Disrupted sleep-wake regulation in the MCI-Park mouse model of Parkinson's disease.

Disrupted sleep has a profound adverse impact on lives of Parkinson's disease (PD) patients and their caregivers. Sleep disturbances are exceedingly common in PD, with substantial heterogeneity in type, timing, and severity. Among the most common sleep-related symptoms reported by PD patients are insomnia, excessive daytime sleepiness, and sleep fragmentation, characterized by interruptions and decreased continuity of sleep. Alterations in brain wave activity, as measured on the electroencephalogram (EEG), also occur in PD, with changes in the pattern and relative contributions of different frequency bands of the EEG spectrum to overall EEG activity in different vigilance states consistently observed. The mechanisms underlying these PD-associated sleep-wake abnormalities are poorly understood, and they are ineffectively treated by conventional PD therapies. To help fill this gap in knowledge, a new progressive model of PD - the MCI-Park mouse - was studied. Near the transition to the parkinsonian state, these mice exhibited significantly altered sleep-wake regulation, including increased wakefulness, decreased non-rapid eye movement (NREM) sleep, increased sleep fragmentation, reduced rapid eye movement (REM) sleep, and altered EEG activity patterns. These sleep-wake abnormalities resemble those identified in PD patients. Thus, this model may help elucidate the circuit mechanisms underlying sleep disruption in PD and identify targets for novel therapeutic approaches.

Forward and reverse genetic approaches to behavior in the mouse.

Modern molecular genetic and genomic approaches are revolutionizing the study of behavior in the mouse. "Reverse genetics" (from gene to phenotype) with targeted gene transfer provides a powerful tool to dissect behavior and has been used successfully to study the effects of null mutations in genes implicated in the regulation of long-term potentiation and spatial learning in mice. In addition, "forward genetics" (from phenotype to gene) with high-efficiency mutagenesis in the mouse can uncover unknown genes and has been used to isolate a behavioral mutant of the circadian system. With the recent availability of high-density genetic maps and physical mapping resources, positional cloning of virtually any mutation is now feasible in the mouse. Together, these approaches permit a molecular analysis of both known and previously unknown genes regulating behavior.

Role of mouse cryptochrome blue-light photoreceptor in circadian photoresponses.

Cryptochromes are photoactive pigments in the eye that have been proposed to function as circadian photopigments. Mice lacking the cryptochrome 2 blue-light photoreceptor gene (mCry2) were tested for circadian clock-related functions. The mutant mice had a lower sensitivity to acute light induction of mPer1 in the suprachiasmatic nucleus (SCN) but exhibited normal circadian oscillations of mPer1 and mCry1 messenger RNA in the SCN. Behaviorally, the mutants had an intrinsic circadian period about 1 hour longer than normal and exhibited high-amplitude phase shifts in response to light pulses administered at circadian time 17. These data are consistent with the hypothesis that CRY2 protein modulates circadian responses in mice and suggest that cryptochromes have a role in circadian photoreception in mammals.

Mutagenesis and mapping of a mouse gene, Clock, essential for circadian behavior.

In a search for genes that regulate circadian rhythms in mammals, the progeny of mice treated with N-ethyl-N-nitrosourea (ENU) were screened for circadian clock mutations. A semidominant mutation, Clock, that lengthens circadian period and abolishes persistence of rhythmicity was identified. Clock segregated as a single gene that mapped to the midportion of mouse chromosome 5, a region syntenic to human chromosome 4. The power of ENU mutagenesis combined with the ability to clone murine genes by map position provides a generally applicable approach to study complex behavior in mammals.

Mammalian circadian autoregulatory loop: a timeless ortholog and mPer1 interact and negatively regulate CLOCK-BMAL1-induced transcription.

We report the cloning and mapping of mouse (mTim) and human (hTIM) orthologs of the Drosophila timeless (dtim) gene. The mammalian Tim genes are widely expressed in a variety of tissues; however, unlike Drosophila, mTim mRNA levels do not oscillate in the suprachiasmatic nucleus (SCN) or retina. Importantly, hTIM interacts with the Drosophila PERIOD (dPER) protein as well as the mouse PER1 and PER2 proteins in vitro. In Drosophila (S2) cells, hTIM and dPER interact and translocate into the nucleus. Finally, hTIM and mPER1 specifically inhibit CLOCK-BMAL1-induced transactivation of the mPer1 promoter. Taken together, these results demonstrate that mTim and hTIM are mammalian orthologs of timeless and provide a framework for a basic circadian autoregulatory loop in mammals.

The circadian clock mutation alters sleep homeostasis in the mouse.

The onset and duration of sleep are thought to be primarily under the control of a homeostatic mechanism affected by previous periods of wake and sleep and a circadian timing mechanism that partitions wake and sleep into different portions of the day and night. The mouse Clock mutation induces pronounced changes in overall circadian organization. We sought to determine whether this genetic disruption of circadian timing would affect sleep homeostasis. The Clock mutation affected a number of sleep parameters during entrainment to a 12 hr light/dark (LD 12:12) cycle, when animals were free-running in constant darkness (DD), and during recovery from 6 hr of sleep deprivation in LD 12:12. In particular, in LD 12:12, heterozygous and homozygous Clock mutants slept, respectively, approximately 1 and approximately 2 hr less than wild-type mice, and they had 25 and 51% smaller increases in rapid eye movement (REM) sleep during 24 hr recovery, respectively, than wild-type mice. The effects of the mutation on sleep are not readily attributable to differential entrainment to LD 12:12 because the baseline sleep differences between genotypes were also present when animals were free-running in DD. These results indicate that genetic alterations of the circadian clock system and/or its regulatory genes are likely to have widespread effects on a variety of sleep and wake parameters, including the homeostatic regulation of sleep.

The mouse Clock mutation behaves as an antimorph and maps within the W19H deletion, distal of Kit.

Clock is a semidominant mutation identified from an N-ethyl-N-nitrosourea mutagenesis screen in mice. Mice carrying the Clock mutation exhibit abnormalities of circadian behavior, including lengthening of endogenous period and loss of rhythmicity. To identify the gene affected by this mutation, we have generated a high-resolution genetic map (> 1800 meioses) of the Clock locus. We report that Clock is 0.7 cM distal of Kit on mouse chromosome 5. Mapping shows that Clock lies within the W19H deletion. Complementation analysis of different Clock and W19H compound genotypes indicates that the Clock mutation behaves as an antimorph. This antimorphic behavior of Clock strongly argues that Clock defines a gene centrally involved in the mammalian circadian system.

High-resolution mapping of a novel genetic locus regulating voluntary physical activity in mice.

Both human beings and animals exhibit substantial inter-individual variation in voluntary physical activity, and evidence indicates that a significant component of this variation is because of genetic factors. However, little is known of the genetic basis underlying central regulation of voluntary physical activity in mammals. In this study, using an F(2) intercross population and interval-specific congenic strains (ISCS) derived from the C57BL/6J strain and a chromosome 13 substitution strain, C57BL/6J-Chr13A/J/NA/J, we identified a 3.76-Mb interval on chromosome 13 containing 25 genes with a significant impact on daily voluntary wheel running activity in mice. Brain expression and polymorphisms between the C57BL/6J and A/J strains were examined to prioritize candidate genes. As the dopaminergic pathway regulates motor movement and motivational behaviors, we tested its function by examining cocaine-induced locomotor responses in ISCS with different levels of activity. The low-activity ISCS exhibited a significantly higher response to acute cocaine administration than the high-activity ISCS. Expression analysis of key dopamine-related genes (dopamine transporter and D1, D2, D3, D4 and D5 receptors) revealed that expression of D1 receptor was higher in the low-activity ISCS than in the high-activity ISCS in both the dorsal striatum and nucleus accumbens. Pathway analysis implicated Tcfap2a, a gene found within the 3.76-Mb interval, involved in the D1 receptor pathway. Using a luciferase reporter assay, we confirmed that the transcriptional factor, Tcfap2a, regulates the promoter activity of the D1 receptor gene. Thus, Tcfap2a is proposed as a candidate genetic regulator of the level of voluntary physical activity through its influence on a dopaminergic pathway.

Photoperiodic responses differ among inbred strains of golden hamsters (Mesocricetus auratus).

Inbred strains of golden hamsters differ in both the free-running period of the circadian rhythm of locomotor activity in constant darkness, and in the phase angle of entrainment of activity to a 14L:10D cycle. To determine whether these differences in circadian entrainment affect photoperiodic time measurement, we measured the critical photoperiod for maintaining testicular function as well as the rate of response for four different inbred strains (MHA/SsLak, LSH/SsLak, BIO 1.5, and BIO 87.20) and an outbred stock (Lak:LVG(SYR)) of golden hamsters. Hamsters of each group were maintained for 12 wk under one of five different LD cycles. Animals of all groups maintained testis size in 14L:10D and 12.5L:11.5D. Significant strain differences were observed in the critical photoperiod for maintaining testis size after 12 wk; the LSH/SsLak inbred strain showed complete testicular regression during exposure to 12L:12D, while little change was observed in any of the other strains under this photoperiod. Some degree of testicular regression was observed in all groups exposed to 11.5L:12.5D, while complete regression was observed in all animals exposed to 6L:18D. The rate of testicular regression differed markedly between the different groups under both 6L:18D and 11.5L:12.5D. The differences in critical photoperiod and rate of testicular regression observed between the various strains could not be correlated with the known strain differences in entrainment or circadian period, indicating that genetic differences in photoperiodic response are not related to the genetic differences in circadian rhythmicity.(ABSTRACT TRUNCATED AT 250 WORDS)

Overview of circadian rhythms.

The daily light-dark cycle governs rhythmic changes in the behavior and/or physiology of most species. Studies have found that these changes are governed by a biological clock, which in mammals is located in two brain areas called the suprachiasmatic nuclei. The circadian cycles established by this clock occur throughout nature and have a period of approximately 24 hours. In addition, these circadian cycles can be synchronized to external time signals but also can persist in the absence of such signals. Studies have found that the internal clock consists of an array of genes and the protein products they encode, which regulate various physiological processes throughout the body. Disruptions of the biological rhythms can impair the health and well-being of the organism.

Reduced light sensitivity of the circadian clock in a hypopigmented mouse mutant.

Pink-eyed dilution (p/p) is a recessive mutation in mice which results in reduced pigmentation of the retinal pigment epithelium, as well as alterations in visual pathways and function. We investigated whether this mutation also affects light information reaching the circadian clock. Entrainment to a 12 h light 12 h dark cycle and the free-running period in constant darkness were not affected by this mutation. Phase shifts in response to 1 h light pulses consisting of bright white light at either circadian time 16 or 24 also did not differ between mutant and wild-type C57BL/6J mice. However, when 5 min, 502 nm light pulses of 1.2 x 10(-1) microW/cm2 or 4 x 10(-2) microW/cm2 were given at circadian time 16, the mutant mice responded with significantly smaller phase shifts than the wild-type mice. When animals were transferred to constant light, the free-running period of wild-type mice was longer than that of mutant mice, a finding which is consistent with a sensitivity difference between mutant and wild-type mice. Horseradish peroxidase tracing of retinal innervation of the hypothalamic suprachiasmatic nuclei (SCN)--the location of a circadian pacemaker--revealed a reduced innervation of the SCN in mutant mice compared with wild-type mice. The total volume of the SCN, as determined by neutral red stain, was also reduced in mutant mice, although not to as great an extent as the retinal innervation. Taken together, these results indicate that while basic characteristics of circadian clock function are not altered by the pink-eyed dilution mutation, the sensitivity of the clock to light is reduced.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacological and genetic approaches for the study of circadian rhythms in mammals.

Two different approaches have been utilized to study the controlling mechanisms that underlie the generation and entrainment of circadian rhythms in mammals. The use of specific drugs to alter the period and/or the phase of circadian rhythms has provided new insights into both the pathways by which environmental information reaches the mammalian circadian pacemaker in the suprachiasmatic nuclei (SCN) and the cellular and neurochemical events within the SCN itself which are involved in circadian rhythmicity. A second approach, which seeks to exploit genetic differences in the properties of the circadian system, holds the promise of eventually defining the cellular and molecular events that are part of the clock itself, the events that underlie the entrainment of the circadian clock by environmental factors, and the expression of overt rhythms driven by the clock. It is anticipated that the pharmacological and genetic approaches to the study of circadian rhythms will complement each other as the underlying physiological mechanisms of the circadian clock system become defined.

Rapid photoperiod-induced increase in detectable GnRH mRNA-containing cells in Siberian hamster.

To determine whether changes in gonadotropin-releasing hormone (GnRH) neurons are early indicators of photostimulation, Siberian hamsters were placed in short days (6:18-h light-dark) at 3 (experiment 1) or 6 (experiment 2) wk of age where they were held for 3 (experiment 1) or 4 (experiment 2) wk. Hamsters were then moved to long photoperiod (16:8-h light-dark). In experiment 1, brains were collected 1-21 days after transfer from short to long days. In experiment 2, brains were collected only on the second morning of long day exposure. Long and short day controls were included in both experiments. Cells containing GnRH mRNA, as visualized by in situ hybridization, were counted. As expected, there were no differences in the number of detectable GnRH mRNA-containing cells among animals chronically exposed to long or short photoperiods. However, on the second morning after transfer from short to long photoperiod, a positive shift in the distribution of GnRH mRNA-containing cells occurred relative to the respective controls in the two experiments. Increases in follicle-stimulating hormone secretion and gonadal growth occurred days later. In conclusion, a rapid but transient increase in the distribution of detectable GnRH mRNA-containing cells is an early step in the photostimulation of the hypothalamic-pituitary-gonadal axis.

Depressive-like behavior and stress reactivity are independent traits in a Wistar Kyoto x Fisher 344 cross.

Depression is a heritable disorder that is often precipitated by stress. Abnormalities of the stress-reactive hypothalamic-pituitary-adrenal (HPA) axis are also common in depressed patients. In animal models, the forced swim test (FST) is the most frequently used test of depressive-like behavior. We have used a proposed animal model of depression, the Wistar Kyoto (WKY) rat, to investigate the relationship as well as the mode of inheritance of FST behaviors and HPA measures. Through reciprocal breeding of WKY and F344 parent strains and brother-sister breeding of the F1 generation, we obtained 486 F2 animals. Parent, F1 and F2 animals were tested in the FST. Blood samples were collected for determination of basal and stress (10-min restraint) plasma corticosterone (CORT) levels, and adrenal weights were measured. We found that all measures were heritable to some extent and that this heritability was highly sex dependent. Both correlation and factor analyses of the F2 generation data demonstrate that FST behavior and HPA axis measures are not directly related. Thus, the underlying genetic components of depressive-like behavior and HPA axis abnormalities are likely to be disparate in the segregating F2 generation of a WKY x F344 cross.

Functional identification of the mouse circadian Clock gene by transgenic BAC rescue.

As a complementary approach to positional cloning, we used in vivo complementation with bacterial artificial chromosome (BAC) clones expressed in transgenic mice to identify the circadian Clock gene. A 140 kb BAC transgene completely rescued both the long period and the loss-of-rhythm phenotypes in Clock mutant mice. Analysis with overlapping BAC transgenes demonstrates that a large transcription unit spanning approximately 100,000 base pairs is the Clock gene and encodes a novel basic-helix-loop-helix-PAS domain protein. Overexpression of the Clock transgene can shorten period length beyond the wild-type range, which provides additional evidence that Clock is an integral component of the circadian pacemaking system. Taken together, these results provide a proof of principle that "cloning by rescue" is an efficient and definitive method in mice.

Positional cloning of the mouse circadian clock gene.

We used positional cloning to identify the circadian Clock gene in mice. Clock is a large transcription unit with 24 exons spanning approximately 100,000 bp of DNA from which transcript classes of 7.5 and approximately 10 kb arise. Clock encodes a novel member of the bHLH-PAS family of transcription factors. In the Clock mutant allele, an A-->T nucleotide transversion in a splice donor site causes exon skipping and deletion of 51 amino acids in the CLOCK protein. Clock is a unique gene with known circadian function and with features predicting DNA binding, protein dimerization, and activation domains. CLOCK represents the second example of a PAS domain-containing clock protein (besides Drosophila PERIOD), which suggests that this motif may define an evolutionarily conserved feature of the circadian clock mechanism.

Automated measurement of mouse freezing behavior and its use for quantitative trait locus analysis of contextual fear conditioning in (BALB/cJ x C57BL/6J)F2 mice.

The most commonly measured mouse behavior in fear conditioning tests is freezing. A technical limitation, particularly for genetic studies, is the method of direct observation used for quantifying this response, with the potential for bias or inconsistencies. We report the use of a computerized method based on latency between photobeam interruption measures as a reliable scoring criterion in mice. The different computer measures obtained during contextual fear conditioning tests showed high correlations with hand-scored freezing; r values ranged from 0.87 to 0.94. Previously reported strain differences between C57BL/6J and DBA/2J in context-dependent fear conditioning were also detected by the computer-based system. In addition, the use of computer-scored freezing of 199 (BALB/cJ x C57BL/6J)F2 mice enabled us to detect a suggestive gender-dependent chromosomal locus for contextual fear conditioning on distal chromosome 8 by QTL analysis. Automation of freeze scoring would significantly increase efficiency and reliability of this learning and memory test.

Genome-wide epistatic interaction analysis reveals complex genetic determinants of circadian behavior in mice.

Genetic heterogeneity underlies many phenotypic variations observed in circadian rhythmicity. Continuous distributions in measures of circadian behavior observed among multiple inbred strains of mice suggest that the inherent contributions to variability are polygenic in nature. To identify genetic loci that underlie this complex behavior, we have carried out a genome-wide complex trait analysis in 196 (C57BL/6J X BALB/cJ)F(2) hybrid mice. We have characterized variation in this panel of F(2) mice among five circadian phenotypes: free-running circadian period, phase angle of entrainment, amplitude of the circadian rhythm, circadian activity level, and dissociation of rhythmicity. Our genetic analyses of these phenotypes have led to the identification of 14 loci having significant effects on this behavior, including significant main effect loci that contribute to three of these phenotypic measures: period, phase, and amplitude. We describe an additional locus detection method, genome-wide genetic interaction analysis, developed to identify locus pairs that may interact epistatically to significantly affect phenotype. Using this analysis, we identified two additional pairs of loci that have significant effects on dissociation and activity level; we also detected interaction effects in loci contributing to differences of period, phase, and amplitude. Although single gene mutations can affect circadian rhythms, the analysis of interstrain variants demonstrates that significant genetic complexity underlies this behavior. Importantly, most of the loci that we have detected by these methods map to locations that differ from the nine known clock genes, indicating the presence of additional clock-relevant genes in the mammalian circadian system. These data demonstrate the analytical value of both genome-wide complex trait and epistatic interaction analyses in further understanding complex phenotypes, and point to promising approaches for genetic analysis of such phenotypes in other mammals, including humans.

Differential regulation of mammalian period genes and circadian rhythmicity by cryptochromes 1 and 2.

Cryptochromes regulate the circadian clock in animals and plants. Humans and mice have two cryptochrome (Cry) genes. A previous study showed that mice lacking the Cry2 gene had reduced sensitivity to acute light induction of the circadian gene mPer1 in the suprachiasmatic nucleus (SCN) and had an intrinsic period 1 hr longer than normal. In this study, Cry1(-/-) and Cry1(-/-)Cry2(-/-) mice were generated and their circadian clocks were analyzed at behavioral and molecular levels. Behaviorally, the Cry1(-/-) mice had a circadian period 1 hr shorter than wild type and the Cry1(-/-)Cry2(-/-) mice were arrhythmic in constant darkness (DD). Biochemically, acute light induction of mPer1 mRNA in the SCN was blunted in Cry1(-/-) and abolished in Cry1(-/-)Cry2(-/-) mice. In contrast, the acute light induction of mPer2 in the SCN was intact in Cry1(-/-) and Cry1(-/-)Cry2(-/-) animals. Importantly, in double mutants, mPer1 expression was constitutively elevated and no rhythmicity was detected in either 12-hr light/12-hr dark or DD, whereas mPer2 expression appeared rhythmic in 12-hr light/12-hr dark, but nonrhythmic in DD with intermediate levels. These results demonstrate that Cry1 and Cry2 are required for the normal expression of circadian behavioral rhythms, as well as circadian rhythms of mPer1 and mPer2 in the SCN. The differential regulation of mPer1 and mPer2 by light in Cry double mutants reveals a surprising complexity in the role of cryptochromes in mammals.