TY - JOUR
AU - Rath, Martin Fredensborg
AB - Abstract A molecular circadian oscillator resides in neurons of the cerebral cortex, but its role is unknown. Using the Cre-LoxP method, we have here abolished the core clock gene Arntl in those neurons. This mouse represents the first model carrying a deletion of a circadian clock component specifically in an extrahypothalamic cell type of the brain. Molecular analyses of clock gene expression in the cerebral cortex of the Arntl conditional knockout mouse revealed disrupted circadian expression profiles, whereas clock gene expression in the suprachiasmatic nucleus was still rhythmic, thus showing that Arntl is required for normal function of the cortical circadian oscillator. Daily rhythms in running activity and temperature were not influenced, whereas the resynchronization response to experimental jet-lag exhibited minor though significant differences between genotypes. The tail-suspension test revealed significantly prolonged immobility periods in the knockout mouse indicative of a depressive-like behavioral state. This phenotype was accompanied by reduced norepinephrine levels in the cerebral cortex. Our data show that Arntl is required for normal cortical clock function and further give reason to suspect that the circadian oscillator of the cerebral cortex is involved in regulating both circadian biology and mood-related behavior and biochemistry. circadian, clock gene, conditional knockout, hippocampus, neocortex Introduction In mammals, the main circadian timekeeper resides in the suprachiasmatic nucleus (SCN) of the hypothalamus (Saper 2013). At the molecular level, the circadian clockwork consists of transcriptional and post-translational autoregulatory feedback loops with oscillating clock gene products encoding transcriptional and post-translational modulators as core elements (Reppert and Weaver 2002). These clock genes include Clock (circadian locomotor output cycles kaput), Arntl (arylhydrocarbon receptor nuclear translocator-like, also known as Bmal1 and Mop3), Per1 (period 1), Per2 (period 2), Per3 (period 3), Cry1 (cryptochrome 1), Cry2 (cryptochrome 2), and Nr1d1 (nuclear receptor 1D1, also known as Rev-Erbα). An increasing amount of evidence suggests that additional clocks or oscillators residing outside the SCN have the capacity to generate peripheral circadian rhythms (Guilding and Piggins 2007). To this end, the expression profiles of all aforementioned core clock genes have been mapped in the rodent neocortex (Rath et al. 2013, 2014) and in the hippocampus (Jilg et al. 2010). All known core clock genes are expressed in the rodent cerebral cortex, and their oscillating nature suggests the presence of a running molecular circadian oscillator. Histological analyses have localized the clock gene products in the neocortex to neurons (Rath et al. 2013, 2014). Lesion of the SCN blocks the rhythmic neocortical expression of clock genes, thus showing that neocortical oscillations are controlled by the SCN (Rath et al. 2013). From a clinical perspective these findings are interesting, because molecular changes in the cerebral cortex of patients diagnosed with major depressive disorder, including differences in the expression profile of clock genes, have been recently reported (Li et al. 2013). However, the specific role of the oscillator of the cerebral cortex in circadian biology and mood-related behavior is unknown. To investigate the role of the circadian clockwork specifically in neurons of the cerebral cortex, conventional global knockout methods (Antoch et al. 1997; Thresher et al. 1998; Zheng et al. 1999, 2001; Bunger et al. 2000) are not viable. In the current report, however, we use cell-specific gene targeting to eliminate the central clock gene Arntl in neurons of the cerebral cortex to generate a cerebral cortex neuron-specific clock gene knockout mouse. Materials and Methods Generation of an Arntl Conditional Knockout Mouse Homozygote Emx1 (empty spiracles 1)-Cre carrying mice (B6.129S2-Emx1tm1(cre)Krj/J; stock number 5628) (Guo et al. 2000; Gorski et al. 2002) and Arntl-flox carrying mice (B6.129S4(Cg)-Arntltm1Weit/J; stock number 7668) (Storch et al. 2007) were obtained from Jackson Laboratories (Charles River, Sulzfeld, Germany). The 2 strains were crossed to generate Emx1-Cre/Arntl-flox heterozygotes; these were crossed to generate Emx1-Cre+/Arntl-flox+/+ offspring. Hemizygote Emx1-Cre+/homozygote Arntl-flox+/+ breeding was subsequently used for generating Emx1-Cre+/Arntl-flox+/+ experimental animals, whereas Emx1-Cre−/Arntl-flox+/+ animals were used for control purposes. The animals were housed under controlled light conditions in a standard 12 h light:12 h dark schedule (12 L:12D), where nothing else is noted, with food and water ad libitum. All animal experiments were performed in accordance with the guidelines of EU Directive 86/609/EEC and the specific experiments in this study were approved by the Danish Council for Animal Experiments (authorization number 2012-DY-2934-00022) and the Faculty of Health and Medical Sciences, University of Copenhagen (authorization number P13-035). Genotyping Genotyping was performed on DNA extracted from tail- or ear-samples by use of the HotSHOT method (Truett et al. 2000). Standard 10 µL PCR reactions (Roche, Hvidovre, Denmark) were performed on 100–1000 ng DNA by use of primer sets with sequences provided by Jackson Laboratories (Bar Harbor, ME): 5′-AAGGTGTGGTTCCAGAATCG-3′/5′-CTCTCCACCAGAAGGCTGAG-3′ (oIMR4170/oIMR4171; Emx1-cre wild-type allele), GCGGTCTGGCAGTAAAAACTATC-3′/5′-GTGAAACAGCATTGCTGTCACTT-3′(oIMR1084/oIMR1085; Emx1-cre mutant allele), 5′-ACTGGAAGTAACTTTATCAAACTG-3′/5′-CTGACCAACTTGCTAACAATTA-3′ (oIMR7525/oIMR7526, Arntl-flox mutant and wild-type alleles). The amplification program included 3 min at 94°C, 31 cycles of 94°C for 30 s, 62°C for 60 s and 72°C for 60 s, and a final step of 72°C for 2 min. This was followed by electrophoresis in a 2% agarose gel. Telemetric Registration Five Emx1-Cre+/Arntl-flox+/+ mice (3 males, 2 females; 2.5–4 months of age) and 5 Emx1-Cre-/Arntl-flox+/+ mice (3 males, 2 females; 2.5–5.5 months of age) had TA-F10 telemetry transmitters (Data Sciences International, St. Paul, MN) implanted in the abdomen through a 1 cm incision in the linea alba. The incision was closed in 2 layers with simple interrupted sutures using Vicryl 5/0 Plus. Animals were anaesthetized with a mixture of Fentanyl (0.315 mg/mL), Fluanisone (10 mg/mL) and Midazolam (5 mg/mL) in a dose of 0.075 mL/10 g body weight s.c. Supplementary intraoperative analgesia consisted of Bupivacaine (0.05 mL, 0.25% s.c.) and Carprofen (5 mg/kg/day s.c.). Animals received Carprofen (5 mg/kg/day s.c.) for 2 days and Buprenorphine (0.05 mg/kg s.c. single dose) postoperatively. Telemetry registrations were initiated 16–27 days post surgery. Spontaneous locomotor activity and body temperature were monitored for 10 s every 10 min in animals implanted with radio transmitters by use of the Dataquest A.R.T. system (Data Sciences International, St. Paul, MN) as previously described (Rovsing et al. 2010). Conditional knockout and control mice were housed in the same room under controlled light conditions, including a standard 12 L:12D for 19 days (lights on at 6.00), experimental jet-lag 12 L:12D for 14 days (lights on at 2.00), standard 12 L:12D for 18 days (lights on at 6.00), DD (constant darkness) for 10 days, standard 12 L:12D for 5 days (lights on at 6.00), and LL (constant light) for 9 days. Analyses of Telemetric Data Actograms and chi square periodograms were generated by use of ActogramJ (Schmid et al. 2011). Cosinor analyses and two-way ANOVA followed by Bonferroni post hoc test were performed by use of Prism 5.0d (GraphPad Software, La Jolla, CA). Period length (tau) and rhythm stability were determined by analyzing chi square based periodograms using the Qp-value as a measure of rhythm robustness (Refinetti 2004). Chronotype of individual animals was determined as the mean median of activity (MoA) (Wicht et al. 2014). Activity on-sets and off-sets calculated in ActogramJ were used for analyzing resynchronization after experimental jet-lag as activity onset relative to lights off or activity offset relative to lights on (Pfeffer et al. 2015). Animal Behavioral Tests Of all, 17 male Emx1-Cre+/Arntl-flox+/+ mice (14–21 weeks of age, 25–38 g) and 14 male Emx1-Cre−/Arntl-flox+/+ mice (14–20 weeks of age, 27–38 g) were subjected to tail suspension test at ZT4-6. The test was performed as previously described (Castagne et al. 2011) with the following modifications: no pharmacological intervention was introduced, tail attachment site was 26 cm above the ground, animals were suspended in an opaque cylinder (diameter 19 cm, height 21.4 cm) and monitored for 6 min. Total period with active movement was registered using a stopwatch. Data were analyzed statistically with unpaired t-test by use of Prism 5.0d (GraphPad Software, La Jolla, CA). The same cohort of animals was used for open field test (2 days before tail suspension test) and forced swim tests (18 and 19 days after the tail suspension test, respectively) (Castagne et al. 2011). Following the forced swim test, animals were sacrificed and brains used for high pressure liquid chromatography. High Pressure Liquid Chromatography To compare the response in brain monoamine contents between genotypes following a exposure to a stressful situation, animals were sacrificed by decapitation directly after the forced swim test (see above) at ZT4-6. Brains were immediately removed and dissected. The neocortex was removed bilaterally from the cingulate gyrus to the rhinal sulcus; the hippocampus was subsequently removed bilaterally. Tissues were immediately frozen on solid CO2. Extraction of monoamines, high pressure liquid chromatography and data analysis was done as previously described (Runegaard et al. 2016) with perchloric acid volume adjusted to 250 μl and the supernatant filtered. Data were analyzed statistically with unpaired t-test by use of Prism 5.0d (GraphPad Software, La Jolla, CA). Immunohistochemistry Animals were perfusion fixed with 4% paraformaldehyde at ZT5. Brains were postfixed overnight in the same fixative, cryoprotected in 25% sucrose and frozen on solid CO2. Coronal cryostat sections (40 µm) were cut and reacted free-floating as previously described (Rath et al. 2007). Sections were incubated in goat anti-ARNTL (BMAL1 N-20) polyclonal IgG (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:200. Specificity controls have been previously published (Rath et al. 2014). The primary antiserum was detected with biotinylated donkey anti-goat IgG (Santa Cruz Biotechnology) diluted 1:500, and the chromogenic reaction was performed in a combination of ABC-Vectastain (Vector Laboratories, Burlingame, CA) and diaminobenzidine. Western Blotting Animals were sacrificed by decapitation at ZT4 and brains were immediately removed; dissected brain structures were frozen on solid CO2. Protein was extracted as previously described (Rath et al. 2016). A sample of 50 µg protein per lane was run in a Nupage 4–12% Bis Tris gel (Life Technologies, Nærum, Denmark) and blotted by use of the XCell SureLock system (Invitrogen, Taastrup, Denmark) as previously described (Rath et al. 2014) . HiMark Pre-Stained HMW Protein Standard (Invitrogen) and Precision Plus Protein Kaliedoscope (Bio-Rad Laboratories, Copenhagen, Denmark) molecular weight markers were run simultaneously. The membrane was blocked in 2% skim milk for 30 min and incubated in goat anti-ARNTL (BMAL1 N-20) polyclonal IgG (Santa Cruz Biotechnology) diluted 1:500 in 2% skim milk at 4°C overnight. The blot was subsequently developed as previously described (Rath et al. 2006). Quantitative Real-Time RT-PCR A total of 24 Emx1-Cre+/Arntl-flox+/+ mice (11 males, 13 females; 13–25 weeks of age) and 24 Emx1-Cre-/Arntl-flox+/+ mice (15 males, 9 females; 13–23 weeks of age) were kept in DD for 2 days before being sacrificed. With 3-h intervals throughout the presumptive day and night, 3 animals per genotype were sacrificed by decapitation under dim red light, the brains were dissected, brain structures removed bilaterally as described above and immediately frozen on solid CO2. RNA isolation, cDNA synthesis and SYBR green based quantitative real-time RT-PCR reactions were performed as previously described (Rath et al. 2014) with the following modifications: The reaction volume was 10 μL and reactions were run on a LightCycler 96 (Roche, Hvidovre, Denmark). For detection of Per1, Per2, Clock, Cry1, Nr1d1, Dbp, actin beta, and glyceraldehyde 3-phosphate dehydrogenase transcripts, primer sets with previously published sequences were used (Rath et al. 2014). For detection of the Per3 transcript, a primer set with the following sequences was used: 5′-ATGCCTGCGCCGTCAGAGTC-3′/5′-ACTCCTGCTGCGCTGTTGCC-3′generating an amplicon corresponding to position 3197–3341 on mouse Per3 mRNA (GenBank accession number NM_011067.3). For detection of the Cry2 transcript, a primer set with the following sequences was used: 5′-CCTCAGCCCCTACCTGCGCT-3′/5′-GGGTTGTTGGTGGCCGCTGT-3′ generating an amplicon corresponding to position 850–1011 on mouse Cry2 mRNA (GenBank accession number NM_009963.4). Radiochemical In Situ Hybridization Overall, 19 Emx1-Cre+/Arntl-flox+/+ mice (9 males, 10 females; 14–17 weeks of age) and 18 Emx1-Cre−/Arntl-flox+/+ mice (12 males, 6 females; 13–19 weeks of age) were sacrificed by decapitation with 4-h intervals throughout the day and night; during the dark period, animals were sacrificed by decapitation under dim red light. Brains were immediately removed and frozen in crushed solid CO2. In situ hybridization was performed on coronal cryostat sections (12 µm) by use of 35S-labeled DNA probes as previously described (Klitten et al. 2008). For detection of Per2 and Nr1d1 transcripts, probes with previously published sequences were used (Rath et al. 2014). For detection of Per1 transcripts, a mixture of probes with the following sequences were used: 5′-TTTCGAAGTGTGTATTCGGATGTGATATGCTCCAAT-3′ corresponding to position 833-798 on mouse Per1 mRNA (GenBank accession number NM_011065.4) and 5′-CGCCTGTCTCTGAGGGCTGACTAATTTAATCACGACAC-3′ corresponding to position 91-54 on mouse Per1 mRNA (GenBank accession number NM_011065.4). For detection of Arntl transcripts, a probe with the following sequence was used: 5′-CATGTTGGTACCAAAGAAGCCAATTCATCAATGAAA-3′ corresponding to position 825–790 on mouse Arntl mRNA (GenBank accession number NM_007489.4); this part of the transcript is encoded by exon 8 of the Arntl gene, which is flanked by LoxP-sites in the Arntl-flox carrying mice (B6.129S4(Cg)-Arntltm1Weit/J) (Storch et al. 2007). Images on X-ray films were digitized and quantified by use of Scion Image Beta 4.0.2 (Scion Frederick, MD, USA). In the neocortex, all 6 layers including both motor and somatosensory areas at Bregma −1.7 mm were quantified; in the hippocampus, the pyramidal layer of the dorsal part of the cornu ammonis (CA1) was used for quantification. Analyses of Gene Expression Data One-way or two-way ANOVA followed by Bonferroni's multiple comparisons post hoc tests and cosinor analyses for estimation of the time of peaking gene expression were performed by use of Prism 5.0d (GraphPad Software, La Jolla, CA). In all statistical analyses, a two-tailed P-value below 0.05 was considered to represent statistical significance. Results Arntl mRNA and ARNTL Protein are Absent From the Cerebral Cortex of the Emx1-Cre+/Arntl-flox+/+ Mouse, but Arntl is Still Rhythmic in the SCN Clock genes, including Arntl, are expressed in neurons of the cerebral cortex (Rath et al. 2013, 2014). To examine the role of Arntl and circadian clock gene function specifically in the cerebral cortex, we generated a cortical neuron-specific Arntl conditional knockout mouse by crossing a strain carrying a floxed Arntl allele (Storch et al. 2007) with a strain expressing Cre under the Emx1-promoter (Guo et al. 2000) (Fig. 1). The selection of the Emx1-Cre strain was based on the known expression profile of the homeobox gene Emx1 (empty spiracles) that is specifically expressed in neurons of the developing cerebral cortex (Simeone et al. 1992; Gulisano et al. 1996). The Emx1-Cre+/Arntl-flox+/+ mouse was visually indistinguishable from wild-type littermates. Figure 1. View largeDownload slide Evaluation of Arntl expression in the neocortex, hippocampus and suprachiasmatic nucleus of the cerebral cortex-specific Arntl conditional knockout mouse (Emx1-Cre+/Arntl-flox+/+). (A) Immunohistochemical detection of ARNTL protein in coronal sections of the neocortex, hippocampus and suprachiasmatic nucleus (SCN) of the control (Emx1-Cre−/Arntl-flox+/+) (left column) and Arntl conditional knockout (Arntl CKO; Emx1-Cre+/Arntl-flox+/+) (right column). Scale bars, 200 μm. (B) Western blot analysis for detection of ARNTL in protein extracts of different parts of the brain of Arntl conditional knockout mice (Emx1-Cre+/Arntl-flox+/+) and controls (Emx1-Cre-/Arntl-flox+/+). Mouse ARNTL protein (NP_031515.1) has a predicted molecular weight of 68.7 kDa. Arrows indicate molecular weight markers. Specificity controls have been previously published (Rath et al. 2014). (C) In situ hybridization for detection of Arntl transcripts in the brain of the Arntl conditional knockout mouse (Emx1-Cre+/Arntl-flox+/+) and the control mouse (Emx1-Cre−/Arntl-flox+/+). Arntl mRNA is below detectable levels in the cerebral cortex of the Arntl conditional knockout mouse (upper images), but the transcript is present in the suprachiasmatic nucleus (SCN; lower images). Displayed images represent maximal levels of expression. Scale bars, 1 mm. (D) Quantitative analysis of in situ hybridization for detection of Arntl transcripts in the neocortex, hippocampus and suprachiasmatic nucleus (SCN) of the Arntl conditional knockout mouse (Emx1-Cre+/Arntl-flox+/+; solid line, closed circles). For comparison, Arntl transcript levels in the neocortex and hippocampus of the control mouse (Emx1-Cre−/Arntl-flox+/+) are displayed (dashed line, open circles). Arntl CKO, Arntl conditional knockout mouse (Emx1-Cre+/Arntl-flox+/+). Figure 1. View largeDownload slide Evaluation of Arntl expression in the neocortex, hippocampus and suprachiasmatic nucleus of the cerebral cortex-specific Arntl conditional knockout mouse (Emx1-Cre+/Arntl-flox+/+). (A) Immunohistochemical detection of ARNTL protein in coronal sections of the neocortex, hippocampus and suprachiasmatic nucleus (SCN) of the control (Emx1-Cre−/Arntl-flox+/+) (left column) and Arntl conditional knockout (Arntl CKO; Emx1-Cre+/Arntl-flox+/+) (right column). Scale bars, 200 μm. (B) Western blot analysis for detection of ARNTL in protein extracts of different parts of the brain of Arntl conditional knockout mice (Emx1-Cre+/Arntl-flox+/+) and controls (Emx1-Cre-/Arntl-flox+/+). Mouse ARNTL protein (NP_031515.1) has a predicted molecular weight of 68.7 kDa. Arrows indicate molecular weight markers. Specificity controls have been previously published (Rath et al. 2014). (C) In situ hybridization for detection of Arntl transcripts in the brain of the Arntl conditional knockout mouse (Emx1-Cre+/Arntl-flox+/+) and the control mouse (Emx1-Cre−/Arntl-flox+/+). Arntl mRNA is below detectable levels in the cerebral cortex of the Arntl conditional knockout mouse (upper images), but the transcript is present in the suprachiasmatic nucleus (SCN; lower images). Displayed images represent maximal levels of expression. Scale bars, 1 mm. (D) Quantitative analysis of in situ hybridization for detection of Arntl transcripts in the neocortex, hippocampus and suprachiasmatic nucleus (SCN) of the Arntl conditional knockout mouse (Emx1-Cre+/Arntl-flox+/+; solid line, closed circles). For comparison, Arntl transcript levels in the neocortex and hippocampus of the control mouse (Emx1-Cre−/Arntl-flox+/+) are displayed (dashed line, open circles). Arntl CKO, Arntl conditional knockout mouse (Emx1-Cre+/Arntl-flox+/+). To confirm deletion of Arntl in the cerebral cortex, immunohistochemical analyses were performed (Fig. 1A). ARNTL protein was strongly expressed in the neocortex and hippocampus of the Emx1-Cre−/Arntl-flox+/+ control mouse, but it was only detectable in very few neocortical neurons and undetectable in the hippocampus of the Emx1-Cre+/Arntl-flox+/+ conditional knockout mouse. These results were confirmed by western blotting, which showed a very weak band in neocortical extracts from Emx1-Cre+/Arntl-flox+/+ conditional knockout mice as compared with the neocortex and other tissues of Emx1-Cre−/Arntl-flox+/+ control mice (Fig. 1B). The low levels of ARNTL detected in a limited number of cells in the neocortex of the Emx1-Cre+/Arntl-flox+/+ conditional knockout mouse reflect the reported recombination efficiency of 91% in the cerebral cortex of the Emx1-Cre strain (Guo et al. 2000). In both genotypes, ARNTL protein was detected in the SCN (Fig. 1A). In situ hybridization confirmed the absence of Arntl transcripts in the neocortex and hippocampus of the Emx1-Cre+/Arntl-flox+/+ conditional knockout mouse (Fig. 1C); the hybridization signal in the cerebral cortex of the Emx1-Cre+/Arntl-flox+/+ conditional knockout mouse was similar to background levels and significantly different from that of the of the Emx1-Cre−/Arntl-flox+/+ control mouse (Fig. 1D) in both the neocortex (P < 0.0001, two-way ANOVA) and in the hippocampus (P < 0.0001, two-way ANOVA). Arntl was still detectable in the SCN (Fig. 1C) exhibiting a daily rhythm (Fig. 1D; P < 0.05, one-way ANOVA), thus confirming region-specific abolishment of Arntl expression in the cerebral cortex. Circadian Rhythms in Clock Gene Expression are Disrupted in the Neocortex and Hippocampus of the Emx1-Cre+/Arntl-flox+/+ Conditional Knockout Mouse To determine if deletion of Arntl influenced the molecular oscillating clockwork of the neocortex and hippocampus, quantitative RT-PCR analyses were performed on dissected brain structures of the Emx1-Cre+/Arntl-flox+/+ conditional knockout mouse (Fig. 2; Table 1; Fig. 3; Table 2). Table 1 Quantitative analysis of clock and clock-controlled gene expression in the neocortex of the Emx1-Cre+/Arntl-flox+/+ conditional knockout mouse as determined by quantitative RT-PCR Transcript  Genotype  Difference between genotypes (significance level)  Circadian changes (significance level)  Expression level (mean ± SEM)  Fold change (ratio ± SE)  Sine wave peak (CT; 95% CI)  Per1  Control  **  **  4823.8 (±646.9)  4.48 (±1.10)  15.4 (11.3–19.6)  Arntl CKO  ns  3666.8 (±220.3)  –  –  Per2  Control  ns  **  1601.8 (±251.4)  5.09 (±1.36)  16.8 (14.8–18.9)  Arntl CKO  ns  1660.3 (±91.5)  –  –  Per3  Control  ***  **  1525.5 (±153.4)  2.29 (±0.57)  14.7 (13.7–15.7)  Arntl CKO  ns  630.0 (±27.8)  –  –  Clock  Control  ***  ns  1745.8 (±53.6)  –  –  Arntl CKO  ns  2783.8 (±109.9)  –  –  Nr1d1  Control  ***  ***  3354.7 (±419.1)  2.88 (±0.74)  10.7 (8.8–12.6)  Arntl CKO  ns  1265.6 (±98.7)  –  –  Cry1  Control  ***  ns  505.4 (±49.9)  –  –  Arntl CKO  ns  1218.5 (±36.7)  –  –  Cry2  Control  *  ns  2102.9 (±94.2)  –  –  Arntl CKO  ns  2615.2 (93.8)  –  –  Dbp  Control  ***  **  300.9 (±43.4)  2.69 (±0.34)  12.9 (11.8–14.1)  Arntl CKO  ns  112.9 (±16.4)  –  –  Transcript  Genotype  Difference between genotypes (significance level)  Circadian changes (significance level)  Expression level (mean ± SEM)  Fold change (ratio ± SE)  Sine wave peak (CT; 95% CI)  Per1  Control  **  **  4823.8 (±646.9)  4.48 (±1.10)  15.4 (11.3–19.6)  Arntl CKO  ns  3666.8 (±220.3)  –  –  Per2  Control  ns  **  1601.8 (±251.4)  5.09 (±1.36)  16.8 (14.8–18.9)  Arntl CKO  ns  1660.3 (±91.5)  –  –  Per3  Control  ***  **  1525.5 (±153.4)  2.29 (±0.57)  14.7 (13.7–15.7)  Arntl CKO  ns  630.0 (±27.8)  –  –  Clock  Control  ***  ns  1745.8 (±53.6)  –  –  Arntl CKO  ns  2783.8 (±109.9)  –  –  Nr1d1  Control  ***  ***  3354.7 (±419.1)  2.88 (±0.74)  10.7 (8.8–12.6)  Arntl CKO  ns  1265.6 (±98.7)  –  –  Cry1  Control  ***  ns  505.4 (±49.9)  –  –  Arntl CKO  ns  1218.5 (±36.7)  –  –  Cry2  Control  *  ns  2102.9 (±94.2)  –  –  Arntl CKO  ns  2615.2 (93.8)  –  –  Dbp  Control  ***  **  300.9 (±43.4)  2.69 (±0.34)  12.9 (11.8–14.1)  Arntl CKO  ns  112.9 (±16.4)  –  –  Circadian changes in gene expression for each genotype and difference between genotypes were determined by two-way ANOVA followed by Bonferroni's multiple comparisons post hoc tests; P-values: ***P < 0.001; **P < 0.01; *P < 0.05; ns, not significant. Gene expression level was determined by calculating the overall mean of the mean values at each Circadian Time point analyzed. Fold change in gene expression was determined as the ratio between the highest and lowest value. The Circadian Time (CT) of peak expression was estimated mathematically by fitting the circadian data to a sine wave curve; 95% confidence intervals (95% CI) are given in parentheses. Arntl CKO, Emx1-Cre+/Arntl-flox+/+; control, Emx1-Cre−/Arntl-flox+/+. Table 2 Quantitative analysis of clock and clock-controlled gene expression in the hippocampus of the Emx1-Cre+/Arntl-flox+/+ conditional knockout mouse as determined by quantitative RT-PCR Transcript  Genotype  Difference between genotypes (significance level)  Circadian changes (significance level)  Expression level (mean ± SEM)  Fold change (ratio ± SE)  Sine wave peak (CT; 95% CI)  Per1  Control  ***  **  3127.5 (±174.6)  1.60 (±0.15)  13.7 (11.8–15.6)  Arntl CKO  ns  2360.0 (±94.8)  –  –  Per2  Control  ns  ***  1066.4 (±92.2)  1.98 (±0.16)  16.6 (15.7–17.6)  Arntl CKO  *  985.0 (±52.7)  1.57 (±0.15)  15.4 (13.5–20.0)  Per3  Control  ***  nsa  1093.6 (±63.7)  –  –  Arntl CKO  nsa  381.0 (±39.4)  –  –  Clock  Control  ***  ns  1588.0 (±32.8)  –  –  Arntl CKO  ns  1969.7 (±39.4)  –  –  Nr1d1  Control  ***  ***  3345.1 (±462.3)  3.33 (±0.66)  9.7 (7.6–11.7)  Arntl CKO  ns  1354.1 (148.4)  –  –  Cry1  Control  ***  ns  435.8 (±25.4)  –  –  Arntl CKO  ns  933.8 (32.3)  –  –  Cry2  Control  ***  nsa  1944.6 (±64.3)  –  –  Arntl CKO  nsa  2272.2 (±29.3)  –  –  Dbp  Control  ***  *  277.3 (±31.8)  2.53 (±0.45)  12.3 (11.0–13.5)  Arntl CKO  ns  95.7 (±13.6)  –  –  Transcript  Genotype  Difference between genotypes (significance level)  Circadian changes (significance level)  Expression level (mean ± SEM)  Fold change (ratio ± SE)  Sine wave peak (CT; 95% CI)  Per1  Control  ***  **  3127.5 (±174.6)  1.60 (±0.15)  13.7 (11.8–15.6)  Arntl CKO  ns  2360.0 (±94.8)  –  –  Per2  Control  ns  ***  1066.4 (±92.2)  1.98 (±0.16)  16.6 (15.7–17.6)  Arntl CKO  *  985.0 (±52.7)  1.57 (±0.15)  15.4 (13.5–20.0)  Per3  Control  ***  nsa  1093.6 (±63.7)  –  –  Arntl CKO  nsa  381.0 (±39.4)  –  –  Clock  Control  ***  ns  1588.0 (±32.8)  –  –  Arntl CKO  ns  1969.7 (±39.4)  –  –  Nr1d1  Control  ***  ***  3345.1 (±462.3)  3.33 (±0.66)  9.7 (7.6–11.7)  Arntl CKO  ns  1354.1 (148.4)  –  –  Cry1  Control  ***  ns  435.8 (±25.4)  –  –  Arntl CKO  ns  933.8 (32.3)  –  –  Cry2  Control  ***  nsa  1944.6 (±64.3)  –  –  Arntl CKO  nsa  2272.2 (±29.3)  –  –  Dbp  Control  ***  *  277.3 (±31.8)  2.53 (±0.45)  12.3 (11.0–13.5)  Arntl CKO  ns  95.7 (±13.6)  –  –  Circadian changes in gene expression for each genotype and difference between genotypes were determined by two-way ANOVA followed by Bonferroni's multiple comparisons post hoc tests; P-values: ***P < 0.001; **P < 0.01; *P < 0.05; ns, not significant. Gene expression level was determined by calculating the overall mean of the mean values at each Circadian Time point analyzed. Fold change in gene expression was determined as the ratio between the highest and lowest value. The Circadian Time (CT) of peak expression was estimated mathematically by fitting the circadian data to a sine wave curve; 95% confidence intervals (95% CI) are given in parentheses. Arntl CKO, Emx1-Cre+/Arntl-flox+/+; control, Emx1-Cre−/Arntl-flox+/+. aIn case of Per3 and Cry2, two-way ANOVA identified significant effects of circadian time (P < 0.05), but differences between individual time points were identified by use of Bonferroni's multiple comparisons post hoc test. Figure 2. View largeDownload slide Clock gene expression in the neocortex of the Arntl conditional knockout mouse. Clock and clock-controlled gene transcript levels in the neocortex of the Arntl conditional knockout mouse (Emx1-Cre+/Arntl-flox+/+, Arntl CKO; solid line, closed circles) as determined by quantitative RT-PCR. For comparison, transcript levels in the neocortex of the control mouse (Emx1-Cre−/Arntl-flox+/+) were determined (dashed line, open circles). Mice were kept in DD and each point represents the mean with SEM of 3 mice analyzed. For statistical and cosinor analyses, see Table 1. Figure 2. View largeDownload slide Clock gene expression in the neocortex of the Arntl conditional knockout mouse. Clock and clock-controlled gene transcript levels in the neocortex of the Arntl conditional knockout mouse (Emx1-Cre+/Arntl-flox+/+, Arntl CKO; solid line, closed circles) as determined by quantitative RT-PCR. For comparison, transcript levels in the neocortex of the control mouse (Emx1-Cre−/Arntl-flox+/+) were determined (dashed line, open circles). Mice were kept in DD and each point represents the mean with SEM of 3 mice analyzed. For statistical and cosinor analyses, see Table 1. Figure 3. View largeDownload slide Clock gene expression in the hippocampus of the Arntl conditional knockout mouse. Clock and clock-controlled gene transcript levels in the hippocampus of the Arntl conditional knockout mouse (Emx1-Cre+/Arntl-flox+/+, Arntl CKO; solid line, closed circles) as determined by quantitative RT-PCR. For comparison, transcript levels in the hippocampus of the control mouse (Emx1-Cre−/Arntl-flox+/+) were determined (dashed line, open circles). Mice were kept in DD and each point represents the mean with SEM of 3 mice analyzed. For statistical and cosinor analyses, see Table 2. Figure 3. View largeDownload slide Clock gene expression in the hippocampus of the Arntl conditional knockout mouse. Clock and clock-controlled gene transcript levels in the hippocampus of the Arntl conditional knockout mouse (Emx1-Cre+/Arntl-flox+/+, Arntl CKO; solid line, closed circles) as determined by quantitative RT-PCR. For comparison, transcript levels in the hippocampus of the control mouse (Emx1-Cre−/Arntl-flox+/+) were determined (dashed line, open circles). Mice were kept in DD and each point represents the mean with SEM of 3 mice analyzed. For statistical and cosinor analyses, see Table 2. In the neocortex of the Emx1-Cre−/Arntl-flox+/+ control mouse (Fig. 2; Table 1), the period genes (Per1, Per2, and Per3) were all rhythmic with 2- to 5-fold circadian changes and peaking expression levels reached early in the presumptive night (CT14–CT17); however, rhythmic expression was not detected in the Emx1-Cre+/Arntl-flox+/+ conditional knockout mouse in any of the genes, and the expression levels of Per1 and Per3 were reduced (Table 1). Statistical analyses did not reveal general differences between the genotypes in neocortical Per2 expression, but the gene was rhythmic only in the Emx1-Cre−/Arntl-flox+/+ control mouse (Table 1). Expression of Nr1d1 and the Dbp (D site of albumin promoter binding protein) clock-controlled gene exhibited 2- to 3-fold circadian rhythms with peaking expression at the presumptive day-night transition (CT10-CT13) in the neocortex of the Emx1-Cre−/Arntl-flox+/+ control mouse. However, in the neocortex of the Emx1-Cre+/Arntl-flox+/+ conditional knockout mouse, both Nr1d1 and Dbp were expressed at reduced levels and did not exhibit circadian changes (Fig. 2; Table 1). Clock and cryptochrome genes (Cry1 and Cry2) did not exhibit circadian rhythms in the neocortex of any of the genotypes, but the expression profiles differed significantly between genotypes with increased transcript levels in the Emx1-Cre+/Arntl-flox+/+ conditional knockout mouse (Fig. 2; Table 1). In the hippocampus, Per1 and Per2 were rhythmic with 2-fold circadian changes and peaking expression levels reached early in the presumptive night (CT13–CT17) in the Emx1-Cre−/Arntl-flox+/+ control mouse (Fig. 3; Table 2). Circadian changes in Per3 expression were not detected, but significant differences between genotypes were detected in expression of both Per1 and Per3 with reduced expression levels in the hippocampus of the Emx1-Cre+/Arntl-flox+/+ conditional knockout mouse and arrhythmic expression of Per1 (Fig. 3; Table 2). However, statistical analyses did not reveal differences between the genotypes in hippocampal Per2 expression, which was also rhythmic in the Emx1-Cre+/Arntl-flox+/+ conditional knockout mouse (Table 2). In the hippocampus, as in the neocortex, expression of Nr1d1 and Dbp exhibited 2- to 3-fold circadian rhythms with peaking expression at the presumptive day-night transition (CT9-CT13) in the control mouse. In the hippocampus of the Emx1-Cre+/Arntl-flox+/+ conditional knockout mouse, both Nr1d1 and Dbp were detected at reduced levels and did not exhibit circadian changes (Fig. 3; Table 2). Clock, Cry1 and Cry2 did not exhibit circadian rhythms in the hippocampus in any of the genotypes (P > 0.05, Bonferroni's multiple comparisons post hoc test), but the expression was significantly different between genotypes with increased expression levels of both Clock, Cry1, and Cry2 in the hippocampus of the Emx1-Cre+/Arntl-flox+/+ conditional knockout mouse (Fig. 3; Table 2). In situ hybridization analyses on animals kept in 12 L:12D confirmed the changes in neocortical and hippocampal clock gene expression in the Emx1-Cre+/Arntl-flox+/+ conditional knockout mouse (Fig. 4). Per1 was detectable in the cerebral cortex of both genotypes, but expression levels were reduced in the neocortex (P < 0.0001, two-way ANOVA) and in the hippocampus (P < 0.0001, two-way ANOVA) of the Emx1-Cre+/Arntl-flox+/+ conditional knockout mice as compared with control mice (Fig. 4A). Per2 expression was also seen in the neocortex and hippocampus of both of the Emx1-Cre+/Arntl-flox+/+ conditional knockout mice and controls (Fig. 4B), and although the genotypes differed significantly in Per2 expression in both the neocortex (P < 0.001, two-way ANOVA) and the hippocampus (P < 0.0001, two-way ANOVA), rhythmic expression of Per2 was detectable in both brain structures of both genotypes (P < 0.01, Bonferroni's multiple comparisons post hoc test). Nr1d1 was detectable in both the neocortex and the hippocampus of the control mice (Fig. 4C) exhibiting significant rhythms (P < 0.0001, Bonferroni's multiple comparisons post hoc test), but expression levels of Nr1d1 in the Arntl conditional knockout were reduced significantly in both the neocortex and the hippocampus (P < 0.0001, two-way ANOVA) to levels comparable to background without detectable rhythmicity (P > 0.05, Bonferroni's multiple comparisons post hoc test) (Fig. 4C). In summary, the in situ hybridizations confirmed the results obtained by quantitative RT-PCR. Figure 4. View largeDownload slide Clock gene expression in the neocortex, hippocampus and suprachiasmatic nucleus. In situ hybridization for detection of Per1 (A), Per2 (B) and Nr1d1 (C) transcripts in the brain of the Arntl conditional knockout mice (Emx1-Cre+/Arntl-flox+/+) and control mice (Emx1-Cre−/Arntl-flox+/+). Left column: Representative X-ray images with maximal levels of expression in the neocortex, hippocampus and suprachiasmatic nucleus (SCN) are displayed. Scale bars, 1 mm. Right column: Quantitative analysis of in situ hybridization for detection of Per1 (A), Per2 (B) and Nr1d1 (C) transcripts in the neocortex, hippocampus and suprachiasmatic nucleus (SCN) of the Arntl conditional knockout mouse (Emx1-Cre+/Arntl-flox+/+; solid line, closed circles). For comparison, Arntl transcript levels in the neocortex and hippocampus of the control mouse (Emx1-Cre−/Arntl-flox+/+) are displayed (dashed line, open circles). Arntl CKO, Arntl conditional knockout mouse (Emx1-Cre+/Arntl-flox+/+). Figure 4. View largeDownload slide Clock gene expression in the neocortex, hippocampus and suprachiasmatic nucleus. In situ hybridization for detection of Per1 (A), Per2 (B) and Nr1d1 (C) transcripts in the brain of the Arntl conditional knockout mice (Emx1-Cre+/Arntl-flox+/+) and control mice (Emx1-Cre−/Arntl-flox+/+). Left column: Representative X-ray images with maximal levels of expression in the neocortex, hippocampus and suprachiasmatic nucleus (SCN) are displayed. Scale bars, 1 mm. Right column: Quantitative analysis of in situ hybridization for detection of Per1 (A), Per2 (B) and Nr1d1 (C) transcripts in the neocortex, hippocampus and suprachiasmatic nucleus (SCN) of the Arntl conditional knockout mouse (Emx1-Cre+/Arntl-flox+/+; solid line, closed circles). For comparison, Arntl transcript levels in the neocortex and hippocampus of the control mouse (Emx1-Cre−/Arntl-flox+/+) are displayed (dashed line, open circles). Arntl CKO, Arntl conditional knockout mouse (Emx1-Cre+/Arntl-flox+/+). Daily Rhythms of Clock Gene Expression Persist in the SCN of the Emx1-Cre+/Arntl-flox+/+ Conditional Knockout Mouse In situ hybridization was also performed on sections containing the SCN (Fig. 4). Clock gene expression was detectable in the SCN of the Arntl conditional knockout mouse with significant daily rhythms detected in case of both Per1 (P < 0.0001, one-way ANOVA), Per2 (P < 0.0001, one-way ANOVA) and Nr1d1 (P < 0.0001, one-way ANOVA), showing that a running molecular circadian master clock was still present in the Arntl conditional knockout mouse (Fig. 4). Daily Rhythms in Spontaneous Locomotor Activity and Body Temperature of the Emx1-Cre+/Arntl-flox+/+ Conditional Knockout Mouse are Similar to those of the Control Mouse, But Resynchronization After Experimental Jet-Lag Differs Between Genotypes The spontaneous locomotor activity of the Emx1-Cre+/Arntl-flox+/+ conditional knockout and the Emx1-Cre−/Arntl-flox+/+ control mice was monitored in a standard 12 L:12D light regime (Fig. 5). Both genotypes exhibited similar rhythm robustness (Qp) and period length (tauLD) as revealed by chi-square periodograms (Supplementary Material, Table S1). Cosinor analyses also revealed similar activity profiles (mesor and amplitude), and differences in chronotypes (MoA) between the genotypes were not detected (Supplementary Material, Table S1). These parameter values were maintained in constant darkness (DD), whereas in constant light (LL), both genotypes exhibited a period length (tauLL) above 25 h. In the same mice, body temperature was monitored revealing similar results with no significant differences between genotypes (Supplementary Material, Fig. S1; Supplementary Material, Table S2). Figure 5. View largeDownload slide Locomotor activity. Double plotted actograms of spontaneous locomotor activity measured in a control (Emx1-Cre−/Arntl-flox+/+) and an Arntl conditional knockout (Emx1-Cre+/Arntl-flox+/+) mouse, respectively. The animals were exposed to a standard 12 h light:12 h dark protocol (12 L:12D), 4 h phase advance and 4 h phase delay, constant darkness (DD) and constant light (LL). An activity interval ranging from 0 to 40 cm/min is displayed. Arntl CKO, Arntl conditional knockout mouse (Emx1-Cre+/Arntl-flox+/+). Figure 5. View largeDownload slide Locomotor activity. Double plotted actograms of spontaneous locomotor activity measured in a control (Emx1-Cre−/Arntl-flox+/+) and an Arntl conditional knockout (Emx1-Cre+/Arntl-flox+/+) mouse, respectively. The animals were exposed to a standard 12 h light:12 h dark protocol (12 L:12D), 4 h phase advance and 4 h phase delay, constant darkness (DD) and constant light (LL). An activity interval ranging from 0 to 40 cm/min is displayed. Arntl CKO, Arntl conditional knockout mouse (Emx1-Cre+/Arntl-flox+/+). To investigate the effect of phase advance and phase delay on locomotor activity, the mice were exposed to two 4 h shifts in photophase (Fig. 5). Entrainment was quantified as the difference between lights off and activity onset (Fig. 6) and the difference between lights on and activity offset (Supplementary Material, Fig. S2). The phase advance had a highly significant effect on activity onset relative to lights off (P < 0.0001, two-way ANOVA) (Fig. 6); in both genotypes, significant differences were detected in activity onset relative to lights off on the first two days after changing the light schedule (P < 0.01, Bonferroni's multiple comparisons post hoc test), but full resynchronization was obtained on the third day (P > 0.05, Bonferroni's multiple comparisons post hoc test). The adaptation process differed between genotypes (P < 0.01, two-way ANOVA): on the first day after the phase advance, the activity onset relative to lights off was significantly smaller in the Emx1-Cre+/Arntl-flox+/+ conditional knockout mice than that of the control (P < 0.05, Bonferroni's multiple comparisons post hoc test) (Fig. 6). Figure 6. View largeDownload slide Resynchronization of running activity after experimental 4 h jetlag. Bar graphs display the difference in hours between activity onset and lights off following a phase advance (upper graph) or phase delay (lower graph), respectively. Day -1 represents the last photoperiod prior to changing of the light schedule. Significance levels representing two-tailed P-values based on Bonferroni's multiple comparisons post hoc test following two-way ANOVA analyses are shown: *P < 0.05; ***P < 0.001. Figure 6. View largeDownload slide Resynchronization of running activity after experimental 4 h jetlag. Bar graphs display the difference in hours between activity onset and lights off following a phase advance (upper graph) or phase delay (lower graph), respectively. Day -1 represents the last photoperiod prior to changing of the light schedule. Significance levels representing two-tailed P-values based on Bonferroni's multiple comparisons post hoc test following two-way ANOVA analyses are shown: *P < 0.05; ***P < 0.001. Following a 4 h phase delay, the locomotor activity rhythm of both the controls and the Emx1-Cre+/Arntl-flox+/+ conditional knockout mice as determined by the activity onset relative to lights off was altered (P < 0.0001, two-way ANOVA) (Fig. 6); in both genotypes, significant differences in activity onset relative to lights off were detected on the first day after changing the light schedule (P < 0.05, Bonferroni's multiple comparisons post hoc test), whereas full resynchronization was obtained on the second day (P > 0.05, Bonferroni's multiple comparisons post hoc test). Again the process of entrainment differed between genotypes (P < 0.001, two-way ANOVA), as the activity onset relative to lights off on the first day after the phase delay was significantly larger in the Emx1-Cre+/Arntl-flox+/+ conditional knockout mice than that of the controls (P < 0.001, Bonferroni's multiple comparisons post hoc test) (Fig. 6). Analysis of the activity offset relative to lights on following the phase delay (Supplementary Material, Fig. S2) confirmed that the resynchronization process differed between genotypes (P < 0.001, two-way ANOVA) with activity offset relative to lights on being significantly larger in the Emx1-Cre+/Arntl-flox+/+ conditional knockout mouse on the first day after the phase delay (P < 0.05, Bonferroni's multiple comparisons post hoc test) (Supplementary Material, Fig. S2). The Emx1-Cre+/Arntl-flox+/+ Conditional Knockout Mouse Exhibits Depressive-Like Behavior Patients suffering from major depressive disorder exhibit a marked difference in expression of clock genes, including Arntl, in the cerebral cortex (Li et al. 2013); however, it is unknown if disturbance of the cortical circadian clock is directly involved in the etiology of the disorder. This prompted us to investigate depressive-like behavior in the Emx1-Cre+/Arntl-flox+/+ conditional knockout mouse by use of the tail suspension test (Fig. 7) (Cryan et al. 2005). The phase of immobility was significantly prolonged in the Emx1-Cre+/Arntl-flox+/+ conditional knockout mouse as compared with Emx1-Cre-/Arntl-flox+/+ controls (P < 0.0001, Student's t-test), thus suggesting a depressive-like state in the knockout mouse. Differences between the Emx1-Cre+/Arntl-flox+/+ conditional knockout mouse and the control were not detected in forced swim test or open field test (Supplementary Material, Fig. S3 and Fig. S4); these findings and the telemetric analyses of locomotor activity (Fig. 5) confirm that motor skills of the Emx1-Cre+/Arntl-flox+/+ conditional knockout mouse are not impaired and therefore can be omitted in the interpretation of the tail suspension test. Figure 7. View largeDownload slide Tail suspension test of the cerebral cortex-specific conditional Arntl conditional knockout mouse. The Arntl conditional knockout mouse (Emx1-Cre+/Arntl-flox+/+) was compared with the control (Emx1-Cre−/Arntl-flox+/+) to investigate differences in immobility. The time of activity during inescapable stress was 50% longer in the control as compared with the Arntl CKO indicative of a depressive-like behavior in the latter. This reflected a very highly significant difference between genotypes with a two-tailed P-value of < 0.0001 (***) as revealed by use of Student's t-test. Arntl CKO, Arntl conditional knockout mouse (Emx1-Cre+/Arntl-flox+/+). Figure 7. View largeDownload slide Tail suspension test of the cerebral cortex-specific conditional Arntl conditional knockout mouse. The Arntl conditional knockout mouse (Emx1-Cre+/Arntl-flox+/+) was compared with the control (Emx1-Cre−/Arntl-flox+/+) to investigate differences in immobility. The time of activity during inescapable stress was 50% longer in the control as compared with the Arntl CKO indicative of a depressive-like behavior in the latter. This reflected a very highly significant difference between genotypes with a two-tailed P-value of < 0.0001 (***) as revealed by use of Student's t-test. Arntl CKO, Arntl conditional knockout mouse (Emx1-Cre+/Arntl-flox+/+). Norepinephrine Levels are Reduced in the Neocortex and Hippocampus of the Emx1-Cre+/Arntl-flox+/+ Conditional Knockout Mouse Decreased levels of norepinephrine and other monoamines in the brain have been linked to depression as the basis for the monoamine hypothesis (Bunney and Davis 1965). Our findings of a depressive-like behavioral state in the tail suspension test prompted us to investigate the levels of norepinephrine and serotonin in the neocortex and hippocampus of the Emx1-Cre+/Arntl-flox+/+ conditional knockout mouse. Brain structures dissected from mice exposed to a short-term inescapable stress situation were subjected to high pressure liquid chromatography analyses (Fig. 8). In the Emx1-Cre+/Arntl-flox+/+ conditional knockout mouse, the norepinephrine content was significantly reduced in both the neocortex (P < 0.01, Student's t-test) and the hippocampus (P < 0.05, Student's t-test) as compared with Emx1-Cre−/Arntl-flox+/+ controls, suggesting that the depressive-like state of the Emx1-Cre−/Arntl-flox+/+ controls is accompanied by reductions in brain monoamine content. However, levels of serotonin did not differ between genotypes (Fig. 8). Figure 8. View largeDownload slide Norepinephrine and serotonin in the neocortex and hippocampus of the cerebral cortex-specific Arntl conditional knockout mouse. Monoamine content was analyzed in the neocortex and hippocampus of the Arntl conditional knockout mouse (Emx1-Cre+/Arntl-flox+/+) subjected to an inescapable stress situation. Reduced levels of norepinephrine (NE; upper panel) in the Emx1-Cre+/Arntl-flox+/+ conditional knockout were detected both brain structures (**P < 0.001; *P < 0.05; Student's t-test), whereas serotonin (5-HT) contents (lower panel) did not differ between genotypes (P > 0.05, Student's t-test). 5-HT, serotonin; Arntl CKO, Arntl conditional knockout mouse (Emx1-Cre+/Arntl-flox+/+); NE, norepinephrine. Figure 8. View largeDownload slide Norepinephrine and serotonin in the neocortex and hippocampus of the cerebral cortex-specific Arntl conditional knockout mouse. Monoamine content was analyzed in the neocortex and hippocampus of the Arntl conditional knockout mouse (Emx1-Cre+/Arntl-flox+/+) subjected to an inescapable stress situation. Reduced levels of norepinephrine (NE; upper panel) in the Emx1-Cre+/Arntl-flox+/+ conditional knockout were detected both brain structures (**P < 0.001; *P < 0.05; Student's t-test), whereas serotonin (5-HT) contents (lower panel) did not differ between genotypes (P > 0.05, Student's t-test). 5-HT, serotonin; Arntl CKO, Arntl conditional knockout mouse (Emx1-Cre+/Arntl-flox+/+); NE, norepinephrine. Discussion The presence of a central circadian clock in the SCN of the hypothalamus has been well documented (Stephan and Zucker 1972; Reppert and Weaver 2002). However, we have recently shown the existence of a molecular circadian oscillator in neurons of the rodent cerebral cortex (Rath et al. 2013, 2014). This oscillator is driven by the SCN (Rath et al. 2013), but the possible physiological significance of this peripheral oscillator is unknown. To discern the roles of different neuronal clocks, the core circadian clock gene Arntl has previously been deleted specifically in the forebrain (Izumo et al. 2014; Snider et al. 2016), histaminergic neurons of the hypothalamus (Yu et al. 2014) and in the retina (Storch et al. 2007), respectively. However, in the current study, we present data on a novel conditional knockout mouse, in which the Arntl clock gene was specifically deleted in neurons of the cerebral cortex. The deletion of Arntl heavily disrupted the circadian clock machinery of the neocortex and hippocampus and also proved to influence animal behavior and brain monoamine contents. Arntl is an Essential Component of the Molecular Circadian Oscillator in the Cerebral Cortex The molecular circadian clockwork involves oscillating clock gene products encoding transcriptional modulators (Reppert and Weaver 2002). The circadian profiles of clock gene expression, for example, rhythmicity and circadian time of peak expression, in the cerebral cortex of the control mouse were similar to those previously reported in wild-type mice (Jilg et al. 2010; Rath et al. 2014). Deletion of Arntl had profound effects on the molecular clockwork of both the neocortex and the hippocampus affecting the expression of 7 out of 8 clock genes. Although the complex interplay between different feedback mechanisms and redundancies in the molecular circadian clock make it difficult to predict the outcome of specifically deleting one clock gene, our findings establish Arntl as a central and essential component of the cortical circadian oscillator. Our finding that Per1 and Per3 are both affected in the cerebral cortex of the Arntl conditional knockout mouse with reduced and, in the neocortex, arrhythmic expression is in line with the ARNTL/CLOCK heterodimer complex controlling their expression on a daily basis (Gekakis et al. 1998; Jin et al. 1999). In case of Per2, general differences between genotypes were not detectable; however, rhythmic expression in the Arntl conditional knockout mouse was detected in the hippocampus only. In contrast to this finding, low and arrhythmic expression of Per2 in the SCN of the global Arntl knockout mouse has been reported (Bunger et al. 2000; Pfeffer et al. 2009), suggesting that differences in regulatory mechanisms between the central clock of the SCN and the oscillator of the hippocampus exist. Regional differences within the brain in the molecular effect of global deletion of Per1 have been reported (Feillet et al. 2008), and it is possible that the action of an Arntl paralog (Shi et al. 2010) may be sufficient to drive rhythmic period expression in the hippocampus. The finding that Cry1 and Cry2 are upregulated in the cerebral cortex of the Arntl conditional knockout mouse is in agreement with the ARNTL/CLOCK complex acting as a suppressor on expression of cryptochrome genes (Kondratov et al. 2006). A similar upregulation of cryptochromes has also been reported in the retina-specific Arntl conditional knockout mouse (Storch et al. 2007). In contrast, expression levels of cryptochrome genes along with other clock genes are reduced in the SCN of the Clock knockout (Kume et al. 1999; Debruyne et al. 2006). Another role of the ARNTL/CLOCK heterodimer is to initiate transcription of Nr1d1 (Preitner et al. 2002), and this seems to explain the reduced and nonrhythmic levels of Nr1d1 transcripts in both the neocortex and hippocampus of the Arntl conditional knockout. As part of a negative feedback loop, NR1D1 itself is known to directly repress the expression of Clock (Crumbley and Burris 2011), and this may explain our finding of increased levels of Clock transcripts in the cerebral cortex of the Arntl conditional knockout mouse. As opposed to the other genes analyzed in this study, the Dbp gene is not an intrinsic part of the molecular circadian clock, but a circadian clock-controlled output gene (Lopez-Molina et al. 1997) with a circadian expression profile driven by ARNTL/CLOCK (Ripperger and Schibler 2006). The reduced and arrhythmic expression of Dbp reported here represents a final line of evidence of a malfunctioning circadian oscillator in the cerebral cortex of the Arntl conditional knockout mouse. Arntl in the Cerebral Cortex Does not Influence Traditional Circadian Parameters Normal activity rhythms in the Arntl global knockout mouse under LD have been reported (Bunger et al. 2000), whereas rhythm instability is prominent in DD. A recent investigation of the Arntl global knockout mouse suggested that the knockout mouse has a later chronotype and exhibits reduced rhythm robustness in LD (Pfeffer et al. 2015). These previously reported differences do not seem to be related to the oscillator of the cerebral cortex, which, as we show here, does not influence traditional circadian parameters. The circadian oscillator of the cerebral cortex does not influence the time required to obtain full resynchronization after experimental jet-lag, which is in contrast to the Arntl global knockout, which resynchronizes faster than wild-type animals (Pfeffer et al. 2015), but during the resynchronization period, minor, though significant, differences in initial resynchronization rates between the genotypes are detectable. Changes in the sensitivity of the circadian system to out of phase light have been reported in studies of the global Arntl knockout mouse (Pfeffer et al. 2009); however, in the Arntl conditional knockout mouse of the present study, these initial differences in the resynchronization process are more likely to be a secondary effect of changes in mood-related behavior, as discussed below. Deletion of Arntl in the Cerebral Cortex Induces a Depressive-Like State in Behavior and Norepinephrine Contents Major depressive disorder has been associated with changes in rhythmic clock gene expression in the human brain, including the neocortex and hippocampus (Li et al. 2013). In addition, changes in mood-related behavior have been reported in global clock gene knockout mouse strains (Schnell et al. 2015). With regard to extrahypothalamic clock gene expression in the brain, a number of studies have reported differential expression of clock genes in animal models of depression, including changes in the cerebral cortex (Koresh et al. 2012; Jiang et al. 2013; Calabrese et al. 2016; Christiansen et al. 2016). In the current study, we found that the lack of Arntl in neurons of the hippocampus and neocortex induces a depressive-like behavior. This result adds to a very limited amount of existing evidence (Jiang et al. 2013) in support of the possibility that changes in clock gene expression in the cerebral cortex could be involved as a factor in the etiology of depression. Our finding that the level of norepinephrine was reduced in the neocortex and hippocampus of the Arntl conditional knockout mouse suggests an imbalance in the monoamine system, which would selectively involve norepinephrine, whereas serotonin levels are unaffected. We therefore speculate that the etiology of the observed depressive-like state may involve reduced levels of norepinephrine in the cerebral cortex. Similar reductions in norepinephrine-content of the cerebral cortex have been reported in a rat model of depression (Weiss et al. 1981). Notably, the reduced level of norepinephrine in the cerebral cortex does not imply a local clock-induced alteration of monoamine synthesis in cortical neurons, since norepinephrine is predominantly synthesized in the locus caeruleus of the brain stem. The connection between the local oscillator of the cerebral cortex and changes in behavior and monoamines implies a functional relationship with the deletion of the circadian clockwork being the causal factor; however, the signaling mechanism linking clock gene expression in the cerebral cortex to a response in behavior and neurotransmitter contents is enigmatic. Recent evidence from studies on the hippocampus suggests that PER1 is important for local subcellular signaling in circadian modulation of memory performance via the cAMP response element binding protein (Rawashdeh et al. 2014, 2016); however, even in the SCN, the mechanism linking rhythmic clock gene expression and circadian changes in neuronal firing activity (Herzog et al. 1998; Albus et al. 2002; Nakamura et al. 2002) has not been established. In this context, although the current study clearly shows a causal connection between deletion of Arntl and changes in mood-related behavior and neurotransmitter contents, further studies are needed to unravel the underlying mechanism. Supplementary Material Supplementary material is available at Cerebral Cortex online. Abbreviations Arntl, arylhydrocarbon receptor nuclear translocator-like; CT, circadian time; Clock, circadian locomotor output cycles kaput; Cry, cryptochrome; Dbp, D site of albumin promoter binding protein; DD, dark:dark (constant darkness); Emx1, empty spiracles 1; 12L:12D, 12 h light:12 h dark cycle; LL, light:light (constant light); MoA, median of activity; Nr1d1, nuclear receptor subfamily one group D member 1; Per, period; SCN, suprachiasmatic nucleus; ZT, Zeitgeber time. Funding Lundbeck Foundation (Grant number R108-A10301 to M.F.R.), Mental Health Services of the Capital Region of Denmark (PhD-stipend for T.B. granted to P.W.), the Carlsberg Foundation (Grant number CF15-0515 to M.F.R.), Agnes og Poul Friis Fond (grant number 1208009 to M.F.R.), Brødrene Hartmanns Fond (Grant number 27227 to M.F.R.), and Beckett Fonden (Grant number 44081 to M.F.R.). Notes We wish to thank Rikke Lundorf and Jytte Rasmussen for expert technical assistance. Conflict of Interest: None declared. 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TI - The Circadian Oscillator of the Cerebral Cortex: Molecular, Biochemical and Behavioral Effects of Deleting the Arntl Clock Gene in Cortical Neurons
JO - Cerebral Cortex
DO - 10.1093/cercor/bhw406
DA - 2018-02-01
UR - https://www.deepdyve.com/lp/oxford-university-press/the-circadian-oscillator-of-the-cerebral-cortex-molecular-biochemical-xm0LCSntUn
SP - 644
EP - 657
VL - 28
IS - 2
DP - DeepDyve
ER -