as AVP, may play an additional role in bridging AVP neurons to VIP neurons. Whether the presumed intermediate neurons are specialized cells with a specific neurochemical character or a heterogeneous population remains unknown. GRP or calretinin neurons may be good candidates because they are GABA neurons located between AVP and VIP neurons, have contacts of AVP fibers, and make contacts onto VIP neurons [ 34 ]. On the other hand, because the SCN is full of many types of GABA neurons, the intermediate neurons may be more broadly distributed spatially and neurochemically. In terms of neuronal activity, at first glance, the presumed intermediate neurons are expected to be more active during the night to inhibit VIP neurons and to be inhibited by AVP neurons. However, MUA recordings have shown that SCN neurons are generally more active during the day, with a peak around midday [ 1 ], which apparently contradicts this expectation. Nevertheless, the phase of the firing rhythms of individual SCN neurons is distributed broadly, and indeed, the MUA begins to rise before the (subjective) day and still falls after the onset of the (subjective) night, covering dusk and dawn [ 25 , 41 , 42 ]. During these transitional time windows, disinhibition of VIP neurons by AVP neuronal GABA may be critical to set the locomotor activity onset and offset precisely. Indeed, the VIP-Ca 2+ was high at midday even when AVP neurons did not release GABA ( Avp-Vgat −/− ), and it was low at midnight even when VIP neurons did not have GABA A R ( Vip-GABA A R −/− ), indicating GABA-independent regulation of daily VIP neuronal activities around their peak and trough, which likely includes the TTFL-driven cellular clocks. Recent studies using organotypic SCN cultures have nicely demonstrated the critical role of astrocytes in circadian timekeeping by controlling the extracellular GABA levels through rhythmic GABA uptake, GABA release, and glutamate release to stimulate nerve terminal GABA release [ 19 , 20 , 43 ]. Importantly, astrocytes drive the daily extracellular GABA fluctuation, which is higher at night. Therefore, it is tempting to hypothesize that GABA released from AVP neurons somehow modulates astrocyte activity to reduce GABA levels around VIP neurons. Although most SCN neurons contain GABA and also express GABA A R, they are heterogeneous populations and form assymmetric neural and humoral networks, indicating functional differentiation among cell types. In particular, GABAergic communication between the SCN shell and core is likely to be far from symmetric [ 37 , 44 , 45 ]. Thus, it is essential to pay attention to the direction of GABAergic signaling under analysis when we investigate the function of GABA in the SCN central clock. To this end, Vgat has been deleted to eliminate GABA release specifically in VIP or AVP neurons; the former caused little effect [ 46 , 47 ], while the latter lengthened the activity time of the behavior rhythm [ 25 ]. However, until now there has been no means to inhibit GABA reception in a cell type-specific manner without detailed information on the subtype genes expressed in the cells of interest. Here, we achieved a complete cell type-specific blockade of GABA A R signaling by deleting all three subtype genes of the requisite β-subunit using in vivo genome editing. A similar strategy to simultaneously disrupt multiple subtype genes would be applicable to study the cell type-specific functions of other ion channels with many subtype members, such as AMPA receptors and potassium channels. Moreover, it is noteworthy that the current strategy of recording the VIP-Ca 2+ response to optogenetic stimulation of AVP neurons using a combination of Cre/tTA driver mice and Cre/tTA-dependent AAV vectors allowed us to investigate the GABAergic regulatory network in the direction from AVP neurons to VIP neurons. Although the locomotor activity times were altered, the free-running periods did not change in either Avp-Vgat −/− or Vip-GABA A R −/− mice. Other reported mice with genetic impairments of VGAT or GABA A R also showed free-running periods in the normal range, whereas the amplitudes of the circadian rhythm were reduced to varying degrees [ 48 – 50 ]. We previously showed that clock gene expression rhythms do not alter significantly in either the shell or core of the SCN in Avp-Vgat −/− mice, suggesting that the TTFLs tick normally in the absence of GABA release of AVP neurons [ 25 ]. Thus, AVP neuronal GABA may modify the temporal pattern of VIP neuronal activity with little effect on the TTFL. On the other hand, AVP neurons have been suggested to function as the primary pacesetter cells to determine the SCN ensemble period, which does not require GABA from these neurons [ 10 – 12 , 25 ]. Therefore, AVP neurons may use GABA to set timers that control the phase relationship of evening and morning locomotor activities on their cellular clocks. Thus, AVP neurons may regulate the pacesetting of the SCN ensemble rhythm and the phase-setting of the evening and morning locomotor activities via partially independent transmitter systems. Materials and Methods Animals All experimental procedures were approved by the Kanazawa University Animal Experiment Committee and the Kanazawa University Safety Committee for genetic recombinant experiments. Avp-Cre BAC transgenic (C57BL/6J- Tg(Avp-icre)#Meid /Rbrc, RBRC12048) [ 11 ] and Vip-tTA knock-in (B6(Cg)- Vip em1(tTA2)Miem /Rbrc, RBRC12109) [ 26 ] mice were reported previously. Vip-ires-Cre ( Vip tm1(cre)Zjh / J, JAX:010908) [ 33 ], Vgat flox ( Slc32a1 tm1Lowl /J, JAX:012897) [ 51 ], and Rosa26-LSL-SpCas9-2A-EGFP mice (B6J.129(B6N)- Gt(ROSA)26Sor tm1(CAG-cas9*,-EGFP)Fezh /J, JAX:026175) [ 32 ] were obtained from Jackson Laboratory. All lines were congenic on C57BL/6. We compared the conditional knockouts with controls whose genetic backgrounds were comparable. Avp-Cre , Vip-ires-Cre , Vip-tTA , and Rosa26-LSL-SpCas9-2A-EGFP mice were used in hemizygous or heterozygous condition. We used both male and female mice in our experiment. Mice were maintained under a strict 12-h light/12-h dark cycle in a temperature- and humidity-controlled room and fed ad libitum. Viral vector and surgery The AAV-2 ITR containing plasmids pGP-AAV-CAG-FLEX-jGCaMP7s-WPRE (Addgene plasmid #104495, a gift from Dr. Douglas Kim and GENIE Project) [ 27 ]. pAAV - TRE-jGCaMP7s was described previously [ 52 ]. In addition, we modified this plasmid to make an improved version by using an EcoRI-HindIII fragment of this plasmid containing jGCaMP7s sequence to replace an EcoRI-HindIII fragment containing ChrimsonR-mCherry from pAAV-TRE-ChrimsonR-mCherry (Addgene #92207, a gift from Alice Ting)[ 36 ]. pAAV-U6-gGabrb1∼3-EF1α-DIO-mCherry , plasmids for CRISPR-Cas9-mediated Gabrb1∼3 gene disruption, was generated as follows. The target sites for CRISPR-Cas9 were designed by CRISPOR ( http://crispor.tefor.net/ ) [ 53 ]. Two sequences targeting each Gabrb gene were selected: Gabrb1 , 5’-AAGGATATGACATTCGCTTG- 3’ and 5’-CGCATCCCGACGTCCACCGG-3’; Gabrb2 , 5’-TGACCCTAGTAATATGTCGC-3’ and 5’-ATGTTCATTCCTACGGCCAC-3’; Gabrb3 , 5’-ATTCGCCTGAGACCCGACTT-3’ and 5’-CGACATCGCCAGCATCGACA-3’. Oligonucleotides encoding the guide sequences were cloned into the BbsI and BsaI sites of pX333 (Addgene #64073, a gift from Dr. Andrea Ventura) [ 54 ]. Then, a fragment containing two tandem units of U6-gRNA was amplified by PCR, using the following primers: 5’-agtacgcgTCGAGCATGCTCGAGAATGG-3’ and 5’-agtacgcgtCGGGTACCCCATTTGTCTGC-3’, and cloned into the MluI site of pAAV-EF1a-DIO-mCherry (a gift from Dr. Bryan Roth) as described previously [ 52 ]. pAAV-U6-gGabrb2-EF1α-DIO-mCherry happened to contain two copies of the amplified fragment. pAAV-U6-gControl-EF1α-DIO-mCherry contains spacer sequences from pX333 (Addgene plasmid #64073, a gift from Dr. Andrea Ventura) instead of gRNA sequences for Gabrb3 genes. pAAV - CAG-FLEX-ChrimsonR-mCherry was described previously (9). As a negative control for the optogenetic study, we injected AAV- EF1α-DIO-mCherry or AAV- EF1α-DIO-hM3Dq-mCherry , which was generated with plasmids pAAV-EF1α-DIO-mCherry or pAAV-EF1α-DIO-hM3Dq-mCherry provided by Dr. Bryan Roth, University of North Carolina [ 55 ]. pAAV - TRE-FLEXoff-jGCaMP7s was made by replacing an EcoRI-SpeI fragment containing ChrimsonR-mCherry of pAAV-TRE-ChrimsonR-mCherry with an EcoRI-XbaI fragment containing jGCaMP7s from pGP-AAV-CAG-FLEX-jGCaMP7s-WPRE . pAAV - EF1-rDIO-jGCaMP7s was made by replacing an AscI-NheI fragment containing ChR2-EYFP of pAAV-DIO-hChR2(H134R)-EYFP-WPRE-pA (provided by Dr. Karl Deisseroth, Stanford University) with a jGCaMP7s cDNA fragment amplified by PCR from pGP-AAV-CAG-FLEX-jGCaMP7s-WPRE, using the following primers: 5’-ataggcgcGCCACCATGGGTTCTCATCA-3’ and 5’-gcgactagTCACTTCGCTGTCATCATTTG-3’. Note that gene expression from AAV- TRE-FLEXoff-jGCaMP7s and AAV - EF1-rDIO-jGCaMP7s stops upon Cre-mediated recombination. Recombinant AAV vectors (AAV2-rh10) were produced using a triple- transfection, helper-free method and purified as described previously [ 11 ]. The titers of recombinant AAV vectors were determined by quantitative PCR: AAV- CAG-DIO-jGCaMP7s , 3.4 × 10 13 ; AAV- TRE-jGCaMP7s , 6.3 × 10 11 ; AAV- TRE-jGCaMP7s (improved), 5.8 × 10 12 ; AAV- TRE-FLEXoff-jGCaMP7s , 1.48 × 10 13 ; AAV- EF1-rDIO-jGCaMP7s , 1.5 × 10 13 ; AAV -U6-gGabrb1-EF1α-DIO-mCherry , 6.06 × 10 12 ; AAV -U6-gGabrb2-EF1α-DIO-mCherry , 7.42 × 10 12 ; AAV -U6-gGabrb3-EF1α-DIO-mCherry , 6.56 × 10 12 ; AAV- U6-gControl-EF1α-DIO-mCherry , 2.6 × 10 12 ; AAV- EF1α-DIO-hM3Dq-mCherry , 4.5 × 10 12 ; AAV- CAG-FLEX-ChrimsonR-mCherry , 1.5 × 10 13 ; and AAV- EF1α-DIO-mCherry , 5.2 × 10 12 genome copies/ml. Stereotaxic injection of AAV vectors was performed as described previously [ 11 ]. Two weeks after surgery, we began monitoring the mice for their locomotor activity. In vivo fiber photometry We used 8 Avp-Vgat −/− × Vip-tTA ( Avp-Cre; Vgat flox/flox ; Vip wt/tTA ) mice, 5 control mice ( Avp-Cre; Vgat wt/flox ; Vip wt/tTA ) and 12 Vip-ires-Cre; Rosa26-LSL-SpCas9-2A-EGFP mice for the GABA A R disruption study (5 or 7 for gGabrb1-3 or gControl ). The mice were anesthetized by administering a cocktail of medetomidine (0.3 mg/kg), midazolam (4 mg/kg), and butorphanol (5 mg/kg) and were secured at the stereotaxic apparatus (Muromachi Kikai). Lidocaine (1%) was applied for local anesthesia before making the surgical incision. We drilled small hole in the exposed region of the skull using a dental drill. We injected 0.5 to 1.0 μL of the virus (AAV -U6-gGabrb1-3-EF1α-DIO-mCherry ; AAV-CAG-FLEX-jGCaMP7s mixed (gGabrb1: gGabrb2: gGabrb3: jGCaMP7s=1:1:1:2) or AAV -U6-gControl-EF1α-DIO-mCherry ; AAV-CAG-FLEX-jGCaMP7s mixed or AAV-TRE-jGCaMP7s ) (flow rate = 0.1 μL/min) at the right or bilateral of SCN (posterior: 0.5 mm, lateral: 0.25 mm, depth: 5.7 mm from the bregma) with a 33 G Hamilton Syringe (1701RN Neuros Syringe, Hamilton) to label VIP neurons. Subsequently, we placed an implantable optical fiber (400 μm core, N.A. 0.39, 6 mm, ferrule 2.5 mm, FT400EMT-CANNULA, Thorlabs or RWD) above the SCN (posterior: 0.2 mm, lateral: 0.2 mm, depth: 5.2 to 5.4 mm from the bregma) with self-adhesive resin cement (Super-bond C&B, Sun Medical or RelyX TM Unicem2 Automix, 3M ESPE). The cement was painted black. Atipamezole (0.3 mg/kg) was administered postoperatively to reduce the anesthetized period. The mice were used for experiments 2 to 8 weeks after the virus injection and optical fiber implantation. Their ages ranged from 3 to 14 months old, including both males and females. A single-color fiber photometry system (COME2-FTR, Lucir) was used to record the Ca 2+ signal of SCN neurons in freely moving Avp-Vgat −/− mice ( Figs 1 and S1) [ 12 , 56 ]. Fiber-coupled LED (M470F3, Thorlabs) with LED Driver (LEDD1B, Thorlabs) was used as an excitation blue light source. The light was reflected by a dichroic mirror (495 nm), went through an excitation bandpass filter (472/30 nm), and then to the animal via a custom-made patch cord (400 um core, N.A. 0.39, ferrule 2.5 mm, length 50 cm, COME2-FTR/MF-F400, Lucir) and the implanted optical fiber. We detected the jGCaMP7s fluorescence signal by a photomultiplier through the same optical fibers and an emission bandpass filter (520/36 nm); furthermore, we recorded the signal using Power Lab (AD Instruments) with Lab Chart 8 software (AD Instruments). The excitation blue light intensity was 10 to 30 μW at the tip of the patch cord of the animal side. We recorded the same for 30 s every 10 min for 2 weeks to reduce photobleaching. During the recording, the mouse was housed in a 12-h light-dark cycle for more than 5 days (LD condition) and then moved to continuous darkness for approximately 10 days (DD condition) in a custom-made acrylic cage surrounded by a sound-attenuating chamber. A rotary joint for the patch cord was stopped during the recording to prevent artificial baseline fluctuation. The animal’s locomotor activity was monitored using an infrared sensor (Supermex PAT.P and CompACT AMS Ver. 3, Muromachi Kikai). The detected GCaMP signal was averaged within a 30-s session [ 25 ]. To detrend the gradual decrease of the signal during recording days, ±12 h average from the time (145 points) was calculated as baseline (F). The data were subsequently detrended by the subtraction of F (ΔF). Then, the ΔF/F value was calculated. To determine VIP-Ca 2+ onset and VIP-Ca 2+ offset, ΔF/F were smoothened with a 21-point moving average, then the local maximum and minimum time points within one day were determined. Then, the time points crossing the 50% amplitude (ΔF/F maximum – minimum value) were defined as VIP-Ca 2+ onset and offset, respectively. The middle of the time points between the VIP-Ca 2+ onset and offset were defined as the VIP-Ca 2+ midpoint. Additionally, the intervals between VIP-Ca 2+ onset and offset were defined as high Ca 2+ duration. A double-plotted actogram of jGCaMP7s signal was designed by converting all ΔF to positive values by subtracting the minimum value of ΔF. Subsequently, these values were multiplied by 100 or 1,000 and rounded off. The plots were made via ClockLab (Actimetrics) with normalization in each row. Another dual-color fiber photometry system (FP3002, Neurophotometrics) was used to record the calcium signal of SCN neurons in freely moving Vip-GABA A R −/− mice ( Figure 3 ) [ 12 , 57 , 58 ]. Excitation light sources were a 470-nm LED for detecting calcium-dependent jGCaMP7s fluorescence signal (F470) and a 415-nm LED for calcium-independent isosbestic fluorescence signal (F415). The duration of excitation lights is 50 ms, and the onsets of the excitation timing of LEDs were interleaved. The lights passed through excitation bandpass filters, dichroic mirrors, and then to the animal via fiber-optic patch cords (BBP(4)_400/440/900-0.37_1m_FCM-4xFCM_LAF, MFP_400/440/LWMJ-0.37_1m_FCM-ZF2.5_LAF, Doric Lenses) and the implanted optical fiber. Subsequently, both signals were detected using a CMOS camera through the optical fibers, dichroic mirrors, and emission bandpass filters. The recorded signals were acquired using Bonsai software, with a sampling rate of 10 Hz for each color. The excitation intensities of the 470-nm and 415-nm LED at the animal side’s patch cord tip were from 60 μW to 110 μW. We recorded the same for 30 s every 10 min for 3 weeks to reduce photobleaching. The detected GCaMP signal was averaged within a 30-s session. Ratio (R) was defined as the ratio between F470 and F415 (F470/F415) for calibration and reducing motion artifacts. To detrend the gradual decrease of the signal during recording days, ±12 h average from the time (145 points) was calculated as baseline (R 0 ). The data were subsequently detrended by the subtraction of R 0 (ΔR). Then, the ΔR/ R 0 value was calculated. To determine VIP-Ca 2+ onset and VIP-Ca 2+ offset, ΔR/R 0 were smoothened with a 21-point moving average, then the local maximum and minimum time points within one day were determined. Then, the time points crossing the 50% amplitude (ΔR/R 0 maximum – minimum value) were defined as VIP-Ca 2+ onset and offset, respectively. The middle of the time points between the VIP-Ca 2+ onset and offset were defined as the VIP-Ca 2+ midpoint. Additionally, the intervals between midpont phases were defined as the periods [ 11 ] and the intervals between VIP-Ca 2+ onset and offset were defined as high Ca 2+ duration. A double-plotted actogram of GCaMP signal was designed by converting all R values. The plots were made via ClockLab (Actimetrics). Ca 2+ activity profile analyses were performed via ClockLab (Actimetrics). Before analyzing with the ClockLab, all R or ΔF values were normalized by subtracting the minimum value of R or ΔF, dividing the result by the difference between the maximum and minimum R or ΔF values, and then multiplying by 100 or 1000. Subsequently, the last 5 days in the LD condition and last 5 or 10 days in the DD condition were selected using ClockLab for Ca 2+ activity profile analysis. The resulting profiles were further normalized in the same way to generate the final Ca 2+ activity profile. CT12 was determined as the onset of locomotor activity. VIP-Ca 2+ CT0 was determined as the onset of VIP-Ca 2+ activity. High Ca 2+ duration (20% amplitude) was the interval between VIP-Ca 2+ onset and offset defined as the time points crossing the 20% amplitude of normalized calcium activity profile with VIP-Calcium circadian time, rather than 50% as described above. During the fiber photometry recordings, the animal’s locomotor activity was monitored using an infrared sensor (Supermex PAT.P and CompACT AMS Ver. 3, Muromachi Kikai) in 1-min bins, then 10-min bins were made by analysis. A double-plotted actogram of locomotor activity was also prepared and overlaid on that of the GCaMP. The onset and offset of locomotor activity were determined using the actogram of locomotor activity. Initially, we attempted to automatically detect the onset and offset; however, it was followed by a manual visual inspection and modifications by the experimenter. The intervals between locomotor activity onset and offset were defined as locomotor activity time. We confirmed the jGCaMP7s or gRNA (mCherry) expression and the position of the optical fiber by slicing the brains into 30 or 100 μm coronal sections using a cryostat (Leica). The sections were mounted on glass slides with a mounting medium (VECTASHIELD HardSet with DAPI, H-1500, Vector Laboratories or Dako Fluorescence Mounting Medium, Agilent Technologies) and observed via epifluorescence microscope (KEYENCE, BZ-9000E). Behavioral analyses Male and female Vip-GABA A R −/− ( Vip-ires-Cre; Rosa26-LSL-SpCas9-2A-EGFP injected bilaterally with AAV -U6-gGabrb1∼3-EF1α-DIO-mCherry mixture) and control (injected with control AAV) ( Figure 2C ) mice, aged 8 to 20 weeks, were housed individually in a cage placed in a light-tight chamber (light intensity was approximately 100 lux). Spontaneous locomotor activity (home-cage activity) was monitored by infrared motion sensors (O’Hara) in 1-min bins as described previously [ 12 ]. Actogram, activity profile, and χ 2 periodogram analyses were performed via ClockLab (Actimetrics). The free-running period and amplitude (Qp values) were calculated for the last 10 days in constant darkness (DD) by periodogram. The onset of locomotor activity, defined as CT12, was calculated from the daily activity profile of the same 10 days in DD using the median activity level as a threshold for onset detection ( Fig 2D and 2E ). The onset and offset of locomotor activity used to calculate the locomotor activity time ( Fig 2F ) were determined using the actogram. Initially, we attempted to automatically detect the onset and offset; however, it was followed by a manual visual inspection, along with modifications by the experimenter. In vivo optogenetic stimulation with fiber photometry We used 10 Avp-Cre; Vip-tTA mice ( n = 6 for ChrimsonR, n = 4 for control, both male and female). Some animals in this section are from a previously used cohort [ 12 ]. We injected 1.0 μL of the mixture of viruses (AAV- TRE-jGCaMP7s with AAV- CAG - Flex-ChrimsonR-mCherry or AAV- EF1α - DIO-hM3Dq-mCherry and AAV- TRE-jGCaMP7s ) into the right SCN (posterior: 0.5 mm, lateral: 0.25 mm, depth: 5.7 mm from the bregma) and then implanted an optical fiber (400 μm core, N.A. 0.39, 6 mm, Thorlabs) above the SCN (posterior: 0.2 mm, lateral: 0.2 mm, depth: 5.3 mm from the bregma) with dental cement. The mice were used for experiments more than 2 weeks after the surgery. The dual-color fiber photometry system (FP3002, Neurophotometrics) was used to record the Ca 2+ signal of SCN neurons with optogenetic stimulation in freely moving mice, as described above in “In vivo fiber photometry” ( Fig 4 ) [ 57 , 58 ]. The excitation intensities of the 470-nm and 415-nm LEDs at the tip of patch cord on the animal side ranged 130 μW and 90μW, respectively. Additionally, a 635-nm red laser (inside the fiber photometry system FP3002) was transmitted through the same optical fibers with an intensity of 2 mW. During the 720-s recording every 2 h, optical stimulation (635 nm, 50 ms pulse, 5 Hz, 120 s, 600 pulses) was applied in the middle of the recording. Throughout the experiment, the mice were housed in a custom-made acrylic cage surrounded by a sound-attenuating chamber and maintained in a 12-h LD cycle. The recorded data were interleaved to eliminate artifacts caused by red laser stimulation, and half of it was discarded. Consequently, the final sampling rate for the jGCaMP7s fluorescence signals at the 470-nm light excitation (F470) and the 415-nm excitation (F415) was 5 Hz. Ratio (R) was defined as the ratio between F470 and F415 (F470/F415) for calibration and reducing motion artifacts. The baseline ratio (R0) is the mean R-value of the pre-stimulation period (-30 s - 0 s). ΔR is the difference between the mean R-value of the late phase during the stimulation period (90 s - 120 s, R1) and R0. After the recordings were completed, we confirmed the jGCaMP7s and ChrimsonR- mCherry expressions and the position of the optical fiber by histology. Slice electrophysiology Vip-GABA A R -/- (made as described above) mice were compared to control ( Vip-ires-Cre; Rosa26-LSL-Cas9-2A-EGFP with AAV-EF1a-DIO-mCherry injected, 1.0 μL bilateral of SCN) mice. Both male and female mice aged 8 to 20 weeks were used. Coronal brain slices (250 µm thick), including the SCN, were prepared with a linear-slicer (NLS-MT, Dosaka EM), as described previously [ 59 ]. Under an upright fluorescence microscope (Olympus, BX51WI), we visually identified VIP neurons with EGFP fluorescence in the ventral SCN and non-VIP neurons without the fluorescence in the dorsal SCN. The gRNA AAV-infected VIP neurons were identified by additional mCherry fluorescence. For recording of non-glutamatergic spontaneous postsynaptic currents (PSCs), the slices were continuously perfused with an artificial CSF (ACSF) with the following composition (in mM): 125 NaCl, 2.5 KCl, 2 CaCl2, 1 MgSO4, 26 NaHCO3, 1.25 NaH2PO4, 10 d-glucose, equilibrated with 95% O2 and 5% CO2, kept at 31 ± 1 °C, and further mixed with 10 μM 6-cyano-7-nitroquinoxaline-2,3 dione disodium (CNQX) and 25 μM D-(-)-2-amino-5-phosphonopentanoic acid (D-AP5) to block fast glutamatergic transmission. Whole-cell voltage-clamp recordings were made at −60 mV with borosilicate glass electrodes (5–6 MΩ) filled with an internal solution containing the following components (in mM): 87 CsCl, 20 TEA-Cl 10 HEPES, 10 EGTA, 0.5 CaCl2, 4 MgCl2, 2 QX314, 4 Na-ATP, 0.4 Na-GTP, and 10 phosphocreatine (pH 7.3, adjusted with CsOH). A combination of an EPC10/2 amplifier and Patchmaster software (HEKA) was used to control membrane voltage and data acquisition. Series resistance was compensated routinely by 80%. The non-glutamatergic PSCs completely disappeared in the presence of 10 μM gabazine (SR95531, Tocris), showing that they were mediated by GABA A receptors [ 25 ]. The GABAergic PSCs recorded for 1-3 min were analyzed using the MiniAnalysis program (Synaptosoft). The events were picked by an amplitude threshold of 5 pA and confirmed visually to have a typical PSC waveform. Ex vivo optogenetic stimulation and calcium imaging We used 9 Avp-Cre; Vip-tTA mice ( n = 6 for ChrimsonR, n = 3 for control) for VIP-Ca 2+ recording and 5 Avp-Cre mice for recording Ca 2+ in non-AVP neurons (aged 8 to 20 weeks, including both males and females). We injected 0.6 μL of the mixture of viruses (VIP-Ca 2+ : AAV- TRE-FLEXoff-jGCaMP7s with AAV- CAG - Flex-ChrimsonR-mCherry or AAV- EF1α - DIO-mCherry, non-AVP-Ca 2+ : AAV- EF1α-rDIO-jGCaMP7s with AAV- CAG-Flex-ChrimsonR-mCherry ) into the SCN bilateral (posterior: 0.5 mm, lateral: 0.25 mm, depth: 5.7 mm from the bregma). Coronal brain slices (300 µm thick), including the SCN, were prepared as described in “Slice electrophygiology.” For this experiment, mice were sacrificed at ZT19–20 in darkness under a red light. The slices were placed in an imaging chamber and continuously perfused with the ACSF. Before the recordings started around projected ZT22, the slices were pre-incubated in the experimental environment for at least one hour, which was critical to observe VIP-Ca 2+ response to the optogenetic stimulation. Under the upright fluorescence microscope (Olympus, BX51WI), we visually identified AVP neurons with ChrimsonR-mCherry or mCherry (control) fluorescence in the dorsomedial SCN and VIP neurons with jGCaMP7s fluorescence in the ventral SCN. We imaged jGCaMP7s fluorescence every 5 sec through an optical filter set (an excitation filter 470-495 nm, a dichroic mirror 505 nm and an emission filter 510- 550 nm) and a digital CMOS camera (Prime BSI Express, Teledyne Vision Solutions) with MetaFluor software. After the GCaMP signal had stabilized, we optogenetically stimulated SCN AVP neurons for 2 min (617 nm, 10 Hz, 40 ms duration) through an optic fiber (200 μm core, N.A. 0.39, 6 mm, ferrule 1.25 mm, FT200EMT-CANNULA; Thorlabs) positioned near the AVP neurons and connected to a high-powered LED under the control of Digital Stimulator (WPI DS8000B) and LED driver (THORLABS). Gabazine (10 μM) was applied to ACSF to verify GABA A R involvement. For VIP-Ca 2+ , we measured the fluorescence values of the entire jGCaMP7s-expressing ROIs using MetaFluor software, detrended the traces by division, and calculated the change in fluorescence over baseline fluorescence (ΔF/F 0 %) as (Fi-F 0 ) * 100 / F 0 . The average F values 1 or 2 min before the optogenetic stimulation or blockers perfusion were defined as the baseline (F 0 ). For non-AVP-Ca 2+ , we selected unilateral SCN regions (150 x 260 pixels, 2.6 μm/pixel) for further analysis. Images were analyzed using MATLAB. ΔF/F 0 % values of individual pixels were calculated as described above. The resolution of images was then adjusted to 20.8 μm/pixel, and square regions of interest (ROIs, 4 × 4 pixels) were defined in the middle and ventral of the SCN. M P0 and M T0 are the highest and lowest ΛF/F 0 % values, respectively, of the pre-stimulation period (-60 s ∼ 0 s) in the middle region. M P and M T are the highest and lowest ΛF/F 0 % values during the stimulation period (0 s ∼ 120 s). Correspondingly, V P0 , V T0 , V P , and V T refer to the respective points in the ventral region. Statistical analysis All results are expressed as mean ± SEM. For comparisons of two groups, two-tailed Student’s t test or Welch’s t test was performed. For comparisons of multiple groups with no difference of variance, two-way repeated measures ANOVA followed by post hoc two-tailed Student’s t-test were performed. For comparisons of multiple groups with difference of variance, nonparametric tests, Kruskal–Wallis test with post hoc Dunn’s test were performed. All P values less than 0.05 were considered as statistically significant. Only relevant information from the statistical analysis was indicated in the text and figures. Author contributions Y.P., Y.T. and M.M. designed research; Y.P., Y.T., T.M., M.W., and A.M. performed research; Y.P., Y.T., T.M., and M.W. analyzed data; Y.P., Y.T., and M.M. wrote the paper. Declaration of interests All authors declare they have no competing interests. Supporting Information S1 Fig. Circadian rhythms of the behavior and VIP-Ca 2+ in Avp-Vgat −/− mice S2 Fig. Disruption of GABA A R in SCN VIP neurons by in vivo genome editing S3 Fig. Circadian rhythms of the behavior and VIP-Ca 2+ in Vip-GABA A R −/− mice used for fiber photometry recordings S4 Fig. Response of VIP-Ca 2+ to the optogenetic activation of AVP neurons in SCN slices S5 Fig. Response of non-AVP cells to the optogenetic activation of AVP neurons in coronal SCN slices S6 Fig. A model of how the GABAergic network from AVP neurons to VIP neurons in the SCN sets the activity/rest time of the circadian behavior rhythm Acknowledgements We thank H. Okamoto for the Avp-Cre mouse; S. Horike and T. Daikoku for the Vip-tTA mouse; Z. J. Huang for the Vip-ires-Cre mouse; F. Zhang for the Rosa26-LSL-SpCas9-2A-EGFP mouse; B.B. Lowell for the Vgat flox mouse; Penn Vector Core for pAAV2-rh10 ; D. Kim & GENIE Project for pGP-AAV-CAG-FLEX-jGCaMP7s-WPRE ; A. Ting for pAAV-TRE-ChrimsonR-mCherry ; A. Ventura for pX333 ; B. Roth for pAAV-EF1a-DIO-mCherry and pAAV-EF1α-DIO-hM3Dq-mCherry ; and K. Deisseroth for pAAV-DIO-hChR2(H134R)-EYFP-WPRE-pA ; We thank all lab members, including M. Kawabata and Y. Nishiwaki. This work was supported in part by JSPS KAKENHI Grant Numbers JP24KJ1189 (Y.P.); JP23K06345 (Y.T.); JP22K20738 (A.M.); JP24K02137 (T.M.); JP23K24064; JP25K02440; the Takeda Science Foundation; the Terumo Life Science Foundation; the Research Foundation for Opto-Science and Technology; the Koyanagi Foundation (M.M.); and JST SPRING Grant Number JPMJSP2135 (M.W., Y.P.). References 1. ↵ Hastings MH , Maywood ES , Brancaccio M . Generation of circadian rhythms in the suprachiasmatic nucleus . Nat Rev Neurosci . 2018 ; 19 : 453 – 469 . doi: 10.1038/s41583-018-0026-z OpenUrl CrossRef PubMed 2. ↵ Welsh DK , Takahashi JS , Kay SA . Suprachiasmatic Nucleus: Cell Autonomy and Network Properties . Annu Rev Physiol . 2010 ; 72 : 551 – 577 . doi: 10.1146/annurev-physiol-021909-135919 OpenUrl CrossRef PubMed Web of Science 3. ↵ Aton SJ , Colwell CS , Harmar AJ , Waschek J , Herzog ED . Vasoactive intestinal polypeptide mediates circadian rhythmicity and synchrony in mammalian clock neurons . Nat Neurosci . 2005 ; 8 : 476 – 483 . doi: 10.1038/nn1419 OpenUrl CrossRef PubMed Web of Science 4. Harmar AJ , Marston HM , Shen S , Spratt C , West KM , Sheward WJ , et al. The VPAC2 Receptor Is Essential for Circadian Function in the Mouse Suprachiasmatic Nuclei . Cell . 2002 ; 109 : 497 – 508 . doi: 10.1016/S0092-8674(02)00736-5 OpenUrl CrossRef PubMed Web of Science 5. Colwell CS , Michel S , Itri J , Rodriguez W , Tam J , Lelievre V , et al. Disrupted circadian rhythms in VIP- and PHI-deficient mice. American Journal of Physiology-Regulatory , Integrative and Comparative Physiology . 2003 ; 285 : R939 – R949 . doi: 10.1152/ajpregu.00200.2003 OpenUrl CrossRef PubMed Web of Science 6. ↵ Maywood ES , Reddy AB , Wong GKY , O’Neill JS , O’Brien JA , McMahon DG , et al. Synchronization and Maintenance of Timekeeping in Suprachiasmatic Circadian Clock Cells by Neuropeptidergic Signaling . Current Biology . 2006 ; 16 : 599 – 605 . doi: 10.1016/j.cub.2006.02.023 OpenUrl CrossRef PubMed Web of Science 7. ↵ Collins B , Pierre-Ferrer S , Muheim C , Lukacsovich D , Cai Y , Spinnler A , et al. Circadian VIPergic Neurons of the Suprachiasmatic Nuclei Sculpt the Sleep-Wake Cycle . Neuron . 2020 ; 108 : 486 – 499 .e5. doi: 10.1016/j.neuron.2020.08.001 OpenUrl CrossRef PubMed 8. van der Elst J , De Greve J , Geerts F , De Neve W , Storme G , Willems G . Quantitative study of liver metastases from colon cancer in rats after treatment with cyclosporine A . J Natl Cancer Inst . 1986 ; 77 : 227 – 32 . OpenUrl PubMed Web of Science 9. ↵ Paul S , Hanna L , Harding C , Hayter EA , Walmsley L , Bechtold DA , et al. Output from VIP cells of the mammalian central clock regulates daily physiological rhythms . Nat Commun . 2020 ; 11 : 1453 . doi: 10.1038/s41467-020-15277-x OpenUrl CrossRef PubMed 10. ↵ Mieda M , Okamoto H , Sakurai T . Manipulating the Cellular Circadian Period of Arginine Vasopressin Neurons Alters the Behavioral Circadian Period . Current Biology . 2016 ; 26 : 2535 – 2542 . doi: 10.1016/J.CUB.2016.07.022 OpenUrl CrossRef PubMed 11. ↵ Mieda M , Ono D , Hasegawa E , Okamoto H , Honma Kichi , Honma S , et al. Cellular Clocks in AVP Neurons of the SCN Are Critical for Interneuronal Coupling Regulating Circadian Behavior Rhythm . Neuron . 2015 ; 85 : 1103 – 1116 . doi: 10.1016/J.NEURON.2015.02.005 OpenUrl CrossRef PubMed 12. ↵ Tsuno Y , Peng Y , Horike S , Wang M , Matsui A , Yamagata K , et al. In vivo recording of suprachiasmatic nucleus dynamics reveals a dominant role of arginine vasopressin neurons in circadian pacesetting . PLoS Biol . 2023 ; 21 : e3002281 . doi: 10.1371/journal.pbio.3002281 OpenUrl CrossRef PubMed 13. ↵ Mieda M . The central circadian clock of the suprachiasmatic nucleus as an ensemble of multiple oscillatory neurons . Neurosci Res . 2020 ; 156 : 24 – 31 . doi: 10.1016/j.neures.2019.08.003 OpenUrl CrossRef PubMed 14. ↵ Ono D , Honma Kichi , Yanagawa Y , Yamanaka A , Honma S. Role of GABA in the regulation of the central circadian clock of the suprachiasmatic nucleus . Journal of Physiological Sciences . 2018 ; 68 : 333 – 343 . doi: 10.1007/S12576-018-0604-X/FIGURES/4 OpenUrl CrossRef 15. ↵ Liu C , Reppert SM . GABA synchronizes clock cells within the suprachiasmatic circadian clock . Neuron . 2000 ; 25 : 123 – 128 . doi: 10.1016/S0896-6273(00)80876-4 OpenUrl CrossRef PubMed Web of Science 16. ↵ Freeman GM , Krock RM , Aton SJ , Thaben P , Herzog ED . GABA networks destabilize genetic oscillations in the circadian pacemaker . Neuron . 2013 ; 78 : 799 – 806 . doi: 10.1016/j.neuron.2013.04.003 OpenUrl CrossRef PubMed Web of Science 17. Albus H , Vansteensel MJ , Michel S , Block GD , Meijer JH . A GABAergic mechanism is necessary for coupling dissociable ventral and dorsal regional oscillators within the circadian clock . Curr Biol . 2005 ; 15 : 886 – 93 . doi: 10.1016/j.cub.2005.03.051 OpenUrl CrossRef PubMed Web of Science 18. ↵ Evans JA , Leise TL , Castanon-Cervantes O , Davidson AJ . Dynamic interactions mediated by nonredundant signaling mechanisms couple circadian clock neurons . Neuron . 2013 ; 80 : 973 – 83 . doi: 10.1016/j.neuron.2013.08.022 OpenUrl CrossRef PubMed Web of Science 19. ↵ Patton AP , Morris EL , McManus D , Wang H , Li Y , Chin JW , et al. Astrocytic control of extracellular GABA drives circadian timekeeping in the suprachiasmatic nucleus . Proc Natl Acad Sci U S A . 2023 ; 120 : e2301330120 . doi: 10.1073/PNAS.2301330120/SUPPL_FILE/PNAS.2301330120.SM05.AVI OpenUrl CrossRef PubMed 20. ↵ Brancaccio M , Patton AP , Chesham JE , Maywood ES , Hastings MH . Astrocytes Control Circadian Timekeeping in the Suprachiasmatic Nucleus via Glutamatergic Signaling . Neuron . 2017 ; 93 : 1420 – 1435 .e5. doi: 10.1016/j.neuron.2017.02.030 OpenUrl CrossRef PubMed 21. ↵ Ness N , Díaz-Clavero S , Hoekstra MMB , Brancaccio M. Rhythmic astrocytic GABA production synchronizes neuronal circadian timekeeping in the suprachiasmatic nucleus . EMBO J . 2024 ; 44 : 356 – 381 . doi: 10.1038/s44318-024-00324-w OpenUrl CrossRef PubMed 22. ↵ Wojcik SM , Katsurabayashi S , Guillemin I , Friauf E , Rosenmund C , Brose N , et al. A Shared Vesicular Carrier Allows Synaptic Corelease of GABA and Glycine . Neuron . 2006 ; 50 : 575 – 587 . doi: 10.1016/J.NEURON.2006.04.016/ASSET/EC262458-E959-42D3-98BE-F8A732266890/MAIN.ASSETS/GR8.JPG OpenUrl CrossRef PubMed Web of Science 23. ↵ Ono D , Honma K , Yanagawa Y , Yamanaka A , Honma S . GABA in the suprachiasmatic nucleus refines circadian output rhythms in mice . Commun Biol . 2019 ; 2 : 232 . doi: 10.1038/s42003-019-0483-6 OpenUrl CrossRef PubMed 24. ↵ Bussi IL , Neitz AF , Sanchez REA , Casiraghi LP , Moldavan M , Kunda D , et al. Expression of the vesicular GABA transporter within neuromedin S + neurons sustains behavioral circadian rhythms . Proceedings of the National Academy of Sciences . 2023 ; 120 . doi: 10.1073/pnas.2314857120 OpenUrl CrossRef PubMed 25. ↵ Maejima T , Tsuno Y , Miyazaki S , Tsuneoka Y , Hasegawa E , Islam MT , et al. GABA from vasopressin neurons regulates the time at which suprachiasmatic nucleus molecular clocks enable circadian behavior . Proc Natl Acad Sci U S A . 2021 ; 118 : e2010168118 . doi: 10.1073/PNAS.2010168118/SUPPL_FILE/PNAS.2010168118.SAPP.PDF OpenUrl Abstract / FREE Full Text 26. ↵ Peng Y , Tsuno Y , Matsui A , Hiraoka Y , Tanaka K , Horike SI , et al. Cell Type-Specific Genetic Manipulation and Impaired Circadian Rhythms in Vip tTA Knock-In Mice . Front Physiol . 2022 ; 13 : 895633 . doi: 10.3389/FPHYS.2022.895633/BIBTEX OpenUrl CrossRef 27. ↵ Dana H , Sun Y , Mohar B , Hulse BK , Kerlin AM , Hasseman JP , et al. High-performance calcium sensors for imaging activity in neuronal populations and microcompartments . Nat Methods . 2019 ; 16 : 649 – 657 . doi: 10.1038/s41592-019-0435-6 OpenUrl CrossRef PubMed 28. ↵ Jones JR , Simon T , Lones L , Herzog ED . SCN VIP Neurons Are Essential for Normal Light-Mediated Resetting of the Circadian System . The Journal of Neuroscience . 2018 ; 38 : 7986 – 7995 . doi: 10.1523/JNEUROSCI.1322-18.2018 OpenUrl Abstract / FREE Full Text 29. Mei L , Fan Y , Lv X , Welsh DK , Zhan C , Zhang EE . Long-term in vivo recording of circadian rhythms in brains of freely moving mice . Proceedings of the National Academy of Sciences . 2018 ; 115 : 4276 – 4281 . doi: 10.1073/pnas.1717735115 OpenUrl Abstract / FREE Full Text 30. ↵ Xie L , Xiong Y , Ma D , Shi K , Chen J , Yang Q , et al. Cholecystokinin neurons in mouse suprachiasmatic nucleus regulate the robustness of circadian clock . Neuron . 2023 ; 111 : 2201 – 2217 .e4. doi: 10.1016/j.neuron.2023.04.016 OpenUrl CrossRef PubMed 31. ↵ Jacob TC , Moss SJ , Jurd R . GABAA receptor trafficking and its role in the dynamic modulation of neuronal inhibition . Nat Rev Neurosci . 2008 ; 9 : 331 – 343 . doi: 10.1038/nrn2370 OpenUrl CrossRef PubMed Web of Science 32. ↵ Platt RJ , Chen S , Zhou Y , Yim MJ , Swiech L , Kempton HR , et al. CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer Modeling . Cell . 2014 ; 159 : 440 – 455 . doi: 10.1016/j.cell.2014.09.014 OpenUrl CrossRef PubMed Web of Science 33. ↵ Taniguchi H , He M , Wu P , Kim S , Paik R , Sugino K , et al. A Resource of Cre Driver Lines for Genetic Targeting of GABAergic Neurons in Cerebral Cortex . Neuron . 2011 ; 71 : 995 – 1013 . doi: 10.1016/j.neuron.2011.07.026 OpenUrl CrossRef PubMed Web of Science 34. ↵ Varadarajan S , Tajiri M , Jain R , Holt R , Ahmed Q , LeSauter J , et al. Connectome of the Suprachiasmatic Nucleus: New Evidence of the Core-Shell Relationship . eNeuro . 2018 ; 5 : ENEURO .0205-18.2018. doi: 10.1523/ENEURO.0205-18.2018 OpenUrl Abstract / FREE Full Text 35. ↵ Klapoetke NC , Murata Y , Kim SS , Pulver SR , Birdsey-Benson A , Cho YK , et al. Independent optical excitation of distinct neural populations . Nat Methods . 2014 ; 11 : 338 – 346 . doi: 10.1038/nmeth.2836 OpenUrl CrossRef PubMed Web of Science 36. ↵ Wang W , Wildes CP , Pattarabanjird T , Sanchez MI , Glober GF , Matthews GA , et al. A light- and calcium-gated transcription factor for imaging and manipulating activated neurons . Nat Biotechnol . 2017 ; 35 : 864 – 871 . doi: 10.1038/nbt.3909 OpenUrl CrossRef PubMed 37. ↵ Albus H , Vansteensel MJ , Michel S , Block GD , Meijer JH . A GABAergic Mechanism Is Necessary for Coupling Dissociable Ventral and Dorsal Regional Oscillators within the Circadian Clock . Current Biology . 2005 ; 15 : 886 – 893 . doi: 10.1016/J.CUB.2005.03.051 OpenUrl CrossRef PubMed Web of Science 38. Farajnia S , van Westering TLE , Meijer JH , Michel S . Seasonal induction of GABAergic excitation in the central mammalian clock . Proceedings of the National Academy of Sciences . 2014 ; 111 : 9627 – 9632 . doi: 10.1073/pnas.1319820111 OpenUrl Abstract / FREE Full Text 39. Wagner S , Castel M , Gainer H , Yarom Y . GABA in the mammalian suprachiasmatic nucleus and its role in diurnal rhythmicity . Nature . 1997 ; 387 : 598 – 603 . doi: 10.1038/42468 OpenUrl CrossRef PubMed Web of Science 40. ↵ Irwin RP , Allen CN . GABAergic signaling induces divergent neuronal Ca 2+ responses in the suprachiasmatic nucleus network . European Journal of Neuroscience . 2009 ; 30 : 1462 – 1475 . doi: 10.1111/j.1460-9568.2009.06944.x OpenUrl CrossRef PubMed Web of Science 41. ↵ Nakamura W , Yamazaki S , Nakamura TJ , Shirakawa T , Block GD , Takumi T . In Vivo Monitoring of Circadian Timing in Freely Moving Mice . Current Biology . 2008 ; 18 : 381 – 385 . doi: 10.1016/J.CUB.2008.02.024 OpenUrl CrossRef PubMed Web of Science 42. ↵ Nakamura TJ , Nakamura W , Yamazaki S , Kudo T , Cutler T , Colwell CS , et al. Age-Related Decline in Circadian Output . Journal of Neuroscience . 2011 ; 31 : 10201 – 10205 . doi: 10.1523/JNEUROSCI.0451-11.2011 OpenUrl Abstract / FREE Full Text 43. ↵ Ness N , Díaz-Clavero S , Hoekstra MMB , Brancaccio M . Rhythmic astrocytic GABA production synchronizes neuronal circadian timekeeping in the suprachiasmatic nucleus . EMBO Journal . 2024 [cited 14 Feb 2025]. doi: 10.1038/S44318-024-00324-W/SUPPL_FILE/44318_2024_324_MOESM7_ESM.PDF OpenUrl CrossRef 44. ↵ Myung J , Hong S , DeWoskin D , De Schutter E , Forger DB , Takumi T . GABA-mediated repulsive coupling between circadian clock neurons in the SCN encodes seasonal time . Proceedings of the National Academy of Sciences . 2015 ; 112 . doi: 10.1073/pnas.1421200112 OpenUrl Abstract / FREE Full Text 45. ↵ Evans JA , Leise TL , Castanon-Cervantes O , Davidson AJ . Dynamic Interactions Mediated by Nonredundant Signaling Mechanisms Couple Circadian Clock Neurons . Neuron . 2013 ; 80 : 973 – 983 . doi: 10.1016/j.neuron.2013.08.022 OpenUrl CrossRef PubMed Web of Science 46. ↵ Todd WD , Venner A , Anaclet C , Broadhurst RY , De Luca R , Bandaru SS , et al. Suprachiasmatic VIP neurons are required for normal circadian rhythmicity and comprised of molecularly distinct subpopulations . Nature Communications 2020 11 : 1 . 2020 ; 11 : 1–20. doi: 10.1038/s41467-020-17197-2 OpenUrl CrossRef PubMed 47. ↵ Bussi IL , Neitz AF , Sanchez REA , Casiraghi LP , Moldavan M , Kunda D , et al. Expression of the vesicular GABA transporter within neuromedin S + neurons sustains behavioral circadian rhythms . Proceedings of the National Academy of Sciences . 2023 ; 120 . doi: 10.1073/pnas.2314857120 OpenUrl CrossRef PubMed 48. ↵ Ono D , Honma Kichi , Yanagawa Y , Yamanaka A , Honma S. GABA in the suprachiasmatic nucleus refines circadian output rhythms in mice . Communications Biology 2019 2 : 1 . 2019 ; 2 : 1–12. doi: 10.1038/s42003-019-0483-6 OpenUrl CrossRef 49. Bussi IL , Neitz AF , Sanchez REA , Casiraghi LP , Moldavan M , Kunda D , et al. Expression of the vesicular GABA transporter within neuromedin S+ neurons sustains behavioral circadian rhythms . Proc Natl Acad Sci U S A . 2023 ; 120 : e2314857120 . doi: 10.1073/PNAS.2314857120/SUPPL_FILE/PNAS.2314857120.SAPP.PDF OpenUrl CrossRef PubMed 50. ↵ Granados-Fuentes D , Lambert P , Simon T , Mennerick S , Herzog ED. GABAA receptor subunit composition regulates circadian rhythms in rest–wake and synchrony among cells in the suprachiasmatic nucleus . Proc Natl Acad Sci U S A . 2024 ; 121 : e2400339121 . doi: 10.1073/PNAS.2400339121/SUPPL_FILE/PNAS.2400339121.SM04.MP4 OpenUrl CrossRef PubMed 51. ↵ Tong Q , Ye C-P , Jones JE , Elmquist JK , Lowell BB . Synaptic release of GABA by AgRP neurons is required for normal regulation of energy balance . Nat Neurosci . 2008 ; 11 : 998 – 1000 . doi: 10.1038/nn.2167 OpenUrl CrossRef PubMed Web of Science 52. ↵ Islam MT , Rumpf F , Tsuno Y , Kodani S , Sakurai T , Matsui A , et al. Vasopressin neurons in the paraventricular hypothalamus promote wakefulness via lateral hypothalamic orexin neurons . Current Biology . 2022 ; 32 : 3871 – 3885 .e4. doi: 10.1016/j.cub.2022.07.020 OpenUrl CrossRef PubMed 53. ↵ Concordet J-P , Haeussler M . CRISPOR: intuitive guide selection for CRISPR/Cas9 genome editing experiments and screens . Nucleic Acids Res . 2018 ; 46 : W242 – W245 . doi: 10.1093/nar/gky354 OpenUrl CrossRef 54. ↵ Maddalo D , Manchado E , Concepcion CP , Bonetti C , Vidigal JA , Han Y-C , et al. In vivo engineering of oncogenic chromosomal rearrangements with the CRISPR/Cas9 system . Nature . 2014 ; 516 : 423 – 427 . doi: 10.1038/nature13902 OpenUrl CrossRef PubMed 55. ↵ Armbruster BN , Li X , Pausch MH , Herlitze S , Roth BL . Evolving the lock to fit the key to create a family of G protein-coupled receptors potently activated by an inert ligand . Proceedings of the National Academy of Sciences . 2007 ; 104 : 5163 – 5168 . doi: 10.1073/pnas.0700293104 OpenUrl Abstract / FREE Full Text 56. ↵ Inutsuka A , Yamashita A , Chowdhury S , Nakai J , Ohkura M , Taguchi T , et al. The integrative role of orexin/hypocretin neurons in nociceptive perception and analgesic regulation . Sci Rep . 2016 ; 6 : 29480 . doi: 10.1038/srep29480 OpenUrl CrossRef PubMed 57. ↵ Martianova E , Aronson S , Proulx CD . Multi-Fiber Photometry to Record Neural Activity in Freely-Moving Animals . Journal of Visualized Experiments . 2019 . doi: 10.3791/60278 OpenUrl CrossRef PubMed 58. ↵ Kim CK , Yang SJ , Pichamoorthy N , Young NP , Kauvar I , Jennings JH , et al. Simultaneous fast measurement of circuit dynamics at multiple sites across the mammalian brain . Nat Methods . 2016 ; 13 : 325 – 328 . doi: 10.1038/nmeth.3770 OpenUrl CrossRef PubMed 59. ↵ Hasegawa E , Maejima T , Yoshida T , Masseck OA , Herlitze S , Yoshioka M , et al. Serotonin neurons in the dorsal raphe mediate the anticataplectic action of orexin neurons by reducing amygdala activity . Proc Natl Acad Sci U S A . 2017 ; 114 : E3526 – E3535 . doi: 10.1073/PNAS.1614552114/SUPPL_FILE/PNAS.201614552SI.PDF OpenUrl Abstract / FREE Full Text View the discussion thread. Back to top Previous Next Posted April 29, 2025. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following GABAergic network from AVP neurons to VIP neurons in the suprachiasmatic nucleus sets the activity/rest time of the circadian behavior rhythm Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share GABAergic network from AVP neurons to VIP neurons in the suprachiasmatic nucleus sets the activity/rest time of the circadian behavior rhythm Yubo Peng , Yusuke Tsuno , Takashi Maejima , Mohan Wang , Ayako Matsui , Michihiro Mieda bioRxiv 2025.04.28.650944; doi: https://doi.org/10.1101/2025.04.28.650944 Share This Article: Copy Citation Tools GABAergic network from AVP neurons to VIP neurons in the suprachiasmatic nucleus sets the activity/rest time of the circadian behavior rhythm Yubo Peng , Yusuke Tsuno , Takashi Maejima , Mohan Wang , Ayako Matsui , Michihiro Mieda bioRxiv 2025.04.28.650944; doi: https://doi.org/10.1101/2025.04.28.650944 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 (7635) Biochemistry (17690) Bioengineering (13892) Bioinformatics (41936) Biophysics (21451) Cancer Biology (18588) Cell Biology (25499) Clinical Trials (138) Developmental Biology (13378) Ecology (19899) Epidemiology (2067) Evolutionary Biology (24320) Genetics (15609) Genomics (22506) Immunology (17736) Microbiology (40394) Molecular Biology (17181) Neuroscience (88603) Paleontology (666) Pathology (2832) Pharmacology and Toxicology (4824) Physiology (7641) Plant Biology (15152) Scientific Communication and Education (2045) Synthetic Biology (4294) Systems Biology (9825) Zoology (2271)