Caroline Kopp

Diazepam-induced changes in sleep: Role of the α1 GABAA receptor subtype

Ligands acting at the benzodiazepine (BZ) site of γ-aminobutyric acid type A (GABAA) receptors currently are the most widely used hypnotics. BZs such as diazepam (Dz) potentiate GABAA receptor activation. To determine the GABAA receptor subtypes that mediate the hypnotic action of Dz wild-type mice and mice that harbor Dz-insensitive α1 GABAA receptors [α1 (H101R) mice] were compared. Sleep latency and the amount of sleep after Dz treatment were not affected by the point mutation. An initial reduction of rapid eye movement (REM) sleep also occurred equally in both genotypes. Furthermore, the Dz-induced changes in the sleep and waking electroencephalogram (EEG) spectra, the increase in power density above 21 Hz in non-REM sleep and waking, and the suppression of slow-wave activity (SWA; EEG power in the 0.75- to 4.0-Hz band) in non-REM sleep were present in both genotypes. Surprisingly, these effects were even more pronounced in α1(H101R) mice and sleep continuity was enhanced by Dz only in the mutants. Interestingly, Dz did not affect the initial surge of SWA at the transitions to sleep, indicating that the SWA-generating mechanisms are not impaired by the BZ. We conclude that the REM sleep inhibiting action of Dz and its effect on the EEG spectra in sleep and waking are mediated by GABAA receptors other than α1, i.e., α2, α3, or α5 GABAA receptors. Because α1 GABAA receptors mediate the sedative action of Dz, our results provide evidence that the hypnotic effect of Dz and its EEG “fingerprint” can be dissociated from its sedative action.

Homeostatic sleep regulation is preserved in mPer1 and mPer2 mutant mice

A limited set of genes, Clock, Bmal1, mPer1, mPer2, mCry1 and mCry2, has been shown to be essential for the generation of circadian rhythms in mammals. It has been recently suggested that circadian genes might be involved in sleep regulation. We investigated the role of mPer1 and mPer2 genes in the homeostatic regulation of sleep by comparing sleep of mice lacking mPER1 (mPer1 mutants) or a functional mPER2 (mPer2 mutants), and wild‐type controls (WT) after 6 h of sleep deprivation (SD). Our main result showed that after SD, all mice displayed the typical increase of slow‐wave activity (SWA; EEG power density between 0.75 and 4 Hz) in nonREM sleep, reflecting the homeostatic response to SD. This increase was more prominent over the frontal cortex as compared to the occipital cortex. The genotypes did not differ in the effect of SD on the occipital EEG, while the effect on the frontal EEG was initially diminished in both mPer mutants. Differences between the genotypes were seen in the 24‐h distribution of sleep, reflecting especially the phase advance of motor activity onset observed in mPer2 mutants. While the daily distribution of sleep was modulated by mPer1 and mPer2 genes, sleep homeostasis reflected by the SWA increase after 6‐h SD was preserved in the mPer mutants. The results provide further evidence for the independence of the circadian and the homeostatic components underlying sleep regulation.

Diazepam-induced changes on sleep and the EEG spectrum in mice: role of the α3-GABAA receptor subtype

Benzodiazepines reduce EEG slow‐wave activity in non‐REM sleep by potentiating GABAergic neurotransmission at GABAA receptors via a modulatory binding site. However, the mechanisms of action underlying the effects of benzodiazepines on sleep and the sleep EEG are still unknown. Slow waves during sleep are generated by the corticothalamic system and synchronized by the inhibitory GABAergic neurons of the reticular thalamic nucleus. This region contains exclusively α3‐containing GABAA receptors. We investigated the role of these receptors in the mediation of diazepam effects on the sleep EEG by studying point‐mutated mice in which the α3‐GABAA receptor is diazepam‐insensitive [α3(H126R)]. Sleep was recorded for 12 h after i.p. injection of 3 mg/kg diazepam or vehicle at light onset in α3(H126R) and wild‐type controls (n = 13–17 per genotype). The main effect was a marked reduction of slow‐wave activity (EEG power density in 0.75–4.00 Hz) in non‐REM sleep and a concomitant increase in frequencies above 15.00 Hz in non‐REM sleep and waking in both genotypes. Neither effect of diazepam differed significantly between the genotypes. Despite the exclusive expression of α3‐containing GABAA receptors in the reticular thalamic nucleus, these receptors do not seem to be critical for the mediation of the effects of diazepam on the sleep EEG.

Locomotor activity rhythm in inbred strains of mice: implications for behavioural studies.

Due to the well-known influence of arousal on behavioural responsiveness, this paper focused on the differences in the daily locomotor activity rhythm between inbred strains. The lighting conditions of the environment can provide a regular, defined LD cycle or be constant, e.g. constant dim light or constant darkness. Under a light:dark cycle (daily rhythm) and under free-running conditions, i.e. constant darkness (circadian rhythm), such differences between mouse strains emphasise the relevance of a defined time of day for performing tests in order to obtain reliable behavioural results, and consequently the importance of a well-controlled LD cycle, which constitutes a necessary condition for the stability of daily rhythms. Both the LD cycle in the animal room (during breeding, as well as during the experiments) and the experiment schedule within the LD cycle appear as important factors for a better understanding of behavioural results. This information may be relevant to explain part of the apparently contradictory behavioural results reported in the literature.

Mice deficient for the synaptic vesicle protein Rab3a show impaired spatial reversal learning and increased explorative activity but none of the behavioral changes shown by mice deficient for the Rab3a regulator Gdi1

Rab proteins are small GTPases involved in intracellular trafficking. Among the 60 different Rab proteins described in mammals, Rab3a is the most abundant in brain, where it is involved in synaptic vesicle fusion and neurotransmitter release. Rab3a constitutive knockout mice (Rab3a−/−) are characterized by deficient short‐ and long‐term synaptic plasticity in the mossy fiber pathway and altered circadian motor activity, while no effects on spatial learning have been reported so far for these mice. The goals of this study were to analyse possible behavioral consequences of the lack of synaptic plasticity in the mossy fiber pathway using a broad battery of sensitive behavioral measures that has been used previously to analyse the behavior of Gdi1 mice lacking a protein thought to regulate Rab3a. Rab3a−/− mice showed normal acquisition but moderately impaired platform reversal learning in the water maze including reference memory and episodic‐like memory tasks. A mild deficit in spatial working memory was also observed when tested in the radial maze. Analysis of explorative behavior revealed increased locomotor activity and enhanced exploratory activity in open field, O‐maze, dark/light box and novel object tests. Spontaneous activity in normal home cage settings was unaffected but Rab3a−/− mice showed increased motor activity when the home cage was equipped with a wheel. No differences were found for delayed and trace fear conditioning or for conditioned taste aversion learning. Congruent with earlier data, these results suggest that Rab3a‐dependent synaptic plasticity might play a specific role in the reactivity to novel stimuli and behavioral stability rather than being involved in memory processing. On the other hand, the phenotypic changes in the Rab3a−/− mice bore no relation to the behavioral changes as observed in the Gdi1 mice. Such divergence in phenotypes implies that the putative synaptic interaction between Gdi1 and Rab3a should be reconsidered and re‐analysed.

Influence of estrus cycle and ageing on activity patterns in two inbred mouse strains

Despite the widespread use of inbred mice in research, little is known about aging of the circadian system in female mice, although interactions between female gonadal hormones and circadian rhythms have been established. We investigated the influence of the estrus cycle on circadian aspects of running-wheel activity and changes in the course of aging in female C57BL/6 and C3H/He mice recorded continuously between the ages of 3 and 19 months. In the young, cycling mice the second part of the proestrus night was often, but not consistently, characterized by increased motor activity compared to the remaining estrus cycle nights. After estrus cycling had ceased in the course of ageing, the estrus-dependent day-to-day variability in activity was reduced. The amplitude of the daily rest-activity rhythm decreased progressively after the age of 8 months in C3H/He and 10 months in C57BL/6 mice. The capacity for resynchronisation of activity onset to the LD-cycle was compared in young and old mice after an 8-h phase advance of the LD-cycle. Resynchronisation was significantly slower in old C3H/He mice and unaffected by age in C57BL/6 mice. The circadian period in constant darkness did not change with age in either strain. However, the period was shorter in 17-month old C57BL/6 mice compared to an additional group, which was recorded at the same age, after at least 1-month adaptation to the recording conditions. The results show that the reproductive state as well as ageing influence motor activity patterns of female mice in a strain- and cohort-dependent manner.

Sleep EEG changes after zolpidem in mice.

Zolpidem is a widely used hypnotic that binds preferentially to &agr;1GABAA receptors. We determined the role of these receptors in the effects of zolpidem on sleep in mutant mice carrying zolpidem-insensitive &agr;1GABAA receptors and wild-type controls. Sleep was promoted by zolpidem in both genotypes. In wild-type mice non-REM sleep EEG power was markedly reduced in a broad frequency band >5 or 9 Hz after 5 and 10 mg/kg zolpidem, respectively. In mutants a power reduction appeared at the highest dose only, and was restricted to some low frequencies and the 9–10 Hz bin. We conclude that the effects of zolpidem on the sleep EEG in mice are distinct from the changes typically induced by benzodiazepines, and are primarily mediated by &agr;1GABAA receptors.

The GABAA receptor agonist THIP alters the EEG in waking and sleep of mice

THIP is a GABA(A) agonist with hypnotic properties consisting in reducing sleep latency and prolonging and consolidating sleep. THIP has been reported to increase EEG slow-wave activity (SWA; EEG power in the 0.75-4 Hz band) in non-REM (NREM) sleep in both rats and humans. We investigated the effects of THIP on sleep in C57BL/6 mice. EEG recordings were performed after 2, 4 and 6 mg/kg THIP and saline control. The results were compared with analyses of recordings obtained after 6 h of sleep deprivation (SD) in the same strain of mice. The two higher doses of THIP induced an abnormal EEG pattern both in waking and NREM sleep. The EEG was characterized by sporadic asymmetric high-voltage potentials recurring at a low-frequency (<1 Hz) on the background of a low-amplitude EEG pattern. In contrast, after SD the typical regular synchronous high amplitude delta waves predominated. THIP at 4 and 6 mg/kg led to a prominent enhancement of spectral power in the low-frequency range of the waking and sleep EEG which was much higher than the increase attained after 6 h SD. This effect was particularly prominent in the waking EEG. In NREM sleep the increase of spectral power after THIP reflected the frequency of recurrence of the high-voltage potentials, and was restricted to a narrower frequency band than after SD. The EEG changes after 2mg/kg differed little from saline control. Sleep latency was not affected by the two lower doses of THIP, and was prolonged after 6 mg/kg. REM sleep was suppressed after the two higher doses. In contrast to previous results reported in other species, THIP did not have a hypnotic action in mice. The changes induced by THIP in the waking and sleep EEG differed from those caused by enhanced physiological sleep pressure encountered after SD. Considering the abnormal EEG pattern and the similarity of the spectral changes in the sleep and waking EEG, THIP does not seem to exert a specific effect on mechanisms involved in sleep regulation.

Comparison of the effects of modafinil and sleep deprivation on sleep and cortical EEG spectra in mice.

Modafinil is a wakefulness-promoting substance whose profile differs from that of the classical psychostimulants. It is still unknown whether waking induced by modafinil and wakefulness induced by sleep deprivation differ in terms of their effect on subsequent sleep. To investigate this problem sleep was recorded in two groups of OF1 mice. One group received modafinil (200 mg/kg, i.p.) at light onset which induced a period of wakefulness of approx. 5 h, while animals of the subsequent control group were injected with vehicle and kept awake for an equivalent duration. The effect of the two treatments on sleep was similar. REM sleep was initially reduced and slow-wave activity (SWA; EEG power in the 0.75-4.0 Hz range) in nonREM sleep was enhanced for several hours. The SWA increase was more prominent over the frontal cortex than over the occipital cortex after both treatments. A minor difference was seen at the occipital site where the initial rise of power in the low-frequency range was larger after vehicle combined with enforced waking than after modafinil. The study shows that the homeostatic sleep response following the modafinil-induced wakefulness corresponds largely to the response following a non-pharmacologically induced extended waking episode.

Anxiety in Mice: A Principal Component Analysis Study

Two principal component analyses of anxiety were undertaken investigating two strains of mice (ABP/Le and C57BL/6ByJ) in two different experiments, both classical tests for assessing anxiety in rodents. The elevated plus-maze and staircase were used for the first experiment, and a free exploratory paradigm and light-dark discrimination were used for the second. The components in the analyses produced definitions of four fundamental behavior patterns: novelty-induced anxiety, general activity, exploratory behavior, and decision making. We also noted that the anxious phenotype was determined by both strain and experimental procedure. The relationship between behavior patterns and the use of specific tests plus links with the genetic background are discussed.