Influence of circadian phase and test illumination on pre-clinical models of anxiety

Pre-clinical models of anxiety, particularly the elevated plus-maze (EPM), have been shown to be sensitive to a variety of methodological variations. Recent research has implicated circadian phase of testing in influencing the behavioural profile of 5-HT(1A) ligands on the EPM. The present study investigated the effects of testing animals during the dark and light phases and in light and subjective dark test conditions on baseline behaviour in animal models of anxiety. Eighty singly housed male Sprague-Dawley rats were exposed to a battery of unconditioned, exploratory tests (EPM, open field arena, holeboard) and a new model of extreme anxiety, the unstable elevated exposed plus-maze (UEEPM). Circadian phase of testing failed to consistently alter behaviour on any model. Level of test illumination had no effect on subjects' response to the open field arena, holeboard or UEEPM. Dark testing increased locomotor activity on the EPM (total arm entries, closed arm entries and distance moved) without decreasing open-arm avoidance. The construct of anxiety as measured by a number of different paradigms withstood major intra-laboratory manipulation of circadian phase of testing and illumination of apparatus. It is suggested that the effects of circadian rhythmicity may be confined to the behavioural profiles of serotonergic, particularly 5-HT(1A), ligands on the EPM.     

The development of infants' circadian rest-activity rhythm and mothers' rhythm

In order to know when and how infants obtain their circadian sleep-wake rhythm, infants' developing circadian rest-activity rhythm and mothers' circadian rhythm in the postpartum period were examined using actigraph monitoring. The subjects were 11 primipara and their infants. Actigraphic recordings for the infants and their mothers were made over 3-5 continuous days during the 3rd, 6th, 9th and 12th weeks after birth. A 24-h peak on a mean autocorrelogram of the infants' movements was detected at the 3rd week. The infants' circadian rest-activity rhythm already existed in the 3rd week. The amplitude of this 24-h peak gradually increased from the 6th to 12th week. This may be useful as an index of the development of infants' circadian rest-activity rhythm. An 11-h peak was also observed at the 3rd week. This 11-h peak was thought to be a semi-circadian rhythm. Regarding the mothers, the amplitude of the 24-h peak on the mean autocorrelogram at the 3rd week was the smallest of all other weeks, and it became larger from the 3rd to 12th week. This meant that the mothers' circadian rhythm at the 3rd week was influenced by their interrupted sleep at night to take care of their infants. The mother-infant synchronization is probably the 1st factor in the entrainment of infants' circadian sleep-wake rhythm. In this study, we also propose a novel method for compensating for missing data in autocorrelogram analysis.     

Ultradian rhythm of 100 min in the dark phase EEG of the rat

Using the "variance in statistics" as an index of electroencephalogram (EEG) parameters, we observed the cortico-hippocampal EEG rhythm under a 12:12-h light-dark condition in the rat with chronically implanted electrodes for EEG recording. The above EEG variance was simply measured in real time and on line through a personal computer. It corresponded to EEG slow wave activity and expressed the process of slow wave sleep as described in the two-process model by Borbély et al. Only in the dark phase, mean power spectral density of the EEG variance had a significant peak at about 1/100 cycles/min. This 100-min rhythmicity similar to the basic rest-activity cycle in human beings was observed in rats, particularly in the dark (active) phase for nocturnal animals. We propose that this ultradian 100-min rhythm is essential for the rat to maintain the waking state dominantly over the 12-h dark period.     

Decreased anxiety levels related to aging

The present experiment investigated the effects of aging on emotional behavior, without concomitant influences from any previous behavioral manipulation apart from weighing the rats. Anxiety-related behaviors were measured in the elevated plus-maze (EPM). Performance in the open field (OF) was also assessed to control for any effect of aging on exploratory behavior that could account for changes in emotional behavior. Fifty-two naïve male Wistar rats of 3 (3MO), 17 (17MO), or 24 (24MO) months, were submitted to two sessions (5 min each) of EPM, followed by two sessions (2 min each) of OF, on 4 consecutive days. The main emotional indices (open arm entries, ratio of open arm entries to total entries, time spent in open arms, ratio of time in open arms to time in four arms, open arm ends, and head dips) measured in the EPM indicated a lower level of anxiety in aged (24MO) than in young (3MO) rats, whereas middle-aged (17MO) rats showed intermediate values between those of 3MO and 24MO rats; 3MO rats showed higher general motor activity (number of rearings in closed arms of EPM and in OF, and higher number of areas crossed in OF) than 17MO and 24MO rats. We conclude that aging is associated with a decrease in anxiety and in general motor activity.     

Effects of long-term continuous exposure to light on memory and anxiety in mice

The studies on the relationship between the light/dark cycle and memory function mostly used protocols of acute disruption of the circadian rhythm. The aim of the present study is to verify the effects of long-term continuous exposure to light on memory, anxiety and motor parameters of mice tested in the plus-maze discriminative avoidance task. Mice were conditioned to choose between the two enclosed arms (one aversive and one non-aversive) while avoiding the open arms of a modified elevated plus-maze apparatus. Memory was evaluated by the time spent in the aversive enclosed arm, anxiety was evaluated by the time spent in the open arms and locomotor behavior was evaluated by number of entries in the arms of the maze. The results showed that long-term (35-42 days) continuous light exposure did not modify memory or anxiety parameters but increased locomotor activity. While the increase in locomotor behavior is in line with previous studies, the unexpected absence of alterations in memory and anxiety (reported to be influenced by the circadian rhythm) is discussed.     

Diurnal rhythms of blood pressure, heart rate, and locomotor activity in adult and old male Wistar rats

The diurnal rhythms of systolic blood pressure (SBP), diastolic BP (DBP), heart rate (HR), and spontaneous locomotor activity (SLA) were determined in adult (6-month-old) and old (24-month-old) male Wistar rats by using radiotelemetry. The rhythm parameters (mesor, amplitude, acrophase, and percent rhythm) were analyzed by Fast-Fourier Transform and Cosinor methods. We found that the 12-h mean values of SBP, DBP, HR, and SLA were significantly (p<0.001) higher in the dark phase than in the light phase in both adult and old rats. The nocturnal 12-h mean value of HR was significantly (p<0.01) lower in old than in adult rats. In addition, the differences between diurnal and nocturnal 12-h mean values of SBP and HR were reduced in old rats. There was no significant difference in the 12-h mean values of SBP, DBP, and SLA between old and adult rats. Otherwise, the diurnal HR rhythm amplitude was significantly (p<0.01) reduced in old rats (32+/-2 vs. 46+/-2 bpm). A nearly 1-h delay in SBP and DBP acrophases (03h55 +/- 00h19 vs. 02h57 +/- 00h14 and 03h17 +/- 00h13 vs. 01h53 +/- 00h22, respectively) was found in old rats comparing to adult rats. No significant change in SLA diurnal rhythm was observed in old rats. In conclusion, these results show a decrease in the nocturnal 12-h mean value of HR and an alteration in cardiovascular diurnal rhythms by a 1-h delay of SBP and DBP acrophases and a reduction of HR rhythm amplitude in old Wistar rats.     

Withdrawal effects from progesterone and estradiol relate to individual risk-taking and explorative behavior in female rats

Withdrawal from progesterone and estradiol has been used as an animal model of premenstrual syndrome (PMS) and premenstrual dysphoric disorder (PMDD). In the clinical population individual sensitivity to sex steroid hormones, personality and heredity influence PMS/PMDD. Understanding the phenotypic risk factors of PMS/PMDD and drug development requires an animal model which incorporates individual steroid sensitivity. The main objective of this study was to investigate whether the individual trait of risk-taking and exploration influence the severity of PEWD in female rats. Thirty-two female Wistar rats in their diestrus phase were tested in the open field (OF) and divided into high responders (HR) and low responders (LR). Injections were given i.p. twice daily for 6 days, either 5 mg/kg progesterone combined with 10 µg/kg 17β-estradiol, or vehicle (sesame oil). After a 24-hour withdrawal the animals were tested in the elevated plus maze (EPM). Blood samples for CORT analysis were collected after both behavioral tests. The HR rats withdrawn from progesterone and estradiol, spent less time on the EPM open arms and had lower CORT levels than the HR controls. The LR group showed no differences in EPM behavior and CORT levels during PEWD. The controls showed a stable trait of risk-taking and exploration, indicated by behavioral and CORT level correlations between the OF and EPM tests. These findings show that female rats with the trait of risk-taking and explorative behavior (HR) are more affected by PEWD.     

Influence of physical training on the Syrian hamster's melatonin rhythm

Melatonin secretion can be influenced by acute exercise. Using female Syrian hamsters we assessed the influence of physical exercise in the serum melatonin rhythm. Melatonin concentrations were determined by RIA. After a gradually increased training program in which experiments were performed at the end of the light phase of the photoperiod (14-h light:10-h dark; lights on at 21:00) animals were killed immediately after exercise (14 h light) and at different time points including 11h after light and 3, 6, 8 and 9h after dark. No differences in melatonin concentrations immediately after exercise practice between control and trained animals were observed. A slower melatonin nighttime increase in exercised hamsters, with significantly lower levels 6h after dark in comparison with controls was found. The peak value appeared 8h after dark but high melatonin levels were prolonged in exercised hamsters, showing significantly higher melatonin levels 9h after. In conclusion, our results indicate for the first time that Syrian hamsters submitted to a training program of increased activity at the end of the light phase, the rest period, showed an altered melatonin rhythm, resembling the response observed in humans.     

GIRK2 deficient mice. Evidence for hyperactivity and reduced anxiety

G-protein activated inwardly rectifying potassium channel (GIRK2)-deficient (null mutant) mice were examined in three tests for anxiety: the elevated plus-maze, light/dark box and "canopy" test. In the elevated plus-maze test, GIRK2 null mutant mice spent a higher percentage of time in the open arms and showed a higher number of total entries. A short (6 days) period of social isolation decreased anxiety and also increased the total activity in GIRK2 mutant mice. However, the increase of total activity in GIRK2 null mutant mice was mostly due to an increase in the number of entries into the open arms. The behavior of the wild-type animals was not substantially changed after social isolation. In the light/dark box, GIRK2 homozygous (-/-) mice demonstrated a higher level of locomotion and a higher number of rearings in the light area. In the "canopy" test, GIRK2 mutant mice displayed an increased locomotion in the exposed area and a strong trend to decrease in the number of stretched attend postures (SAP) in the most secure "canopy" area. GIRK2 heterozygous (+/-) animals showed behavioral changes intermediate between wild-type and null mutants only in the elevated plus-maze test after social isolation. In all other tests, GIRK2 heterozygous (+/-) animals did not differ from wild-type mice. Taken together, this data demonstrates that GIRK2 null mutant mice have reduced anxiety with signs of hyperactivity. We suggest that the functional block of dopamine D3 receptors may be a reason for this phenotype.     

Anxiety profile in morphine-dependent and withdrawn rats: effect of voluntary exercise

Withdrawal from chronic opiates is associated with an increase in anxiogenic-like behaviours, but the anxiety profile in the morphine-dependent animals is not clear. Thus, one of the aims of the present study was to examine whether morphine-dependent rats would increase the expression of anxiogenic-like behaviours in novel and stressful conditions. Additionally, recent studies have shown that voluntary exercise can reduce anxiety levels in rodents. Therefore, another aim of this study was to examine the effect of voluntary exercise on the anxiety profile in both morphine-dependent animals and animals experiencing withdrawal. Rats were injected with bi-daily doses (10 mg/kg, at 12 h intervals) of morphine over a period of 10 days in which they were also allowed voluntary exercise. Following these injections, anxiety-like behaviours were tested in the elevated plus-maze (EPM) model and the light/dark (L/D) box. We found reductions in time spent in, and entries into, the EPM open arms and reductions in time spent in the lit side of the L/D box for both sedentary morphine-dependent and withdrawn rats as compared to the sedentary control groups. The exercising morphine-dependent and withdrawn rats exhibited an increase in EPM open arm time and entries and L/D box lit side time as compared with the sedentary control groups. We conclude that voluntary exercise decreases the severity of the anxiogenic-like behaviours in both morphine-dependent and withdrawn rats. Thus, voluntary exercise could be a potential natural method to ameliorate some of the deleterious behavioural consequences of opiate abuse.     

Exposure to a novel stimulus reduces anxiety level in adult and aging rats

Male Wistar rats aged 3, 15 and 24 months were isolated and housed individually for 72 h prior to being subjected to inanimate objects (two objects per rat, each 1.5 cm in diameter and 4 cm in length, made of surgical gauze). Following the exposure to the objects, rats were subsequently tested in an elevated plus-maze. The inanimate objects induced locomotor activity, chewing and transportation of the object. This changed to social interaction and play-like behavioral activity in rats of all ages that were kept in small groups with a latency of 1 to 2 min. One hour after the start of exposure, the animals were tested in the elevated plus-maze to measure anxiety behavior. It was found that all age groups spent significantly more time in the open arm of the elevated plus-maze than the corresponding controls. Latencies to first entry into open arms were also significantly lowered. The number of entries to the open or to the dark arm, however, did not show statistical difference, indicating that the novel object-induced activity failed to exert influence on gross motor activity in the elevated plus-maze. In conclusion, the stimulation elicited by the exposure to novel stimulus (inanimate objects) resulted in a significant reduction of anxiety level both in adult and in aging rats.     

D-cycloserine enhances memory consolidation in the plus-maze retest paradigm

Prior undrugged exposure to the elevated plus-maze (EPM) alters future behavioral strategy as well as responsivity to conventional anxiolytic agents. This EPM retest phenomenon appears to be dependent upon learning the spatial configuration of the maze on initial exposure and, in particular, the location of the relatively safe enclosed arms. As posttraining administration of the glycineB receptor partial agonist, D-cycloserine (DCS), has been shown to enhance the consolidation of many forms of memory, we have examined the effects of this compound on the EPM retest effect in male mice. The results of Experiment 1 confirmed that 5 min undrugged exposure to the EPM completed abolishes the anxiolytic efficacy of chlordiazepoxide (CDP; 15 mg/kg) on 24 hr retest. In Experiment 2, posttraining administration of DCS (7.5 and 15 mg/kg), but not CDP (15 mg/kg) or DCS (30 mg/kg), significantly and selectively increased time spent in the enclosed arms (and reciprocally decreased open arm exploration) on 24 hr retest, a finding consistent with an enhancement of consolidation. Experiment 3 used a modified EPM retest protocol to assess the effects of posttraining DCS (15 mg/kg) on behavioral responses to CDP (15 mg/kg) challenge on 24 hr retest. Using a 1-min prior exposure regimen that did not compromise the anxiolytic efficacy of CDP in control mice, the results showed that posttraining administration of DCS abolished the anxiolytic response to CDP challenge. These data strongly suggest that the EPM retest effect involves glycineB/NMDA receptor-dependent neuroplasticity. Further studies will be required to identify the neural circuitry involved.     

Robust circadian rhythm in heart rate and its variability: influence of exogenous melatonin and photoperiod

Heart rate (HR) and heart rate variability (HRV) undergo marked fluctuations over the 24-h day. Although controversial, this 24-h rhythm is thought to be driven by the sleep-wake/rest-activity cycle as well as by endogenous circadian rhythmicity. We quantified the endogenous circadian rhythm of HR and HRV and investigated whether this rhythm can be shifted by repeated melatonin administration while exposed to an altered photoperiod. Eight healthy males (age 24.4 +/- 4.4 years) participated in a double-blind cross-over design study. In both conditions, volunteers were scheduled to 16 h-8 h rest : wake and dark : light cycles for nine consecutive days preceded and followed by 29-h constant routines (CR) for assessment of endogenous circadian rhythmicity. Melatonin (1.5 mg) or placebo was administered at the beginning of the extended sleep opportunities. For all polysomnographically verified wakefulness periods of the CR, we calculated the high- (HF) and low- (LF) frequency bands of the power spectrum of the R-R interval, the standard deviation of the normal-to-normal (NN) intervals (SDNN) and the square root of the mean-squared difference of successive NN intervals (rMSSD). HR and HRV variables revealed robust endogenous circadian rhythms with fitted maxima, respectively, in the afternoon (16:36 hours) and in the early morning (between 05:00 and 06:59 hours). Melatonin treatment phase-advanced HR, HF, SDNN and rMSSD, and these shifts were significantly greater than after placebo treatment. We conclude that endogenous circadian rhythmicity influences autonomic control of HR and that the timing of these endogenous rhythms can be altered by extended sleep/rest episodes and associated changes in photoperiod as well as by melatonin treatment.     

Dynamic regulation of polysialylated neural cell adhesion molecule in the suprachiasmatic nucleus

The suprachiasmatic nucleus (SCN) prominently expresses polysialic acid (PSA), a carbohydrate polymer that is attached to neural cell adhesion molecule (NCAM) and promotes changes in cell interactions. Previous studies have shown that expression of PSA is important for circadian rhythm stability under constant darkness, and for photic entrainment of the SCN circadian clock. In the present study, immunoblot analyses of the Syrian hamster SCN revealed marked diurnal fluctuations in PSA under a 24-h light/dark cycle. PSA levels were reduced by >90% during the mid-to-late dark phase, and were elevated to maximal daytime levels approximately 1 h after lights-on. A similar pattern of PSA fluctuation persisted under constant darkness. Exposure of animals under a 24-h light/dark cycle to a 30-min light pulse during the late dark phase dramatically increased SCN contents of PSA within 60 min, and these values returned to basal levels 1-2 h later. There was no effect of light-on expression of PSA in the hippocampus. Parallel studies revealed changes in the NCAM-180 isoform that carries PSA in the brain, suggesting that regulation of PSA may include protein as well as carbohydrate-associated mechanisms. Immunohistological analysis revealed light-induced enhancement of PSA in the SCN subregion containing calbindin D(28K) cells. PSA staining was also closely associated with the majority of SCN cells expressing light-inducible Fos protein. This rhythmic, light-inducible expression of PSA within the SCN suggests that dynamic cell interactions are important for the photic regulation of circadian clock phase.     

Emotional behavior in middle-aged rats: Implications for geriatric psychopathologies

Clinical findings reveal that middle-aged patients are more susceptible to suffer from psychiatric disorders than older ones. However, little is known about the emotional behavior of aging rodents. This study aimed to investigate behavioral alterations in male middle-aged Wistar rats in the open-field (OF) test (at illuminated and dimly light conditions), elevated plus maze (EPM), forced swimming (FST) and inhibitory avoidance task (IA). In the EPM, middle-aged rats displayed reduced percentages of the time spent in and entries into open arms. The ambulatory activity measured in the OF under dimly light conditions was identical among groups. However, under illuminated conditions, a reduction in the number of crossings was detected in older rats, reinforcing that aged animals display a genuine anxiogenic-like phenotype. Additionally, aged rats showed an increase in the immobility time in the FST, and a reduction in the latency to step down the platform in the IA. A negative correlation was found between the immobility time and latency to step down the platform, suggesting a relationship between depressive-behavior and cognitive impairment in old rats. Altogether, male middle-aged rats are more anxious, depressed, and display aversive memory impairments. These observations contribute to investigate biological mechanisms and therapeutic interventions for geriatric anxiety and depression.     

Effect of a short photoperiod on circadian rhythms of body temperature and motor activity in old rats

Circadian rhythms of body temperature and motor activity were documented in young and old rats (four 8-week-old and five 22-month-old male Wistars, implanted with telemetric probes and housed in a chronobiological facility) under two different photoperiod conditions. The animals were maintained in a light:dark (LD) cycle of 12 h each (LD 12:12) for 4 weeks and then exposed to a LD 6:18 cycle for 7 weeks to assess the effect of age on the desynchronization of the temporal structure of the rhythms. In old rats under LD 12:12, the power of the 24-h component and the circadian amplitude of body temperature and motor activity were markedly lower than in the young and both rhythms were phase-advanced. After the shift to LD 6:18, the circadian rhythmicity was maintained for both variables and the same phase delay (+5+/-1 h) was observed in both age groups, as was a gradual expansion of the patterns of both functions with the longer night. The photoperiod reduction (6 weeks under LD 6:18) did not modify the power of the 24-h component of body temperature and motor activity in old rats. In young rats, however, the power and amplitude of the 24-h component of motor activity rhythm fell to the levels of those in old rats, while the power of the 24-h component of body temperature rhythm and the amplitude did not change. Our data show that the circadian rhythm of motor activity, but not of body temperature, responds age dependently to a photoperiod reduction.     

Influence of diurnal phase on startle response in adult rats exposed to dexamethasone in utero

Depression and pathological anxiety disorders are among the most prevalent neurological diseases in the world and can be precipitated and exacerbated by stress. Prenatal stress alters both behavioral and endocrine responses to stressful stimuli in later life. We have previously observed increased basal acoustic startle response (ASR) in Wistar rats exposed to stress or dexamethasone (DEX) in utero when tested during the light phase of the circadian rhythm, and decreased prepulse inhibition (PPI) in similar animals tested during the dark phase of the cycle. We speculated that this observation of increased basal startle might be influenced by diurnal phase. In the present study, adult female Sprague Dawley rats, stressed prenatally with DEX (200 μg/kg, gestational days 14-21) and postnatally by blood sampling under restraint, were tested for the ASR during both circadian phases (light and dark). Basal startle was increased in animals tested both during the light and the dark phases of the cycle. We hereby replicated our earlier findings in a new strain and laboratory, thus strengthening the validity of our model regarding prenatal stress effects on ASR in female offspring. Our results indicate that observation of increased basal ASR is not solely dependent on diurnal phase. We found no difference in hippocampal glucocorticoid and mineralcorticoid receptor expression between groups.     

Circadian rhythm dissociation in an environment with conflicting temporal information.

The relative contributions of light-dark (LD) cycles and feeding (EF) cycles in providing temporal information to the circadian time-keeping system were examined in chair-acclimatized squirrel monkeys (Saimiri sciureus). The circadian rhythms of drinking, colonic temperature, urine volume, and urinary potassium excretion were measured with the LD and EF cycles providing either conflicting phases or periods. In conflicting phase experiments, animals were exposed to 24-h LD cycles consisting of 12 h of 600 lx followed by 12 h of less than 1 ls and concurrent 24-h EF cycles in which the animals ate for 3 h and then fasted for 21 h. One group had food available at the beginning and a second group at the end of the light period. In conflicting period experiments, monkeys were exposed to 23-h LD cycles (LD 11.5:11.5) and 24-h EF cycles (EF 3:21). Analysis of the rhythms showed that both phase and period information were conveyed to the drinking and urinary rhythms by the EF cycle, and to the temperature rhythm by the LD cycle.     

The ontogeny of anxiety-like behavior in rats from adolescence to adulthood

In human beings, susceptibility to anxiety disorders can be relatively high during adolescence. Understanding the ontogeny of anxiety-like behavior in laboratory rodents has implications for developing anxiolytic drugs that are suitable for this age group. Given the dearth of information about adolescent rodents, this study examined the response of both male and female adolescent, late adolescent, young adult, and older adult rats to three tests of anxiety-like behavior: the emergence test (ET), open field (OF), and elevated plus-maze (EPM). The results showed that adolescent rats exhibited a higher anxiety-like response than adults on each test; the amount of locomotion in the OF and percentage of time spent on the open arms of the EPM increased across the age groups, while older adult rats made the fewest start box re-entries in the ET. These results support the hypothesis that adolescent rats have a more pronounced response to stressors than do adults.