Chiara M. Portas

Serotonin and the sleep/wake cycle: special emphasis on microdialysis studies

Several areas in the brainstem and forebrain are important for the modulation and expression of the sleep/wake cycle. Even if the first observations of biochemical events in relation to sleep were made only 40 years ago, it is now well established that several neurotransmitters, neuropeptides, and neurohormones are involved in the modulation of the sleep/wake cycle. Serotonin has been known for many years to play a role in the modulation of sleep, however, it is still very controversial how and where serotonin may operate this modulation. Early studies suggested that serotonin is necessary to obtain and maintain behavioral sleep (permissive role on sleep). However, more recent microdialysis experiments provide evidence that the level of serotonin during W is higher in most cortical and subcortical areas receiving serotonergic projections. In this view the level of extracellular serotonin would be consistent with the pattern of discharge of the DRN serotonergic neurons which show the highest firing rate during W, followed by a decrease in slow wave sleep and by virtual electrical silence during REM sleep. This suggests that during waking serotonin may complement the action of noradrenaline and acetylcholine in promoting cortical responsiveness and participate to the inhibition of REM-sleep effector neurons in the brainstem (inhibitory role on REM sleep). The apparent inconsistency between an inhibitory and a facilitatory role played by serotonin on sleep has at least two possible explanations. On the one hand serotonergic modulation on the sleep/wake cycle takes place through a multitude of post-synaptic receptors which mediate different or even opposite responses; on the other hand the achievement of a behavioral state depends on the complex interaction between the serotonergic and other neurotransmitter systems. The main aim of this commentary is to review the role of brain serotonin in relation to the sleep/wake cycle. In particular we highlight the importance of microdialysis for on-line monitoring of the level of serotonin in different areas of the brain across the sleep/wake cycle.

Effects of chronic mild stress on sexual behavior, locomotor activity and consumption of sucrose and saccharine solutions

Many symptoms of human depressive disorders are also observed in animals after exposure to unpredictable stressors. The chronic mild stress (CMS) paradigm was developed in order to better model the human situation by using chronic mild stressors over a longer period. It is claimed that the model induces anhedonia in the animals, a core symptom of depression in humans. Despite the fact that the CMS model has a high degree of face validity, there are a number of laboratories in which the establishment of the model is less reliably observed. We have examined behavior (sexual activity and open field activity) together with hedonic measures (sucrose and saccharine intake) after exposure to CMS. CMS decreased male sexual activity (e.g. reduced capability to ejaculate) and increased activity in an open field test. The hedonic measures showed diverging results after CMS in our laboratory. Sucrose consumption was reduced, while saccharine consumption did not show a comparable change. It is concluded that CMS induces comparable alterations to some depression-like symptoms in humans. Saccharine consumption is not a reliable indicator of the hedonic responsiveness to CMS.

On-line detection of extracellular levels of serotonin in dorsal raphe nucleus and frontal cortex over the sleep/wake cycle in the freely moving rat

We used in vivo microdialysis coupled with polygraphic recording to monitor 5-hydroxytryptamine levels in the dorsal raphe nucleus and frontal cortex across waking, slow-wave sleep and rapid eye-movement sleep. Male Sprague-Dawley rats were prepared with electroencephalogram and electromyogram electrodes. Microdialysis probes were placed in dorsal raphe nucleus and/or frontal cortex. Dialysate samples were manually collected during polygraphically-defined behavioural states and the level of serotonin was assayed by means of microbore high-performance liquid chromatography separation and electrochemical detection. Samples from microdialysis probes histologically localized to the dorsal raphe nucleus and frontal cortex showed different levels of extracellular 5-hydroxytryptamine in waking, slow-wave sleep and rapid eye-movement sleep. In dorsal raphe nucleus the extracellular level of serotonin was highest in waking, decreased in slow-wave sleep to 69% and in rapid eye-movement sleep to 39% of waking mean level (waking 3.2 +/- 0.9; slow-wave sleep 2.2 +/- 0.8; rapid eye-movement sleep 1.3 +/- 0.4 fmol/sample). Mean extracellular levels of serotonin in frontal cortex displayed a similar pattern (waking 1.7 +/- 0.4; slow-wave sleep 1.0 +/- 0.3; rapid eye-movement 0.5 +/- 0.05 fmol/sample). In frontal cortex, rapid eye-movement sleep samples were only obtained in three animals. Our findings are consistent with previous results in cats, and suggest that in rats also, extracellular 5-hydroxytryptamine levels in dorsal raphe nucleus and frontal cortex across the sleep/wake cycle might reflect serotonergic neuronal activity. The findings stress the importance of controlling for behavioural state when investigating neurochemical correlates of serotonergic function.

Chronic mild stress affects sucrose intake and sleep in rats.

Depression in humans is associated with sleep abnormalities of three types: altered rapid eye movement (REM) sleep, fragmented sleep, and reduced delta sleep. In an animal model of depression, chronic exposure to mild stressors (CMS, e.g. periods of soiled cage, reversed light/dark cycle, grouped housing, food and/or water deprivation) causes behavioral and hormonal changes which, in humans, often are associated with depression. In the CMS model, a reduced sucrose intake has been defined as one of the core symptoms of depression, anhedonia, although this finding is not consistent among various laboratories. In the present study, we investigated if the CMS procedure, in our laboratory, would cause decreased sucrose intake and, also, give sleep changes similar to what is found in depressed patients. Exposure to CMS decreased sucrose intake in our rats. The largest effect was obtained after 2 weeks of the stress protocol. CMS rats spent more time in REM sleep and showed more fragmented sleep compared to their baseline recording, while there were no changes in the control rats. Increased sleep fragmentation in CMS rats was particularly evident by increased number of arousals, and increased REM sleep and slow-wave-sleep-1 (SWS-1) episodes. The duration of sleep stage episodes was decreased. The amount of slow-wave-sleep-2 (SWS-2) was not decreased, however SWS-2 in percent of total SWS was reduced. Correlation analysis showed that animals that had less consumption of sucrose spent more time in REM sleep and had increased number of REM sleep episodes. In this study, CMS appears to be a model of depression.

Extracellular levels of serotonin and GABA in the hippocampus after chronic mild stress in rats. A microdialysis study in an animal model of depression

One of the most established hypotheses of depression focuses on alteration of the serotonergic (5-HT) function. Recent evidence suggests that serotonergic involvement in depression may be modulated by the action of gamma-hydroxybutyric acid (GABA). Furthermore, altered GABAergic function is also evident in depressed patients and in animal models of depression. Disturbed sleep is characteristic of patients with mood disorders. The most pronounced changes of the 5-HT firing activity occur during sleep. Hence, the present paper reports a study on simultaneously measurement of hippocampal levels of serotonin and GABA during waking and sleep in the chronic mild stress (CMS) animal model of depression. The neurotransmitter findings are accompanied by depression-like symptoms (e.g. sleep alterations and reduced sucrose intake, a putative indicator of anhedonia in rodents). Our results show that animals exposed to CMS had lower hippocampal GABA levels compared to controls. In addition, after CMS there was a lack of 5-HT stage-dependency. A subgroup (five out of eight animals) showed a consistent increase in 5-HT levels in slow wave sleep and REM sleep. We also observed that this increase occurred in those animals regarded as most anhedonic (lowest intake of sucrose solution). Moreover, REM sleep was positively correlated with anhedonia. No interaction between 5-HT and GABA was found in the hippocampus. The data suggest that both GABAergic and serotonergic systems may be simultaneously but independently involved in depression. The alteration in 5-HT function may represent a link between depression-like behaviour and sleep abnormalities found in depressed patients.

A double exposure to social defeat induces sub-chronic effects on sleep and open field behaviour in rats.

Social defeat, resulting from the fight for a territory is based on the resident-intruder paradigm. A male rat intruder is placed in the territory of an older, bigger and more aggressive male resident and is defeated. In the present study, a double exposure to social defeat increased sleep fragmentation due to an increased amount of waking and slow-wave-sleep-1 (SWS-1) episodes. Also, social defeat increased the amount of slow-wave-sleep-2 (SWS-2). In repeated exposures to an open field, socially defeated rats showed low central activity and persistent defecation indicating high emotionality. The strongest effects of social defeat on sleep and open field behaviour were seen sub-chronically after stress. Social defeat did not induce changes in rapid eye movement (REM) sleep (e.g. total amount, latency), sleep latency, sexual activity, body weight or adrenal weight. A negative correlation between habituation in open field central activity and total sleep fragmentation indicates a commonality of effects of social defeat on both behaviour and sleep.

Transient changes in frontal alpha asymmetry as a measure of emotional and physical distress during sleep.

The aim of this project was to identify measurable physiological responses linked to the human experience of distress in a non-communicative state. Since sleep is a state of sensory-motor detachment in which subjects cannot report how they feel or what they experience, it can be considered a suitable model of non communicative states. A transitory distress-related response has consistently been reported in healthy awake subjects in the form of power spectrum alpha band changes in frontal brain areas. Hence, changes in frontal alpha asymmetry (FAA) were analysed in 16 volunteers before and after administration of distressful aversive stimuli during sleep. Transient physical or emotional distress was achieved by presentation of conditioned (to electroshocks or affective images) or directly-applied aversive stimuli (electroshocks). Frontal beta asymmetry (FBA) was also evaluated for comparisons. Power spectrum data were assessed from frontal electroencephalographic (EEG) leads (F3/F4). Galvanic skin response (GSR) and heart rate (HR) were also simultaneously monitored. To test the possibility that the short lasting stimulations would have long-term physiological effects, we included saliva cortisol sampling, evaluation of dream emotional valence and a standard assessment of sleep quality measurements. Aversive stimulation of diverse nature, compared to neutral valence stimuli, produced measurable changes in FAA (left>right) and in GSR evoked responses in both stage 2 (S2) and rapid eye movement (REM) sleep suggesting implicit processing of experiences with negative valence. Noticeably, dream content evaluation showed a trend towards increased negative emotionality. No FBA was observed, confirming the specificity of FAA responses. Saliva cortisol, and sleep parameters were not significantly affected by the protocol. This is, to our knowledge, the first time that an aversive imaging conditioning protocol has been successfully applied during sleep.

The effect of GABAA antagonist bicuculline on dorsal raphe nucleus and frontal cortex extracellular serotonin: A window on SWS and REM sleep modulation

We investigated the effects of the perfusion of gamma-aminobutyric acid(A) antagonist bicuculline in the dorsal raphe nucleus, on brain 5-hydroxytryptamine level and on sleep. Perfusion of 25 and 50 microM bicuculline into the dorsal raphe nucleus dose-dependently increased dorsal raphe nucleus 5-hydroxytryptamine level during sleep and wakefulness. Frontal cortex 5-hydroxytryptamine level was not affected by either 25 or 50 microM perfusion. 25 microM bicuculline produced only minimal effects on sleep. 50 microM decreased rapid eye movement sleep, slow wave sleep 1 and 2 and increased waking. Sleep changes leveled out towards the end of the bicuculline perfusion despite serotonin levels were still elevated. This suggests that an adaptation mechanism may take place in order to counteract the high serotonergic output, producing uncoupling between serotonin level and behavioural state. The results support the notion that gamma-aminobutyric acid is a strong modulator of dorsal raphe nucleus serotonergic neurons, and that this modulation is important in the regulation of slow wave sleep, rapid eye movement sleep and waking.

Involvement of the 5-HT1A and the 5-HT1B receptor in the regulation of sleep and waking

The involvement of the 5-HT1A and the 5-HT1B receptor in the regulation of sleep and waking is complex due to a multitude of presynaptic and/or postsynaptic actions also involving other neurotransmitter systems. Both receptors produce an important inhibitory feed back to the serotonergic raphe neurons. Overall, most studies support the possibility that stimulation of postsynaptic 5-HT1A receptors, e.g., via systemic administration of a high dose of agonists increases wakefulness and decreases sleep. Local administration of agonists in dorsal raphe nucleus mainly produces a response similar to the “low-dose” systemic administration, decreasing wakefulness and increasing rapid eye movement (REM) sleep via disinhibition of mesopontine REM sleep promoting neurons. Systemic administration of 5-HT1B receptors agonists consistently increases wakefulness and decreases REM sleep, as do the 5-HT1A agonists. The mechanism by which 5-HT1B receptors affect state modulation remain elusive. The general arousing effects of 5-HT1A and 5-HT1B agonists should also be considered in relation to the multiple, largely redundant, neurotransmitter systems that maintain arousal. Finally, 5-HT1A and 5-HT1B receptor are important modulators of the circadian rhythm largely by affecting the response of the suprachiasmatic nucleus to light and the secretion of melatonin from the pineal gland. The development of more selective ligands seems crucial to further explore the role of these receptors in state modulation.

Increased extracellular 5-HT but no change in sleep after perfusion of a 5-HT1A antagonist into the dorsal raphe nucleus of rats

Aim:  The 5‐HT1A receptor antagonist 4‐Iodo‐N‐[2‐[4‐(methoxyphenyl)‐1‐piperazinyl]ethyl]‐N‐2‐pyridinyl‐benzamide hydrochloride (p‐MPPI) (10 μm) was perfused into the dorsal raphe nucleus (DRN) to study simultaneously the effects of the drug on the DRN and frontal cortex extracellular serotonin (5‐hydroxytryptamine, 5‐HT) levels and concurring behavioural states.