Disrupted Circadian Rhythms in a Mouse Model of Schizophrenia

Summary Sleep and circadian rhythm disruption has been widely observed in neuropsychiatric disorders including schizophrenia [1] and often precedes related symptoms [2]. However, mechanistic basis for this association remains unknown. Therefore, we investigated the circadian phenotype of blind-drunk (Bdr), a mouse model of synaptosomal-associated protein (Snap)-25 exocytotic disruption that displays schizophrenic endophenotypes modulated by prenatal factors and reversible by antipsychotic treatment [3, 4]. Notably, SNAP-25 has been implicated in schizophrenia from genetic [5–8], pathological [9–13], and functional studies [14–16]. We show here that the rest and activity rhythms of Bdr mice are phase advanced and fragmented under a light/dark cycle, reminiscent of the disturbed sleep patterns observed in schizophrenia. Retinal inputs appear normal in mutants, and clock gene rhythms within the suprachiasmatic nucleus (SCN) are normally phased both in vitro and in vivo. However, the 24 hr rhythms of arginine vasopressin within the SCN and plasma corticosterone are both markedly phase advanced in Bdr mice. We suggest that the Bdr circadian phenotype arises from a disruption of synaptic connectivity within the SCN that alters critical output signals. Collectively, our data provide a link between disruption of circadian activity cycles and synaptic dysfunction in a model of neuropsychiatric disease.


Supplemental Figures and Tables
The rest/activity profiles of a human subject with schizophrenia illustrates that circadian reversal and activity fragmentation, often accredited to schizophrenia, is also observed in Bdr mice compared to the more stable profile of controls in each case. For details of the human subjects, see Supplemental Experimental Procedures.

Figure S3. Video Tracking Data, Related to Figure 3
Bdr and wild-type mice were observed over a 24-hr 12:12 LD cycle (N = 6). There was no significant difference in the total distance travelled (p = 0.5648, t-test) (A), although Bdr mice travelled proportionally more in the light phase and less in the dark phase (2-way ANOVA main effect of genotype: p = 0.0033) (B). This phase advance of activity is also reflected in the total distance travelled plotted over 24 hr (C) and quantified by a significant increase in the distance travelled by Bdr mice compared to controls in the last 2 hr of the light phase (ZT10-12) compared with a significant decrease towards the end of the dark phase (ZT18-20) (2-way ANOVA effect of genotype: p = 0.0011; Bonferroni post-hoc tests: ZT8-10 p = 0.8067 , ZT10-12 p = 0.0249 , ZT18-20 p = 0.0321) (D). Mice of both genotypes showed a robust masking of activity by light at ZT14 to ZT15 (2-way ANOVA main effect of genotype: p = 0.976) (E). As a correlate of sleep, there was no difference in total immobility over 24 hr between genotypes (p = 0.9284, t-test) (F). All data are expressed as mean ± SEM. Assessment of immobile episodes and therefore sleep fragmentation in the light phase did not show any significant differences between genotypes (data not shown). show comparable mRNA abundance between Bdr and wild-type cohorts; there was no effect of genotype on SCN clock gene expression at any zeitgeber time (ZT) (2-way ANOVA main effect of genotype: p = 0.2647). (E) Nts (Neurotensin) expression is significantly increased at ZT11 in Bdr mice (N = 4, p = 0.02864, ANOVA) compared to the wild-types with comparable levels at ZT11. (F) Bdr mice show comparable Tac1 (substance P) expression to wild-type controls at both ZT6 and ZT11. All data expressed as mean ± SEM.  All statistically significant differences over 1.5-fold are shown. Down-regulation of a transcript in Bdr versus WT is indicated using a negative symbol.

Circadian Screening
Age-matched (12 week) male Bdr (Bdr/+) animals (N = 12) with littermate male controls (+/+) were housed in cages (44 x 26 x 12 cm) fitted with running wheels maintained at constant temperature and humidity, with ad libitum food and water. 6 cages were arranged in light-controlled chambers with externally controlled fluorescent tubes, supplemented with ND filters to bring the lighting environment down to the required intensity at cage level, as assessed with a cage bottom positioned lux meter. A standardised behavioural screen was developed to examine common circadian paradigms ( Figure S1C and Table S1). Data analysis was carried out using 10 min bins of wheel running activity using Clocklab (Actimetrics) and Matlab software. Non-parametric assessments of rhythmicity were carried out using 6 min bins of wheel running activity using Sleep analysis software (Cambridge Neurotechnology Ltd). Negative masking was examined in age-matched (14 week) male Bdr and +/+ littermate control animals (N = 6) using a 10 lux and 150 lux one hr light pulse at ZT14. For the type II phase delay, following entrainment to a 150 lux LD cycle, age-matched (12 week) male Bdr and +/+ littermate control animals (N = 9-10) were subjected to a day of complete darkness from ZT0 with a 150 lux 1 hr light pulse administered at CT16. This Aschoff type II protocol has been previously used for transgenic mice [2] due to the ease of incorporating it within the circadian screen and to minimise the handling of the animals prior to the light pulse. The phase shift was determined by eye-fitting a line through at least 5 consecutive days of the activity onset in LD preceding the light pulse and in DD following the light pulse [3]. All additional assays were carried out on mice under the same conditions but with no access to running wheels.

Nonparametric Circadian Rhythm Analyses (NPCRA)
The nonparametric analysis of circadian rhythms as originally carried out by Van Someren et al. [4] and was performed using Actiwatch Sleep Analysis software (Cambridge Neurotechnology). Intradaily stability and interdaily variability were assessed using 10 day activity profiles exported from the Actimetrics Clocklab software package in 6 min bins. This method has been previously used to assess circadian rhythmicity in a transgenic mouse model of Huntington's disease [5].

Video Tracking
Age-matched 12 week old male Bdr (N = 6) and wild-type (N = 6) littermate control mice were assessed for activity by video tracking in home cages (44 x 26 x 12 cm) using a method that has been proven to correlate almost identically to EMG / telemetry methods [6]. An infrared-capable camera (Sentient) was fitted above each cage for continuous 24-hr recording within light controlled chambers. Mice had free access to food and water, but were always in the field of view of the camera. ANYmaze software (Stoelting) recorded activity parameters including distance travelled. Immobile time was defined as a period of immobility of more than 40 seconds and used as a sleep correlate [6]. All mice were entrained to a 12:12 LD (150 lux) cycle for 10 days, followed by a 2 day 12:12 LD (150 lux) habituation period in the cages equipped for video tracking. All mice were subjected to 24 hr of 12:12 LD baseline recording following this 2 day habituation period. The subsequent 24 hr involved a one hr 150 lux light pulse at ZT14 to measure the suppression of activity by light.

Pupillometry
Relative pupil constriction was assessed in 12 week female Bdr and wild-type (N = 6) mice. Following one hr of dark adaption, the left eye was dilated with atropine and stimulated via a ganzfeld with 480nm (10HBW Andover filtered xenon arc light source) light of 14.6 log quanta. The consensual response of the right eye was recorded using an infrared camera over 27 s. The images were captured at a frame rate of 5-10 fps using a Prosilica gigabit ethernet camera and the data was captured in the form of image files via software provided by BRSL Ltd. running through a CVB platform (Stemmer Imaging). Relative pupil constriction (%) was calculated for each individual mouse by taking the smallest constricted area as a percentage of the preconstricted maximum pupil size. The pupil area per frame captured was calculated using Image J software (NIH).

Suprachiasmatic Nucleus (SCN) Regional Microarray and qPCR
Age-matched (12 week) male Bdr and wild-type animals (N = 5) were individually housed and entrained to a 12:12 LD cycle at 150 lux for 10 days prior to the day of tissue collection. Following cervical dislocation, brains were sectioned on a steel matrix between bregma 0 and 1 mm posterior. Tissue punches were taken using a 1 mm sample corer (FST Ltd.) from regions of the SCN and cortex and then snapfrozen. Total RNA was extracted using a phenol chloroform method in combination with the RNeasyMicro kit (Qiagen) and the quality was assessed on a 2100 BioAnalyzer using the RNA 6000 Pico Assay (Agilent Technologies). SYBR green master mix (Invitrogen) qPCR reactions were run on a Step One Real Time PCR System (Applied Biosystems) with two housekeeping genes (Psmb2 and Gps1). Tissue validation was undertaken using the specific SCN marker Six6 to assess gene expression levels in the SCN compared to the control cortical sample, normalised to housekeeping gene expression. All Ct values were transformed using 2 -Ct prior to target gene expression being normalised to housekeeping gene expression for each independent sample, presented as relative RNA abundance. For the microarray studies, RNA integrity was first assessed on a BioAnalyzer; all samples had a RNA Integrity Number (RIN) ≥ 8 (Agilent Laboratories). Labelled sense ssDNA for hybridization was generated from 110ng starting RNA with the Affymetrix GeneChip WT sense target labeling and control reagents kit (Affymetrix) according to the manufacturer's instructions. Sense ssDNA was fragmented and the distribution of fragment lengths was measured on the BioAnalyser. The fragmented ssDNA was labeled and hybridized to the Affymetrix Mouse Gene 1.0 ST Array (Affymetrix). Chips were processed on an Affymetrix GeneChip Fluidics Station 450 and Scanner 3000. Cel files were generated using Command Console (Affymetrix). Arrays were RMA normalized in GeneSpring GX 11.0 and differentially expressed genes were identified using a Welch t-test with a pvalue cut off of ≤0.05 and a fold change difference of ≥1.5.

In Situ Hybridisation and Light Induction Studies
An independent cohort of age-matched (12 week) male Bdr and wild-type animals (N = 4) were entrained to a 12:12 LD cycle at 150 lux for 10 days prior to the day of tissue collection at ZT6 and 11. Brains were dissected and snap frozen on dry ice in OCT (VWR), followed by sectioning at 12µM. Regions of Avp, Vip and c-fos (18-502, 290-1318 and 560-1463 bp of GenBank accession numbers NM_009732, NM_011702 and NM_010234 respectively) were used for digoxigenin-labeled riboprobe synthesis and hybridization as described previously [7]. Probes were hybridised for 8 hr for Avp and 6 hr for Vip and c-fos. For the light induction studies, an independent cohort of age-matched (12 week) male Bdr and wild-type animals (N = 4) were entrained to a 12:12 LD cycle at 150 lux for 10 days. Brains were dissected either before or after a 30 min light pulse at 150 lux at ZT16 on day 11 [8].

Avp Immunoshistochemistry
An independent cohort of age-matched (12 week) male Bdr and WT animals (N = 4) were entrained to a 12:12 LD cycle at 150 lux for 10 days prior to the day of tissue collection at ZT 2, 6, 10, 14, 18 and 22. Dissected brains were snap frozen on dry ice, followed by sectioning at 12µM. Sections were post-fixed in 4% paraformaldehyde for 4 hr prior to immunostaining. After washes in phosphate buffered saline (PBS), sections were incubated in 3% hydrogen peroxide in methanol and washed again in PBS. Sections were then blocked in 5% normal goat serum with 0.5% Triton X-100, followed by incubation with primary antibodies in the same solution (guinea-pig anti-AVP (Peninsula) 1:5000, 48 hr 4 o C). Biotinylated secondary antibody (2 hr, room temperature) and avidin-biotin complex (ABC) (1 hr, room temperature) treatment was carried out using Vectastain ABC method (Vectorlabs). Staining was visualised using diaminobenzidine (DAB) as the substrate for 2 min. Sections were dehydrated and mounted using Histomount (National Diagnostics). For each animal two sections representing the mid-caudal aspect of the SCN were analyzed bilaterally. Sections were analysed using Axiovision 4.6 (Axiovision) and Image J software (NIH). The SCN was measured within a 200 × 400 μm grid and the signal intensity was calculated after correction for the background.

24-Hr Plasma Corticosterone Profile
Age-matched (12 week) male Bdr and wild-type animals (N = 8) were entrained to a 12:12 LD cycle at 150 lux for 10 days prior blood collection at ZT2, 6, 10, 14, 18 and 22. Plasma corticosterone measurements were calculated using the corticosterone EIA kit (Immunodiagnostic Systems) according to the manufacturer's instructions.

PER2:Luc SCN Organotypic Culture
Brains were removed from pups of at least 10 days of age and sectioned as reported previously [9]. Bioluminescent emissions from PER2::Luc SCN slices were recorded by PMTs for at least 10 days of recording as described previously [9]. Waveforms of rhythmic bioluminescence emission from slices were analyzed in BRASS software (Paul E. Brown and Andrew J. Millar).

Statistics
Statistics for circadian wheel running, all qPCR analysis, IHC and ISH quantification were carried out using the following methods. All statistics were carried out using SPSS v16.0. Student's t-tests were used in cases of direct comparison across one categorical variable (e.g. genotype), and one dependent measurement (Type II phase shift, nonparametric circadian wheel running analyses and pupillometry). Multivariate one-way ANOVAs were used in the case of one categorical variable (e.g. genotype or tissue type), and multiple dependent measurements (e.g. activity across multiple light cycles or gene expression across multiple ZT times). The effects of genotype (circadian wheel running activity, SCN qPCR target gene expression, ISH AVP expression, IHC AVP expression and corticosterone plasma concentration) or tissue type (SCN qPCR Six6 tissue validation) were assessed using pair-wise planned comparisons and these p-values are displayed in the figure legends.
A multivariate two-way ANOVA was used to assess qPCR SCN clock gene expression allowing the comparison of two categorical variables (genotype and clock gene) across multiple dependent variables (gene expression at different ZT times). The effects of genotype were assessed using pair-wise planned comparisons and these p-values are displayed in the figure legends. Statistical analysis of videotracking was carried out using repeated measures two-way ANOVAs (between subjects factor of genotype, within subjects factor of ZT time in 10 min bins and dependent variables of distance travelled or immobile time) with main effects of genotype being followed up with planned comparisons and Bonferroni post-hoc analysis of comparisons which were not accounted for in the experimental design. For direct comparison of total activity or immobility, within a given time frame, Student's t-tests were performed.

Human Data
Human rest-activity patterns are referred to as part of a larger study with two representative examples one from a non-institutionalised patient fulfilling DSM-IV criteria for schizophrenia and another from a healthy unemployed control ( Figure S2) [10]. The patient was stable with respect to psychopathology and social function before entering and during the study. No subject had a history of impaired vision, head injury or significant substance abuse, and the control had no history of any medical condition, including psychiatric illness. Rest-activity and ambient light exposure was monitored simultaneously using a wrist-worn actigraph (Actiwatch-Light, Cambridge Neurotechnology, Ltd) and analysed using CNT Sleep analysis 5.54 software. Recordings were taken from the non-dominant wrist for 6 weeks continuously with a 2-min sampling rate under a self imposed light dark cycle in a home environment. Healthy control: female, 46 years, recording started in August. Patient with disrupted rhythms: male, 34 years, Olanzapine (20mg), recording started in November. All subjects were recruited in West London, United Kingdom. The study protocol was approved by the Ealing and West London Mental Health Trust Local Research Ethics Committee. All participants gave written informed consent.