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-HT(1A) receptor antagonist 4-Iodo-N-[2-[4-(methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinyl-benzamide hydrochloride (p-MPPI) (10 microM) 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. METHODS: Waking, slow wave sleep and rapid eye movement sleep were determined by polygraphic recordings during microdialysis perfusion and extracellular sample collection. The samples were analysed by microbore high-performance liquid chromatography coupled with electrochemical detection for analysis of 5-HT. RESULTS: p-MPPI perfusion into the DRN (n = 6) produced a sixfold 5-HT increase in the DRN during all behavioural states. The increased 5-HT level was most likely related to the blockage of 5-HT(1A) receptors in the DRN by p-MPPI. No significant effect was seen on sleep. CONCLUSION: Despite the dramatic increase in DRN extracellular 5-HT produced by p-MPPI, only a transient and nonsignificant effect on sleep was recorded. It is suggested that the usual coupling between 5-HT level and behavioural state may be lost when an excessive serotonergic output is pharmacologically achieved.

Effects of sleep deprivation on extracellular serotonin in hippocampus and frontal cortex of the rat.

Sleep deprivation improves the mood of depressed patients, but the exact mechanism behind this effect is unclear. An enhancement of serotonergic neurotransmission has been suggested. In this study, we used in vivo microdialysis to monitor extracellular serotonin in the hippocampus and the frontal cortex of rats during an 8 h sleep deprivation period. These brain regions were selected since both have been implicated in depression. The behavioral state of the animal was continuously monitored by polygraphic recordings during the experiment. Sleep deprivation produced a gradual decline in extracellular serotonin levels, both in the hippocampus and in the frontal cortex. In order to investigate whether the reduction in serotonin was due to other factors than sleep deprivation, i.e. time of day effect, another experiment was performed. Here animals were allowed to sleep during most of the recording period. This experiment showed the expected changes in extracellular serotonin levels: consistently higher levels in the awake, non-sleep deprived animals compared to during sleep, but no time of day effect. The reduction in extracellular serotonin during sleep deprivation may suggest that serotonin does not play a major role in the mood-elevating effect of sleep deprivation. However, since 5-HT levels are strongly behavioral state dependent, by eliminating sleep, there may be a net increase in serotonergic neurotransmission during the sleep deprivation period.

Adenosinergic modulation of basal forebrain and preoptic/anterior hypothalamic neuronal activity in the control of behavioral state.

This review describes a series of animal experiments that investigate the role of endogenous adenosine (AD) in sleep. We propose that AD is a modulator of the sleepiness associated with prolonged wakefulness. More specifically, we suggest that, during prolonged wakefulness, extracellular AD accumulates selectively in the basal forebrain (BF) and cortex and promotes the transition from wakefulness to slow wave sleep (SWS) by inhibiting cholinergic and non-cholinergic wakefulness-promoting BF neurons at the AD A1 receptor. New in vitro data are also compatible with the hypothesis that, via presynaptic inhibition of GABAergic inhibitory input, AD may disinhibit neurons in the preoptic/anterior hypothalamus (POAH) that have SWS-selective activity and Fos expression. Our in vitro recordings initially showed that endogenous AD suppressed the discharge activity of neurons in the BF cholinergic zone via the AD A1 receptor. Moreover, in identified mesopontine cholinergic neurons, AD was shown to act post-synaptically by hyperpolarizng the membrane via an inwardly rectifying potassium current and inhibition of the hyperpolarization-activated current, I(h). In vivo microdialysis in the cat has shown that AD in the BF cholinergic zone accumulates during prolonged wakefulness, and declines slowly during subsequent sleep, findings confirmed in the rat. Moreover, increasing BF AD concentrations to approximately the level as during sleep deprivation by a nucleoside transport blocker mimicked the effect of sleep deprivation on both the EEG power spectrum and behavioral state distribution: wakefulness was decreased, and there were increases in SWS and REM sleep. As predicted, microdialyis application of the specific A1 receptor antagonist cyclopentyltheophylline (CPT) in the BF produced the opposite effects on behavioral state, increasing wakefulness and decreasing SWS and REM. Combined unit recording and microdialysis studies have shown neurons selectively active in wakefulness, compared with SWS, have discharge activity suppressed by both AD and the A1-specific agonist cyclohexyladenosine (CHA), while discharge activity is increased by the A1 receptor antagonist, CPT. We next addressed the question of whether AD exerts its effects locally or globally. Adenosine accumulation during prolonged wakefulness occurred in the BF and neocortex, although, unlike in the BF, cortical AD levels declined in the 6th h of sleep deprivation and declined further during subsequent recovery sleep. Somewhat to our surprise, AD concentrations did not increase during prolonged wakefulness (6 h) even in regions important in behavioral state control, such as the POAH, dorsal raphe nucleus, and pedunculopontine tegmental nucleus, nor did it increase in the ventrolateral/ventroanterior thalamic nucleii. These data suggest the presence of brain region-specific differences in AD transporters and/or degradation that become evident with prolonged wakefulness, even though AD concentrations are higher in all brain sites sampled during the naturally occurring (and shorter duration) episodes of wakefulness as compared to sleep episodes in the freely moving and behaving cat. Might AD also produce modulation of activity of neurons that have sleep selective transcriptional (Fos) and discharge activity in the preoptic/anterior hypothalamus zone? Whole cell patch clamp recordings in the in vitro horizontal slice showed fast and likely GABAergic inhibitory post-synaptic potentials and currents that were greatly decreased by bath application of AD. Adenosine may thus disinhibit and promote expression of sleep-related neuronal activity in the POAH. In summary, a growing body of evidence supports the role of AD as a mediator of the sleepiness following prolonged wakefulness, a role in which its inhibitory actions on the BF wakefulness-promoting neurons may be especially important.

How does the brain sustain a visual percept?

Perception involves the processing of sensory stimuli and their translation into conscious experience. A novel percept can, once synthesized, be maintained or discarded from awareness. We used event-related functional magnetic resonance imaging to separate the neural responses associated with the maintenance of a percept, produced by single-image, random-dot stereograms, from the response evoked at the onset of the percept. The latter was associated with distributed bilateral activation in the posterior thalamus and regions in the occipito-temporal, parietal and frontal cortices. In contrast, sustained perception was associated with activation of the pre-frontal cortex and hippocampus. This observation suggests that sustaining a visual percept involves neuroanatomical systems which are implicated in memory function and which are distinct from those engaged during perceptual synthesis.

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.

Auditory processing across the sleep-wake cycle: simultaneous EEG and fMRI monitoring in humans.

We combined fMRI and EEG recording to study the neurophysiological responses associated with auditory stimulation across the sleep-wake cycle. We found that presentation of auditory stimuli produces bilateral activation in auditory cortex, thalamus, and caudate during both wakefulness and nonrapid eye movement (NREM) sleep. However, the left parietal and, bilaterally, the prefrontal and cingulate cortices and the thalamus were less activated during NREM sleep compared to wakefulness. These areas may play a role in the further processing of sensory information required to achieve conscious perception during wakefulness. Finally, during NREM sleep, the left amygdala and the left prefrontal cortex were more activated by stimuli having special affective significance than by neutral stimuli. These data suggests that the sleeping brain can process auditory stimuli and detect meaningful events.

A specific role for the thalamus in mediating the interaction of attention and arousal in humans.

The physiological basis for the interaction of selective attention and arousal is not clearly understood. Here we present evidence in humans that specifically implicates the thalamus in this interaction. We used functional magnetic resonance imaging to measure brain activity during the performance of an attentional task under different levels of arousal. Activity evoked in the ventrolateral thalamus by the attentional task changed as a function of arousal. The highest level of attention-related thalamic activity is seen under conditions of low arousal (secondary to sleep deprivation) compared with high arousal (secondary to caffeine administration). Other brain regions were also active during the attentional task, but these areas did not change their activity as a function of arousal. Control experiments establish that this pattern of changes in thalamic activity cannot be accounted for by nonspecific effects of arousal on cerebral hemodynamics. We conclude that the thalamus is involved in mediating the interaction of attention and arousal in humans.

Volumetric evaluation of the thalamus in schizophrenic male patients using magnetic resonance imaging.

BACKGROUND: The thalamus, an important subcortical brain region connecting limbic and prefrontal cortices, has a significant role in sensory and cortical processing. Although inconsistently, previous studies have demonstrated neuroanatomical abnormalities in the thalamus of schizophrenic patients. METHODS: This structural magnetic resonance imaging study, based on segmentation of contiguous coronal 1.5-mm images, compared thalamic brain volumes of 15 chronic, male schizophrenic patients with 15 normal controls matched on age, sex, handedness, and parental socioeconomic status. RESULTS: There were no significant differences between patients and controls in thalamic volumes, right or left, adjusted for total brain volume; however, there were significantly different correlations of thalamic volumes with prefrontal white matter and lateral ventricles among patients, but not among controls. Thalamic volumes among patients were also significantly correlated with bizarre behavior, hallucinations, and thought disorder. CONCLUSIONS: Findings suggest that connectivity between thalamic nuclei and prefrontal cortical areas are abnormal in chronic male schizophrenic patients. In addition, ventricular enlargement may be, in part, due to subtle reduction in thalamic volume and/or in volume of thalamocortical and corticothalamic fibers secondary to thalamic abnormalities. Finally, correlations with positive symptomatology underscore the role of the thalamus in gating or filtering of sensory information and coordination of cortical processing.

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.

Role of adenosine in behavioral state modulation: a microdialysis study in the freely moving cat.

There is considerable evidence to suggest that the activity of forebrain and mesopontine cholinergic neurons is intimately involved in electroencephalographic arousal. Furthermore, our previous in vitro investigation suggested that both cholinergic systems are under a powerful tonic inhibitory control by endogenous adenosine. We thus examined the in vivo effect, on electrographically defined behavioral states, of microdialysis perfusion of adenosine into the cholinergic zones of the substantia innominata of the basal forebrain and the laterodorsal tegmental nucleus of freely moving cats. Localized perfusion of adenosine into either the basal forebrain or the laterodorsal tegmental nucleus caused a marked alteration in sleep-wake architecture. Adenosine (300 microM) perfused into either the basal forebrain or laterodorsal tegmental nucleus produced a dramatic decrease in waking, to about 50% of the basal level. Perfusion into the basal forebrain resulted in a significant increase in rapid eye movement sleep, while slow wave sleep was unchanged. In contrast, adenosine perfusion into the laterodorsal tegmental nucleus produced an increase of both slow wave sleep and rapid eye movement sleep, the magnitude of which were proportional to the decrease in waking. Electroencephalographic power spectral analysis showed that adenosine perfusion into the basal forebrain increased the relative power in the delta frequency band, whereas higher frequency bands (theta, alpha, beta and gamma) showed a decrease. These data strongly support the hypothesis that adenosine might play a key role as an endogenous modulator of wakefulness and sleep. The decrease in wakefulness may be directly related to the inhibition of cholinergic neurons of the basal forebrain and the laterodorsal tegmentum. The increase in rapid eye movement sleep is a novel but robust effect whose origin, at present, is uncertain. The observation that local perfusion of adenosine into either the basal forebrain or the laterodorsal tegmental nucleus dramatically decreases wakefulness suggests that these areas might represent a major site of action of the xanthine stimulants (adenosine antagonists) found in coffee and tea.

Microdialysis perfusion of 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) in the dorsal raphe nucleus decreases serotonin release and increases rapid eye movement sleep in the freely moving cat.

In vivo microdialysis was used to analyze the role of dorsal raphe nucleus (DRN) neurons in regulating the sleep-waking cycle. Measurements of extracellular serotonin (5-HT) were made in the DRN of freely moving adult cats before and during microdialysis perfusion of 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), a selective 5-HT1A receptor agonist, in artificial CSF. Behavioral state alterations were measured by simultaneous polygraphic recordings. During waking and artificial CSF perfusion of probes histologically localized to the DRN, extracellular 5-HT was 4 fmol/7.5 micro L dialysate sample. With the addition of 8-OH-DPAT (10 microM in artificial CSF) to the perfusate, 5-HT levels in the same state decreased 50%, to 2 fmol/sample (p < 0.01), presumably through 5-HT1A autoreceptor-mediated inhibition of serotonergic neural activity. Concomitantly, this 8-OH-DPAT perfusion produced a short latency, threefold increase in rapid eye movement (REM) sleep, from 10 to 30% of the total recorded time (p < 0.05), whereas waking was not significantly affected. In contrast, and suggesting DRN specificity, 8-OH-DPAT delivery through a probe in the aqueduct did not increase REM sleep but rather tended to increase waking and decrease slow wave sleep. The data on REM sleep provide the first biochemically validated and direct evidence that suppression of DRN serotonergic activity increases REM sleep, and furnish a key complement to our laboratory's in vitro data indicating that mesopontine cholinergic neurons, a target of DRN projections, are inhibited by 5-HT. The 8-OH-DPAT-induced reduction of DRN 5-HT is consistent with the hypothesis that the concomitant REM sleep disinhibition is mediated by DRN serotonergic projections to mesopontine cholinergic neurons, which other data implicate in REM sleep production.

Behavioral state-related changes of extracellular serotonin concentration in the dorsal raphe nucleus: a microdialysis study in the freely moving cat.

For direct measurement of the extracellular concentration of serotonin (5-HT) in the dorsal raphe nucleus (DRN) over the sleep-wake cycle we used the technique of in vivo microdialysis in six freely moving, naturally sleeping cats whose behavioral state was polygraphically determined. Perfusate samples from microdialysis probes histologically localized to the DRN showed the following significantly different levels of extracellular 5-HT, with the numbers in parentheses indicating successively the mean value in fmol/5 microliters perfusate sample, the % level relative to waking, and the sample n: waking (4.02, 100%, n = 38) > slow wave sleep (2.02, 50%, n = 30) > REM sleep (1.61, 38%, n = 17). These data, to our knowledge the first direct DRN 5-HT measurements during behavioral state changes, directly parallel the levels of serotonergic neuronal action potential activity and suggest that DRN extracellular 5-HT is determined by this action potential activity through synaptic release by recurrent axonal collaterals in the DRN.

Effect of ethanol on extracellular 5-hydroxytryptamine output in rat frontal cortex.

The effect of acute administration of ethanol on the release of 5-hydroxytryptamine (5-HT) in the frontal cortex of "Sardinian alcohol preferring" rats, "Sardinian non-preferring" rats and control rats was investigated using in vivo microdialysis. At the dosage of 2.5 g/kg i.p. ethanol increased the level of 5-HT in the dialysate by approximately 80 +/- 20% of basal values in the Sardinian alcohol preferring rats whereas no effect was observed in Sardinian non-preferring and control rats.

Effect of p-chlorophenylalanine on release of 5-hydroxytryptamine from the rat frontal cortex in vivo.

1. Rats were given p-chlorophenylalanine (PCPA, 150 mg kg-1, i.p.) to inhibit partially 5-hydroxytryptamine (5-HT) synthesis so that its concentration in the frontal cortex fell by about half. The effects of this treatment on frontal cortex dialysate 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) concentrations were determined before and after stimulation by increasing K+ concentration in the perfusion fluid by 100 mM for 20 min. Rates of 5-HT synthesis as indicated by the effects of 3-hydroxybenzylhydrazine (NSD 1015, 150 mg kg-1, i.p.) on frontal cortex tissue and dialysate 5-hydroxytryptophan (5-HTP) and dialysate 5-HIAA were also measured in rats that had not been stimulated with K+. 2. Dialysate 5-HT and 5-HIAA concentrations of both vehicle- and PCPA-treated rats fell into major (group 1) and minor (group 2) populations statistically distinguishable from each other by the high 5-HT and low 5-HIAA values of the latter group. 3. In group 1 animals, PCPA decreased both the dialysate 5-HT concentration and its rise following stimulation by K+ in proportion with the decrease of 5-HT in frontal cortex tissue. 5-HIAA fell more markedly than 5-HT and in similar proportion in both tissue and dialysate. The fall of dialysate 5-HIAA on stimulation by K+ was also attenuated to the same degree. The elevated 5-HT/5-HIAA ratios after PCPA treatment imply increased conservation of the depleted 5-HT stores. 4. PCPA decreased the above 5-HIAA values and the effects of NSD 1015 on tissue 5-HTP or dialysate 5-HIAA concentrations in similar proportion. However, PCPA had little effect on corresponding dialysate 5-HTP values. 5. The results are discussed with respect to relationships between synthesis, storage and release of 5-HT. They indicate that (under the conditions of the present study) the availability of 5-HT to receptors is directly proportional to total vesicular stores under both basal conditions and during neuronal firing.

Brain dialysis and dopamine: does the extracellular concentration of dopamine reflect synaptic release?

We considered the drug-induced circling behaviour of rats monolaterally lesioned with kainic acid (KA) as a marker of the dopamine (DA) concentration in the synaptic space. D-Amphetamine produced a dose-related increase in the DA concentration of the dialysate from an intact striatum and a proportional number of ipsilateral circlings. Pargyline or L-dihydroxyphenylalanine (L-Dopa), alone or in combination with benserazide, increased the concentration of DA to a similar or even higher level than d-amphetamine, but failed to elicit a circling response. Apomorphine given after these drugs at the peak of DA accumulation always induced circling behaviour. The results suggest that released DA may undergo different inactivation processes before reaching the dialysis probe, and that these processes may be differentially affected by drug treatments. Alternatively, it may be suggested that DA can be released into the synaptic space, in a functional manner, or into the interstitial fluid, from where it cannot reach the synaptic receptors.