Circadian and acamprosate modulation of elevated ethanol drinking in mPer2 clock gene mutant mice

The PER2 clock gene modulates ethanol consumption, such that mutant mice not expressing functional mPer2 have altered circadian behavior that promotes higher ethanol intake and preference. Experiments were undertaken to characterize circadian-related behavioral effects of mPer2 deletion on ethanol intake and to explore how acamprosate (used to reduce alcohol dependence) alters diurnal patterns of ethanol intake. Male mPer2 mutant and WT (wild-type) mice were entrained to a 12:12?h light-dark (12L:12D) photocycle, and their locomotor and drinking activities were recorded. Circadian locomotor measurements confirmed that mPer2 mutants had an advanced onset of nocturnal activity of about 2?h relative to WTs, and an increased duration of nocturnal activity (p < .01). Also, mPer2 mutants preferred and consumed more ethanol and had more daily ethanol drinking episodes vs. WTs. Measurements of systemic ethanol using subcutaneous microdialysis confirmed the advanced rise in ethanol intake in the mPer2 mutants, with 24-h averages being ～60 vs. ～25?mM for WTs (p < .01). A 6-day regimen of single intraperitoneal (i.p.) acamprosate injections (300?mg/kg) at zeitgeber time (ZT) 10 did not alter the earlier onset of nocturnal ethanol drinking in the mPer2 mutants, but reduced the overall amplitude of drinking and preference (both p < .01). Acamprosate also reduced these parameters in WTs. These results suggest that elevated ethanol intake in mPer2 mutants may be a partial consequence of an earlier nighttime activity onset and increase in nocturnal drinking activity. The suppressive action of acamprosate on ethanol intake is not due to an altered diurnal pattern of drinking, but rather a decrease in the number of daily drinking bouts and amount of drinking per bout.     

Daily restricted feeding resets the circadian clock in the suprachiasmatic nucleus of CS mice

Circadian rhythms in clock gene expressions in the suprachiasmatic nucleus (SCN) of CS mice and C57BL/6J mice were measured under a daily restricted feeding (RF) schedule in continuous darkness (DD), and entrainment of the SCN circadian pacemaker to RF was examined. After 2-3 wk under a light-dark cycle with free access to food, animals were released into DD and fed for 3 h at a fixed time of day for 3-4 wk. Subsequently, they returned to having free access to food for 2-3 wk. In CS mice, wheel-running rhythms entrained to RF with a stable phase relationship between the activity onset and feeding time, and the rhythms started to free run from the feeding time after the termination of RF. mPer1, mPer2, and mBMAL1 mRNA rhythms in the SCN showed a fixed phase relationship with feeding time, indicating that the circadian pacemaker in the SCN entrained to RF. On the other hand, in C57BL/6J mice, wheel-running rhythms free ran under RF, and clock gene expression rhythms in the SCN showed a stable phase relation not to feeding time but to the behavioral rhythms, indicating that the circadian pacemaker in the SCN did not entrain. These results indicate that the SCN circadian pacemaker of CS mice is entrainable to RF under DD and suggest that CS mice have a circadian clock system that can be reset by a signal associated with feeding time.     

The VPAC(2) receptor is essential for circadian function in the mouse suprachiasmatic nuclei

The neuropeptides pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are implicated in the photic entrainment of circadian rhythms in the suprachiasmatic nuclei (SCN). We now report that mice carrying a null mutation of the VPAC(2) receptor for VIP and PACAP (Vipr2(-/-)) are incapable of sustaining normal circadian rhythms of rest/activity behavior. These mice also fail to exhibit circadian expression of the core clock genes mPer1, mPer2, and mCry1 and the clock-controlled gene arginine vasopressin (AVP) in the SCN. Moreover, the mutants fail to show acute induction of mPer1 and mPer2 by nocturnal illumination. This study highlights the role of intercellular neuropeptidergic signaling in maintenance of circadian function within the SCN.     

mPer2 antisense oligonucleotides inhibit mPER2 expression but not circadian rhythms of physiological activity in cultured suprachiasmatic nucleus neurons

Various day-night rhythms, observed at molecular, cellular, and behavioral levels, are governed by an endogenous circadian clock, predominantly functioning in the hypothalamic suprachiasmatic nucleus (SCN). A class of clock genes, mammalian Period (mPer), is known to be rhythmically expressed in SCN neurons, but the correlation between mPER protein levels and autonomous rhythmic activity in SCN neurons is not well understood. Therefore, we blocked mPer translation using antisense phosphothioate oligonucleotides (ODNs) for mPer1 and mPer2 mRNAs and examined the effects on the circadian rhythm of cytosolic Ca2+ concentration and action potentials in SCN slice cultures. Treatment with mPer2 ODNs (20microM for 3 days) but not randomized control ODNs significantly reduced mPER2 immunoreactivity (-63%) in the SCN. Nevertheless, mPer1/2 ODNs treatment inhibited neither action potential firing rhythms nor cytosolic Ca2+ rhythms. These suggest that circadian rhythms in mPER protein levels are not necessarily coupled to autonomous rhythmic activity in SCN neurons.     

Differential effects of two period genes on the physiology and proteomic profiles of mouse anterior tibialis muscles

The molecular components that generate and maintain circadian rhythms of physiology and behavior in mammals are present both in the brain (suprachiasmatic nucleus; SCN) and in peripheral tissues. Examination of mice with targeted disruptions of either mPer1 or mPer2 has shown that these two genes have key roles in the SCN circadian clock. Here we show that loss of the clock gene mPer2 affects forced locomotor performance in mice without altering muscle contractility. A proteomic analysis revealed that the anterior tibialis muscles of the mPer2 knockout mice had higher levels of glycolytic enzymes such as triose phosphate isomerase and enolase than those of either the wild type or mPer1 knockout mice. In addition, the level of expression of HSP90 in the mPer2 mutant mice was also significantly higher than in wildtype mice. These results suggest that the reduced locomotor endurance of the mPer2 knockout mice reflects a greater dependence on anaerobic metabolism under stress conditions, and that the two canonical clock genes, mPer1 and mPer2, play distinct roles in the physiology of skeletal muscle.     

Sleep rhythmicity and homeostasis in mice with targeted disruption of mPeriod genes

In mammals, sleep is regulated by circadian and homeostatic mechanisms. The circadian component, residing in the suprachiasmatic nucleus (SCN), regulates the timing of sleep, whereas homeostatic factors determine the amount of sleep. It is believed that these two processes regulating sleep are independent because sleep amount is unchanged after SCN lesions. However, because such lesions necessarily damage neuronal connectivity, it is preferable to investigate this question in a genetic model that overcomes the confounding influence of circadian rhythmicity. Mice with disruption of both mouse Period genes (mPer)1 and mPer2 have a robust diurnal sleep-wake rhythm in an entrained light-dark cycle but lose rhythmicity in a free-run condition. Here, we examine the role of the mPer genes on the rhythmic and homeostatic regulation of sleep. In entrained conditions, when averaged over the 24-h period, there were no significant differences in waking, slow-wave sleep (SWS), or rapid eye movement (REM) sleep between mPer1, mPer2, mPer3, mPer1-mPer2 double-mutant, and wild-type mice. The mice were then kept awake for 6 h (light period 6-12), and the mPer mutants exhibited increased sleep drive, indicating an intact sleep homeostatic response in the absence of the mPer genes. In free-run conditions (constant darkness), the mPer1-mPer2 double mutants became arrhythmic, but they continued to maintain their sleep levels even after 36 days in free-running conditions. Although mPer1 and mPer2 represent key elements of the molecular clock in the SCN, they are not required for homeostatic regulation of the daily amounts of waking, SWS, or REM sleep.     

Circadian and Acamprosate Modulation of Elevated Ethanol Drinking in mPer2 Clock Gene Mutant Mice

The PER2 clock gene modulates ethanol consumption, such that mutant mice not expressing functional mPer2 have altered circadian behavior that promotes higher ethanol intake and preference. Experiments were undertaken to characterize circadian-related behavioral effects of mPer2 deletion on ethanol intake and to explore how acamprosate (used to reduce alcohol dependence) alters diurnal patterns of ethanol intake. Male mPer2 mutant and WT (wild-type) mice were entrained to a 12:12?h light-dark (12L:12D) photocycle, and their locomotor and drinking activities were recorded. Circadian locomotor measurements confirmed that mPer2 mutants had an advanced onset of nocturnal activity of about 2?h relative to WTs, and an increased duration of nocturnal activity (p < .01). Also, mPer2 mutants preferred and consumed more ethanol and had more daily ethanol drinking episodes vs. WTs. Measurements of systemic ethanol using subcutaneous microdialysis confirmed the advanced rise in ethanol intake in the mPer2 mutants, with 24-h averages being ～60 vs. ～25?mM for WTs (p < .01). A 6-day regimen of single intraperitoneal (i.p.) acamprosate injections (300?mg/kg) at zeitgeber time (ZT) 10 did not alter the earlier onset of nocturnal ethanol drinking in the mPer2 mutants, but reduced the overall amplitude of drinking and preference (both p < .01). Acamprosate also reduced these parameters in WTs. These results suggest that elevated ethanol intake in mPer2 mutants may be a partial consequence of an earlier nighttime activity onset and increase in nocturnal drinking activity. The suppressive action of acamprosate on ethanol intake is not due to an altered diurnal pattern of drinking, but rather a decrease in the number of daily drinking bouts and amount of drinking per bout. (Author correspondence: jglass@kent.edu)     

Increased ethanol intake and preference in cyclin D2 knockout mice

Inhibitory effects of passive ethanol exposure on brain neurogenesis have been extensively documented in animal models. In contrast, a role of brain neurogenesis in ethanol self-administration has not been addressed, as yet. The aim of this study was to assess intake of, and preference for, ethanol solutions [2-16% (v/v)] in a mouse model of adult neurogenesis deficiency based on permanent knockout (KO) of cyclin D2 (Ccnd2). Wild type (WT) and Ccnd2 KO mice did not differ in 2% and 4% ethanol intake. The KO group consumed significantly more ethanol in g/kg when offered with 8% or 16% ethanol as compared with the WT controls. The WT and KO mice did not differ in 2% ethanol preference, but the KO group showed a significantly higher preference for 4-16% ethanol. Animal and human studies have suggested that the low level of response to the sedative/hypnotic effects of alcohol is genetically associated with enhanced alcohol consumption. However, in this study, there were no between-genotype differences in ethanol-induced loss of righting reflex. Previous reports have also suggested that high ethanol intake is genetically associated with the avidity for sweets and better acceptance of bitter solutions. However, the KO and WT mice consumed similar amounts of saccharin solutions and the KOs consumed less quinine (i.e. bitter) solutions as compared with the WTs. In conclusion, these results may indicate that Ccnd2 and, possibly, brain neurogenesis are involved in central regulation of ethanol intake in mice.     

Temporal expression profiles of ceramide and ceramide-related genes in wild-type and <Emphasis Type="Italic">mPer1/mPer2</Emphasis> double knockout mice

Most living organisms exhibit circadian rhythms in physiology and behavior. These oscillations are generated by an endogenous circadian clock and control many biological processes. Ceramide has attracted attention as a signal mediator in diverse cell processes including cell death and differentiation. The relationships between ceramide expression levels and the circadian clock have not previously been investigated. To determine if there are circadian variations in the content of ceramide, we measured ceramide concentrations in the livers of wild-type (WT) and mPer1/mPer2 double knockout (DKO) mice. The ceramide concentration in WT mice was dramatically increased at Zeitgeber Time 9 (ZT9; 9 h after lights-on time) and ZT21 but no rhythmicity in ceramide expression was seen in DKO mice. Because ceramide can be generated by the hydrolysis of sphingomyelin via sphingomyelinase (SMase), or by ceramide synthase (CerS)-mediated synthesis, we assayed the expression patterns of ceramide-related genes using real-time PCR. CerS2 expression levels showed a biphasic pattern of expression in WT mice but no rhythmicity in DKO mice. While the neutral SMase (nSMase) and acidic SMase (aSMase) mRNA in WT mice were expressed in a circadian manner, the correlation between the expression levels of these SMases with times of day was weak in DKO mice. Collectively, our findings suggest that both SMases and CerS2 mRNA expression are regulated by the presence of mPer1/mPer2 circadian clock genes in vivo, and imply that ceramide may play a vital role in circadian rhythms and physiology.     

Urocortin-1 within the centrally-projecting Edinger-Westphal nucleus is critical for ethanol preference

Converging lines of evidence point to the involvement of neurons of the centrally projecting Edinger-Westphal nucleus (EWcp) containing the neuropeptide Urocortin-1 (Ucn1) in excessive ethanol (EtOH) intake and EtOH sensitivity. Here, we expanded these previous findings by using a continuous-access, two-bottle choice drinking paradigm (3%, 6%, and 10% EtOH vs. tap water) to compare EtOH intake and EtOH preference in Ucn1 genetic knockout (KO) and wild-type (WT) mice. Based on previous studies demonstrating that electrolytic lesion of the EWcp attenuated EtOH intake and preference in high-drinking C57BL/6J mice, we also set out to determine whether EWcp lesion would differentially alter EtOH consumption in Ucn1 KO and WT mice. Finally, we implemented well-established place conditioning procedures in KO and WT mice to determine whether Ucn1 and the corticotropin-releasing factor type-2 receptor (CRF-R2) were involved in the rewarding and aversive effects of EtOH (2 g/kg, i.p.). Results from these studies revealed that (1) genetic deletion of Ucn1 dampened EtOH preference only in mice with an intact EWcp, but not in mice that received lesion of the EWcp, (2) lesion of the EWcp dampened EtOH intake in Ucn1 KO and WT mice, but dampened EtOH preference only in WT mice expressing Ucn1, and (3) genetic deletion of Ucn1 or CRF-R2 abolished the conditioned rewarding effects of EtOH, but deletion of Ucn1 had no effect on the conditioned aversive effects of EtOH. The current findings provide strong support for the hypothesis that EWcp-Ucn1 neurons play an important role in EtOH intake, preference, and reward.     

Cloning and characterization of rat casein kinase 1epsilon

Genes differentially expressed in the subjective day and night in the rat suprachiasmatic nucleus (SCN) were surveyed by differential display. A gene homologous to human casein kinase 1epsilon (CK1epsilon) was isolated, which initially appeared to be expressed in the suprachiasmatic nucleus (SCN) in a circadian manner. We here describe the cDNA cloning of the rat CK1epsilon and characterization of the protein products. The rCK1epsilon is predominantly expressed in the brain including the SCN, binds and phosphorylates mPer1, mPer2, and mPer3 in vitro, and translocates mPer1 and mPer3, but not mPer2, to the cell nucleus depending on its kinase activity when coexpressed with these Per proteins in COS-7 cells.     

Ghrelin knockout mice show decreased voluntary alcohol consumption and reduced ethanol-induced conditioned place preference

Recent work suggests that stomach-derived hormone ghrelin receptor (GHS-R1A) antagonism may reduce motivational aspects of ethanol intake. In the current study we hypothesized that the endogenous GHS-R1A agonist ghrelin modulates alcohol reward mechanisms. For this purpose ethanol-induced conditioned place preference (CPP), ethanol-induced locomotor stimulation and voluntary ethanol consumption in a two-bottle choice drinking paradigm were examined under conditions where ghrelin and its receptor were blocked, either using ghrelin knockout (KO) mice or the specific ghrelin receptor (GHS-R1A) antagonist “JMV2959”. We showed that ghrelin KO mice displayed lower ethanol-induced CPP than their wild-type (WT) littermates. Consistently, when injected during CPP-acquisition, JMV2959 reduced CPP-expression in C57BL/6 mice. In addition, ethanol-induced locomotor stimulation was lower in ghrelin KO mice. Moreover, GHS-R1A blockade, using JMV2959, reduced alcohol-stimulated locomotion only in WT but not in ghrelin KO mice. When alcohol consumption and preference were assessed using the two-bottle choice test, both genetic deletion of ghrelin and pharmacological antagonism of the GHS-R1A (JMV2959) reduced voluntary alcohol consumption and preference. Finally, JMV2959-induced reduction of alcohol intake was only observed in WT but not in ghrelin KO mice. Taken together, these results suggest that ghrelin neurotransmission is necessary for the stimulatory effect of ethanol to occur, whereas lack of ghrelin leads to changes that reduce the voluntary intake as well as conditioned reward by ethanol. Our findings reveal a major, novel role for ghrelin in mediating ethanol behavior, and add to growing evidence that ghrelin is a key mediator of the effects of multiple abused drugs.     

Methylation analyses on promoters of mPer1, mPer2, and mCry1 during perinatal development

Recent studies revealed dramatic changes in circadian clock genes’ expression during the perinatal period. In this study, we characterized DNA methylation for three clock genes mPer1, mPer2, and mCry1 at their selected promoter regions during development. Results for the suprachiasmatic nucleus (SCN) and liver (at embryonic day 19, postnatal day 1 and postnatal day 7) were compared to those of sperm. Few methylations were detected for the mPer2 and mCry1 promoters. The 3rd E-box region of the mPer1 promoter exhibited methylation only in sperm. Significant demethylation was observed in the 4th E-box region of the mPer1 promoter between E19 and P1 in the SCN but not in liver tissue. This demethylation state was maintained at P7 for the SCN. Luciferase reporter assays using in vitro methylated promoters revealed an inhibitory effect of promoter methylation on mPer1 expression. The results suggested that epigenetic mechanisms such as DNA methylation might contribute to the developmental expression of clock genes.     

Reorganization of the suprachiasmatic nucleus coding for day length

In mammals, the suprachiasmatic nucleus (SCN), the circadian pacemaker, receives light information via the retina and functions in the entrainment of circadian rhythms and in phasing the seasonal responses of behavioral and physiological functions. To better understand photoperiod-related alterations in the SCN physiology, we analyzed the clock gene expression in the mouse SCN by performing in situ hybridization and real-time monitoring of the mPer1::luc bioluminescence. Under long photoperiod (LP) conditions, the expression rhythms of mPer1 and Bmal1 in the caudal SCN phase-led those in the rostral SCN; further, within the middle SCN, the rhythms in the ventrolateral (VL)-like subdivision advanced compared with those in the dorsomedial (DM)-like subdivision. The mPer1::luc rhythms in the entire coronal slice obtained from the middle SCN exhibited 2 peaks with a wide peak width under LP conditions. Imaging analysis of the mPer1::luc rhythms in several subdivisions of the rostral, middle, caudal, and horizontal SCN revealed wide regional variations in the peak time in the rostral half of the SCN under LP conditions. These variations were not due to alterations in the waveform of a single SCN neuronal rhythm. Our results indicate that LP conditions induce phase changes in the rhythms in multiple regions in the rostral half of the SCN; this leads to different circadian waveforms in the entire SCN, coding for day length.     

IMPAIRED EXPRESSION OF THE mPer2 CIRCADIAN CLOCK GENE IN THE SUPRACHIASMATIC NUCLEI OF AGING MICE

The expression of circadian clock genes was investigated inthe suprachiasmatic nuclei (SCN) of young adult and old laboratory mice. Sampleswere taken at two time points, which corresponded to the expected maximum(circadian time 7 [CT7]) or minimum (CT21) of mPermRNA expression. Whereas the young mice had a stable and well-synchronizedcircadian activity/rest cycle, the rhythms of old animals were less stableand were phase advanced. The expression of mPer1mRNA and mPer2 mRNA was rhythmic in bothgroups, with peak values at CT7. The levels of mClockand mCry1 mRNA were not different dependingon the time of day and did not vary with age. In contrast, an age-dependentdifference was found in the case of mPer2(but not mPer1) mRNA expression, with themaximum at CT7 significantly lower in old mice. The decreased expression of mPer2 may be relevant for the observed differencesin the overt activity rhythm of aged mice. (ChronobiologyInternational, 18(3), 559–565, 2001)