Sleep active cortical neurons expressing neuronal nitric oxide synthase are active after both acute sleep deprivation and chronic sleep restriction.

Non-rapid eye movement (NREM) sleep electroencephalographic (EEG) delta power (~0.5-4 Hz), also known as slow wave activity (SWA), is typically enhanced after acute sleep deprivation (SD) but not after chronic sleep restriction (CSR). Recently, sleep-active cortical neurons expressing neuronal nitric oxide synthase (nNOS) were identified and associated with enhanced SWA after short acute bouts of SD (i.e., 6h). However, the relationship between cortical nNOS neuronal activity and SWA during CSR is unknown. We compared the activity of cortical neurons expressing nNOS (via c-Fos and nNOS immuno-reactivity, respectively) and sleep in rats in three conditions: (1) after 18-h of acute SD; (2) after five consecutive days of sleep restriction (SR) (18-h SD per day with 6h ad libitum sleep opportunity per day); (3) and time-of-day matched ad libitum sleep controls. Cortical nNOS neuronal activity was enhanced during sleep after both 18-h SD and 5 days of SR treatments compared to control treatments. SWA and NREM sleep delta energy (the product of NREM sleep duration and SWA) were positively correlated with enhanced cortical nNOS neuronal activity after 18-h SD but not 5days of SR. That neurons expressing nNOS were active after longer amounts of acute SD (18h vs. 6h reported in the literature) and were correlated with SWA further suggest that these cells might regulate SWA. However, since these neurons were active after CSR when SWA was not enhanced, these findings suggest that mechanisms downstream of their activation are altered during CSR.

c-Fos protein expression is increased in cholinergic neurons of the rodent basal forebrain during spontaneous and induced wakefulness.

It has been proposed that cholinergic neurons of the basal forebrain (BF) may play a role in vigilance state control. Since not all vigilance states have been studied, we evaluated cholinergic neuronal activation levels across spontaneously occurring states of vigilance, as well as during sleep deprivation and recovery sleep following sleep deprivation. Sleep deprivation was performed for 2h at the beginning of the light (inactive) period, by means of gentle sensory stimulation. In the rodent BF, we used immunohistochemical detection of the c-Fos protein as a marker for activation, combined with labeling for choline acetyl-transferase (ChAT) as a marker for cholinergic neurons. We found c-Fos activation in BF cholinergic neurons was highest in the group undergoing sleep deprivation (12.9% of cholinergic neurons), while the spontaneous wakefulness group showed a significant increase (9.2%), compared to labeling in the spontaneous sleep group (1.8%) and a sleep deprivation recovery group (0.8%). A subpopulation of cholinergic neurons expressed c-Fos during spontaneous wakefulness, when possible confounds of the sleep deprivation procedure were minimized (e.g., stress and sensory stimulation). Double-labeling in the sleep deprivation treatment group was significantly elevated in select subnuclei of the BF (medial septum/vertical limb of the diagonal band, horizontal limb of the diagonal band, and the magnocellular preoptic nucleus), when compared to spontaneous wakefulness. These findings support and provide additional confirming data of previous reports that cholinergic neurons of BF play a role in vigilance state regulation by promoting wakefulness.

Sleep fragmentation elevates behavioral, electrographic and neurochemical measures of sleepiness.

Sleep fragmentation, a feature of sleep apnea as well as other sleep and medical/psychiatric disorders, is thought to lead to excessive daytime sleepiness. A rodent model of sleep fragmentation was developed (termed sleep interruption, SI), where rats were awakened every 2 min by the movement of an automated treadmill for either 6 or 24 h of exposure. The sleep pattern of rats exposed to 24 h of SI resembled sleep of the apneic patient in the following ways: sleep was fragmented (up to 30 awakening/h), total rapid eye movement (REM) sleep time was greatly reduced, non-rapid eye movement (NREM) sleep episode duration was reduced (from 2 min, 5 s baseline to 58 s during SI), whereas the total amount of NREM sleep time per 24 h approached basal levels. Both 6 and 24 h of SI made rats more sleepy, as indicated by a reduced latency to fall asleep upon SI termination. Electrographic measures in the recovery sleep period following either 6 or 24 h of SI also indicated an elevation of homeostatic sleep drive; specifically, the average NREM episode duration increased (e.g. for 24 h SI, from 2 min, 5 s baseline to 3 min, 19 s following SI), as did the NREM delta power during recovery sleep. Basal forebrain (BF) levels of extracellular adenosine (AD) were also measured with microdialysis sample collection and high performance liquid chromatography detection, as previous work suggests that increasing concentrations of BF AD are related to sleepiness. BF AD levels were significantly elevated during SI, peaking at 220% of baseline during 30 h of SI exposure. These combined findings imply an elevation of the homeostatic sleep drive following either 6 or 24 h of SI, and BF AD levels appear to correlate more with sleepiness than with the cumulative amount of prior wakefulness, since total NREM sleep time declined only slightly. SI may be partially responsible for the symptom of daytime sleepiness observed in a number of clinical disorders, and this may be mediated by mechanisms involving BF AD.

Differential effect of orexins (hypocretins) on serotonin release in the dorsal and median raphe nuclei of freely behaving rats.

Orexin (hypocretin)-containing neurons in the perifornical hypothalamus project to widespread regions of the brain, including the dorsal and median raphe nuclei [Peyron C, Tighe DK, van den Pol AN, de Lecea L, Heller HC, Sutcliffe JG, Kilduff TS (1998) Neurons containing hypocretin (orexin) project to multiple neuronal systems. J Neurosci 18:9996-10015; Wang QP, Koyama Y, Guan JL, Takahashi K, Kayama Y, Shioda S (2005) The orexinergic synaptic innervation of serotonin- and orexin 1-receptor-containing neurons in the dorsal raphe nucleus. Regul Pept 126:35-42]. Orexin-A or orexin-B was infused by reverse microdialysis into the dorsal raphe nucleus or median raphe nucleus of freely behaving rats, and extracellular serotonin was simultaneously collected by microdialysis and analyzed by high-performance liquid chromatography. We have found that orexin-A produced a dose-dependent increase of serotonin in the dorsal raphe nucleus, but not in the median raphe nucleus. However, orexin-B elicited a small but significant effect in both the dorsal raphe nucleus and median raphe nucleus. Orexins may have regionally selective effects on serotonin release in the CNS, implying a unique interaction between orexins and serotonin in the regulation of activities including sleep-wakefulness.

Effects on serotonin of (-)nicotine and dimethylphenylpiperazinium in the dorsal raphe and nucleus accumbens of freely behaving rats.

The aim of this study was to investigate the neurochemical mechanism underlying the effect of nicotine and dimethylphenylpiperazinium (DMPP) on 5-hydroxytryptamine (5-HT) release in the dorsal raphe nucleus and nucleus accumbens of freely behaving rats. For comparison, lobeline, cytisine and RJR-2403 were also investigated. It was found that all drugs, when infused locally, evoked an increase of 5-HT in the dorsal raphe nucleus. However, the magnitudes of the 5-HT increase were comparatively different between the drugs in the ranking of their potency: DMPP>RJR 2403>>nicotine>lobeline>cytisine. Both methyllycaconitine, a nicotinic acetylcholine receptor (nAChR) antagonist and methyllycaconitine, a selective alpha7-containing nAChR antagonist blocked the effects of nicotine and DMPP, suggesting that alpha7 subunit mediated the increases in 5-HT. However, DMPP was reported to increase 5-HT using non-nAChR mechanism [Lendvai B, Sershen H, Lajtha A, Santha E, Baranyi M, Vizi ES (1996) Differential mechanisms involved in the effect of nicotinic agonists DMPP and lobeline to release [3H]5-HT from rat hippocampal slices. Neuropharmacology 35:1769-1777]. To test if 5-HT carriers were involved, a selective 5-HT reuptake inhibitor citalopram (1 microM) was infused into the dorsal raphe nucleus before administration of nicotine or DMPP. As a result, citalopram significantly blocked the effect of DMPP, whereas it had no influence on nicotine. Finally, the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) was used to test whether the increases in 5-HT were depolarization-dependent. Administration of 8-OH-DPAT (0.1 mg/kg, s.c.) produced significant decreases in 5-HT in the animals treated with nicotine. In contrast, the effect of DMPP was not altered by 8-OH-DPAT, suggesting that the increases in 5-HT were independent of cell membrane depolarization. In conclusion, there are different mechanisms involved in nicotine- and DMPP-evoked increases in 5-HT. This is consistent with prior work suggesting DMPP may primarily act on 5-HT carriers.

Adenosinergic inhibition of basal forebrain wakefulness-active neurons: a simultaneous unit recording and microdialysis study in freely behaving cats.

The majority of neurons in the magnocellular basal forebrain are wakefulness-active with highest discharge activity during wakefulness and a marked reduction in activity just before and during the entry to non-rapid eye movement (REM) sleep. We have hypothesized that the reduction of discharge activity of wakefulness-active neurons and a consequent facilitation of the transition from wakefulness to sleep is due to an increase in the extracellular concentration of adenosine during wakefulness. To test the hypothesis, the present study employed microdialysis perfusion of adenosinergic pharmacological agents combined with single unit recording in freely moving cats, so as to determine: 1). if there were dose-dependent effects on behaviorally identified wakefulness-active neurons; 2). whether effects were mediated by the A1 receptor, as contrasted to the A2a receptor; and 3). if effects were specific to wakefulness-active neurons, and not present in sleep-active neurons, those preferentially discharging in nonREM sleep. Both adenosine and the A1 receptor-specific agonist N6-cyclo-hexyl-adenosine reduced the discharge activity of wakefulness-active neurons (n=16) in a dose-dependent manner but had no effect on sleep-active neurons (n=4). The A1 receptor antagonist 8-cyclopentyl-1-3-dimethylxanthine increased the discharge of wakefulness-active neurons (n=5), but the A2a receptor agonist, CGS-16284, had no effect (n=3). Recording sites were histologically localized to the cholinergic basal forebrain. These data support our hypothesis that adenosine acts via the A1 receptor to reduce the activity of wakefulness-promoting neurons, thus providing a cellular mechanism explaining why the increased adenosine concentrations observed in the basal forebrain following prolonged wakefulness act to induce sleep.

Ventrolateral preoptic nucleus contains sleep-active, galaninergic neurons in multiple mammalian species.

The ventrolateral preoptic nucleus (VLPO) is a group of sleep-active neurons that has been identified in the hypothalamus of rats and is thought to inhibit the major ascending monoaminergic arousal systems during sleep; lesions of the VLPO cause insomnia. Identification of the VLPO in other species has been complicated by the lack of a marker for this cell population, other than the expression of Fos during sleep. We now report that a high percentage of the sleep-active (Fos-expressing) VLPO neurons express mRNA for the inhibitory neuropeptide, galanin, in nocturnal rodents (mice and rats), diurnal rodents (degus), and cats. A homologous (i.e. galanin mRNA-containing cell group) is clearly distinguishable in the ventrolateral region of the preoptic area in diurnal and nocturnal monkeys, as well as in humans. Galanin expression may serve to identify sleep-active neurons in the ventrolateral preoptic area of the mammalian brain. The VLPO appears to be a critical component of sleep circuitry across multiple species, and we hypothesize that shrinkage of the VLPO with advancing age may explain sleep deficits in elderly humans.

Extracellular histamine levels in the feline preoptic/anterior hypothalamic area during natural sleep-wakefulness and prolonged wakefulness: an in vivo microdialysis study.

Increased activity of the histaminergic neurons of the posterior hypothalamus has been implicated in the facilitation of behavioral wakefulness. Recent evidence of reciprocal projections between the sleep-active neurons of the preoptic/anterior hypothalamus and the histaminergic neurons of the tuberomammillary nucleus suggests that histaminergic innervation of the preoptic/anterior hypothalamic area may be of particular importance in the wakefulness-promoting properties of histamine. To test this possibility, we used microdialysis sample collection in the preoptic/anterior hypothalamic area of cats during natural sleep-wakefulness cycles, 6 h of sleep deprivation induced by gentle handling/playing, and recovery sleep. Samples were analyzed by a sensitive radioenzymatic assay. Mean basal levels of histamine in microdialysate during periods of wakefulness (1.155+/-0.225 pg/microl) did not vary during the 6 h of sleep deprivation. However, during the different sleep states, dramatic changes were observed in the extracellular histamine levels of preoptic/anterior hypothalamic area: wakefulness>non-rapid eye movement sleep>rapid eye movement sleep. Levels of histamine during rapid eye movement sleep were lowest (0.245+/-0.032 pg/microl), being significantly lower than levels during non-rapid eye movement sleep (0.395+/-0.081 pg/microl) and being only 21% of wakefulness levels. This pattern of preoptic/anterior hypothalamic area extracellular histamine levels across the sleep-wakefulness cycle closely resembles the reported single unit activity of histaminergic neurons. However, the invariance of histamine levels during sleep deprivation suggests that changes in histamine level do not relay information about sleep drive to the sleep-promoting neurons of the preoptic/anterior hypothalamic area.

Microdialysis perfusion of orexin-A in the basal forebrain increases wakefulness in freely behaving rats.

Recent work indicates that the orexin/hypocretin-containing neurons of the lateral hypothalamus are involved in control of REM sleep phenomena, but site-specific actions in control of wakefulness have been less studied. Orexin-containing neurons project to both brainstem and forebrain regions that are known to regulate sleep and wakefulness, including the field of cholinergic neurons in the basal forebrain (BF) that is implicated in regulation of wakefulness, and includes, in the rat, the horizontal limb of the diagonal band, the substantia innominata, and the magnocellular preoptic region. The present study used microdialysis perfusion of orexin-A directly in the cholinergic BF region of rat to test the hypothesis that orexin-A enhances W via a local action in the BF. A significant dose-dependent increase in W was produced by the perfusion of three doses of orexin-A in the BF (0.1, 1.0, and 10.0 microM), with 10.0 microM producing more than a 5-fold increase in wakefulness, which occupied 44% of the light (inactive) phase recording period. Orexin-A perfusion also produced a significant dose-dependent decrease in nonREM sleep, and a trend-level decrease in REM sleep. The results clearly demonstrate a potent capacity of orexin-A to induce wakefulness via a local action in the BF, and are consistent with previous work indicating that the BF cholinergic zone neurons have a critical role in the regulation of EEG activation and W. The data suggest further that orexin-A has a significant role in the regulation of arousal/wakefulness, in addition to the previously described role of orexin in the regulation and expression of REM sleep and REM sleep-related phenomena.

Adenosine as a biological signal mediating sleepiness following prolonged wakefulness.

Recent reports from our laboratory have shown that extracellular adenosine levels selectively increase in basal forebrain during prolonged wakefulness in cats and rats. Furthermore, microdialysis perfusion of adenosine into the basal forebrain (BF) increased sleepiness and decreased wakefulness in both the species, whereas perfusion of the A(1)-receptor-selective antagonist, cyclopentyl-1, 3-dimethylxanthine resulted in increased wakefulness, an observation similar to that found with caffeine or theophylline administration. The selective participation of the A(1) subtype of the adenosine receptor in mediating the effects of adenosine in the BF was further examined by the technique of single unit recording performed in conjunction with microdialysis perfusion of selective agonists and antagonists. Perfusion of the A(1) agonist cyclohexyladenosine, inhibited the activity of wake-active neurons in the basal forebrain. The effect of prolonged wakefulness-induced increases in adenosine levels were further investigated by determining the changes in the BF in the levels of A(1) receptor binding and the levels of its mRNA. We observed that A(1) receptor mRNA levels increase after 6 h of sleep deprivation. One of the transcription factors that showed increased DNA-binding activity was nuclear factor kappaB (NF-kappaB) and may regulate the expression of A(1) mRNA. We observed, using a gel shift assay, that the DNA-binding activity of NF-kappaB increased following 3 h of sleep deprivation. This was further supported by the increased appearance of NF-kappaB protein in the nuclear extracts and the consequent disappearance of cytoplasmic protein inhibitor kappaB (I-kappaB). Together our results reviewed in this report suggest that the somnogenic effects of adenosine in the BF area may be mediated by the A(1) subtype of adenosine receptor, and its expression might be regulated by induction in the NF-kappaB protein as its transcription factor. This positive feedback might mediate some of long-duration effects of sleep deprivation, including 'sleep debt'.

Brain site-specificity of extracellular adenosine concentration changes during sleep deprivation and spontaneous sleep: an in vivo microdialysis study.

Previous data suggested that increases in extracellular adenosine in the basal forebrain mediated the sleep-inducing effects of prolonged wakefulness. The present study sought to determine if the state-related changes found in basal forebrain adenosine levels occurred uniformly throughout the brain. In vivo microdialysis sample collection coupled to microbore high-performance liquid chromatography measured extracellular adenosine levels in six brain regions of the cat: basal forebrain, cerebral cortex, thalamus, preoptic area of hypothalamus, dorsal raphe nucleus and pedunculopontine tegmental nucleus. In all these brain regions extracellular adenosine levels showed a similar decline of 15 to 20% during episodes of spontaneous sleep relative to wakefulness. Adenosine levels during non-rapid eye movement sleep did not differ from rapid eye movement sleep. In the course of 6h of sleep deprivation, adenosine levels increased significantly in the cholinergic region of the basal forebrain (to 140% of baseline) and, to a lesser extent in the cortex, but not in the other regions. Following sleep deprivation, basal forebrain adenosine levels declined very slowly, remaining significantly elevated throughout a 3-h period of recovery sleep, but elsewhere levels were either similar to, or lower than, baseline. The site-specific accumulation of adenosine during sleep deprivation suggests a differential regulation of adenosine levels by as yet unidentified mechanisms. Moreover, the unique pattern of sleep-related changes in basal forebrain adenosine level lends strong support to the hypothesis that the sleep-promoting effects of adenosine, as well as the sleepiness associated with prolonged wakefulness, are both mediated by adenosinergic inhibition of a cortically projecting basal forebrain arousal system.

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.

A comparison of the effects of amphetamine and low doses of apomorphine on operant force production, inter-response times and response duration in rat.

RATIONALE: Low doses of apomorphine (APO), a non-selective dopamine (DA) agonist, are thought to suppress motor activity via the preferential activation of DA autoreceptors, which effectively reduces DA tone. OBJECTIVES: The suppressant effects on operant responding of low doses of apomorphine were explored and compared with the effects of amphetamine (AMP), an indirect DA agonist. METHODS: In an operant task, rats were trained to press sequentially three separate beams under the following different behavioral requirements: low-force beam (1 g50 g), and a long-duration beam (response duration>2 s). Inter-response times and kinetic measures, such as peak force, the rate of rise of force and response duration, were recorded. Following training, performance was assessed after systemic injection of low doses of APO (0.01, 0.03 and 0.1 mg/kg, s.c.) and AMP (0.1, 0.3 and 1.0 mg/kg, i.p.). RESULTS: APO decreased peak force for the high-force and the long-duration beams by decreasing the rate of rise of force, but did not affect performance success on the low-force beam or response duration on the long-duration beam. This indicates that APO impaired the ability to generate high forces but did not interfere with the memory or execution of an overall motor plan. Low doses of APO also increased the times taken to switch from one response to the next and to visit the tray when food was present. In contrast, AMP at 1.0 mg/kg shortened both the time taken to switch between responses and the time spent visiting the food tray. CONCLUSIONS: Low doses of APO interfered with response initiation and execution, suggesting that dopamine acts as a "gating" system, enabling certain processes to be carried out in an efficient and automated manner.

REM sleep enhancement and behavioral cataplexy following orexin (hypocretin)-II receptor antisense perfusion in the pontine reticular formation.

Orexin (hypocretin)-containing neurons of the hypothalamus project to brainstem sites that are involved in the neural control of REM sleep, including the locus coeruleus, the dorsal raphe nucleus, the cholinergic zone of the mesopontine tegmentum, and the pontine reticular formation (PRF). Orexin knockout mice exhibit narcolepsy/cataplexy, and a mutant and defective gene for the orexin type II receptor is present in dogs with an inherited form of narcolepsy/cataplexy. However, the physiological systems mediating these effects have not been described. We reasoned that, since the effector neurons for the majority of REM sleep signs, including muscle atonia, were located in the PRF, this region was likely implicated in the production of these orexin-related abnormalities. To test this possibility, we used microdialysis perfusion of orexin type II receptor antisense in the PRF of rats. Ten to 24 hours after antisense perfusion, REM sleep increased two- to three-fold during both the light period (quiescent phase) and the dark period (active phase), and infrared video showed episodes of behavioral cataplexy. Moreover, preliminary data indicated no REM-related effects following perfusion with nonsense DNA, or when perfusion sites were outside the PRF. More work is needed to provide precise localization of the most effective site of orexin-induced inhibition of REM sleep phenomena.

Behavioral state-related changes of extracellular serotonin concentration in the pedunculopontine tegmental nucleus: a microdialysis study in freely moving animals.

Neurons of the cholinergic mesopontine tegmentum preferentially discharge during REM sleep and are thought to promote this state. It has been hypothesized they are inhibited during wakefulness by serotonergic input. The present study used the microdialysis sampling procedure coupled to microbore HPLC to measure extracellular serotonin levels in the pedunculopontine tegmental nucleus (PPT) in naturally sleeping cats. Extracellular serotonin levels were found to be highest during periods of wakefulness, lower during slow wave sleep, and lowest during periods of REM sleep. During wakefulness serotonin levels (mean A+/-SEM) measured in 10 A microliter samples were 1.14 A+/- 0.13 fmol/sample, whereas during slow wave sleep levels declined significantly to 72% of the wakefulness baseline (0.85 A +/- 0.11 fmol/sample), and dropped further to 45% of the wakefulness baseline in REM samples (0.52 A +/- 0.10 fmol/sample; all p's<0.003). The decrease in PPT serotonin levels during sleep may be an important determinant in the timing of REM sleep cyclicity. The data support the hypothesis that, during slow wave sleep and REM sleep, the declining levels of serotonin release the PPT REM-promoting neurons from serotonergic inhibition, which, in turn, leads to increases in acetylcholine release in terminal areas, facilitating the emergence of REM sleep.

Behavioral state control through differential serotonergic inhibition in the mesopontine cholinergic nuclei: a simultaneous unit recording and microdialysis study.

Cholinergic neurons of the mesopontine nuclei are strongly implicated in behavioral state regulation. One population of neurons in the cholinergic zone of the laterodorsal tegmentum and the pedunculopontine nuclei, referred to as rapid eye movement (REM)-on neurons, shows preferential discharge activity during REM sleep, and extensive data indicate a key role in production of this state. Another neuronal group present in the same cholinergic zone of the laterodorsal tegmentum and the pedunculopontine nuclei, referred to as Wake/REM-on neurons, shows preferential discharge activity during both wakefulness and REM sleep and is implicated in the production of electroencephalographic activation in both of these states. To test the hypothesis of differential serotonergic inhibition as an explanation of the different state-related discharge activity, we developed a novel methodology that enabled, in freely behaving animals, simultaneous unit recording and local perfusion of neuropharmacological agents using a microdialysis probe adjacent to the recording electrodes. Discharge activity of REM-on neurons was almost completely suppressed by local microdialysis perfusion of the selective 5-HT1A agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT), although this agonist had minimal or no effect on the Wake/REM-on neurons. We conclude that selective serotonergic inhibition is a basis of differential state regulation in the mesopontine cholinergic nuclei, and that the novel methodology combining neurophysiological and neuropharmacological information from the freely behaving animal shows great promise for further insight into the neural basis of behavioral control.

Dopamine depletion in nucleus accumbens influences locomotion but not force and timing of operant responding.

This experiment examined the role of dopamine (DA) in the nucleus accumbens in regulating beam pressing and locomotor responses. Six rats were rewarded with sucrose on a partial reinforcement schedule for pressing force-sensitive beams. Open-field locomotor activity, and the force and timing characteristics of operant motor responses were recorded. It is known that low doses of apomorphine decrease DA tone through activating DA autoreceptors, resulting in suppression of both operant responses and locomotion. Our results showed that DA depletion in the nucleus accumbens, induced by bilateral injection of 6-hydroxydopamine, did not affect the force and timing of operant responses: neither did it reverse the suppressive effects of low doses of apomorphine on the force and timing of operant responses. However, accumbens DA depletion did block the suppressive effect of apomorphine on open-field locomotion. These results were interpreted as support for the hypothesis that the suppressive effects of low doses of apomorphine on locomotion, but not on operant beam pressing, are mediated mainly by DA autoreceptors in the mesolimbic pathway.

Adenosine: a mediator of the sleep-inducing effects of prolonged wakefulness.

Both subjective and electroencephalographic arousal diminish as a function of the duration of prior wakefulness. Data reported here suggest that the major criteria for a neural sleep factor mediating the somnogenic effects of prolonged wakefulness are satisfied by adenosine, a neuromodulator whose extracellular concentration increases with brain metabolism and which, in vitro, inhibits basal forebrain cholinergic neurons. In vivo microdialysis measurements in freely behaving cats showed that adenosine extracellular concentrations in the basal forebrain cholinergic region increased during spontaneous wakefulness as contrasted with slow wave sleep; exhibited progressive increases during sustained, prolonged wakefulness; and declined slowly during recovery sleep. Furthermore, the sleep-wakefulness profile occurring after prolonged wakefulness was mimicked by increased extracellular adenosine induced by microdialysis perfusion of an adenosine transport inhibitor in the cholinergic basal forebrain but not by perfusion in a control noncholinergic region.

The characterization of the effect of locally applied N-methylquipazine, a 5-HT3 receptor agonist, on extracellular dopamine levels in the anterior medial prefrontal cortex in the rat: an in vivo microdialysis study.

In this study, we examined the effect of n-methylquipazine (NMQ), which is a putative 5-hydroxytryptamine3(5-HT3)receptor agonist, on the extracellular concentrations of dopamine (DA) and one of its metabolites, dihydroxyphenylacetic acid (DOPAC), in the anterior medial prefrontal cortex (AmPFc) of awake, freely moving rats. The administration of NMQ via the perfusion fluid produced a concentration-dependent (10-1,000 microM) increase in extracellular DA levels in the AmPFc. In contrast, NMQ produced a decrease in the extracellular concentrations of DOPAC. The increase in extracellular DA levels returned to baseline after the removal of NMQ from the perfusate. The increase in extracellular DA levels in the AmPFc produced by 100 microM of NMQ was markedly attenuated by either the coadministration of tetrodotoxin (1 microM), which inhibits axonal impulse flow, or the depletion of extracellular Ca2+ by removing CaCl2 and adding EDTA to the perfusate. The intradialysate administration of the 5-HT3 antagonist BRL 46470A produced a concentration-dependent (10-1,000 microM) decrease in extracellular DA levels, and this effect was reversible on removal from the perfusate. In contrast, ondansetron (500 and 1,000 microM), which is another 5-HT3 receptor antagonist, produced a transient increase followed by a sustained decrease in extracellular DA levels. The preinfusion of 10 microM of BRL 46470 followed by coperfusion of BRL 46470A with 50 or 100 microM of NMQ via the dialysis probe did not significantly attenuate the increase of NMQ in extracellular DA levels in the AmPFc. The administration of the selective 5-HT2 receptor MDL 100907 (1 mg/kg, i.p.) also did not alter the increase in basal DA levels produced by 100 microM of NMQ. The pretreatment of rats with alpha-methyl-p-tyrosine produced a significant attenuation in the NMQ-induced increase in extracellular DA levels, suggesting that the elevation by NMQ of DA levels is dependent on newly synthesized stores of DA. Overall, these results suggest that the increase in AmPFc DA levels by NMQ is probably not mediated by its interaction with the 5-HT3 receptor.

Effect of acute and chronic fluoxetine on extracellular dopamine levels in the caudate-putamen and nucleus accumbens of rat.

Recent studies indicate that an increase in serotonergic (5-HT) activity in the nucleus accumbens (NAc) produces an increase in dopamine (DA) release, providing a possible mechanism for the involvement of DA in the therapeutic action of selective serotonin reuptake inhibitor (SSRI) antidepressants. However, acutely administered fluoxetine (2.5, 5.0, or 10.0 mg/kg, i.p.) failed to elevate extracellular levels of DA, or its metabolites in the NAc or caudate-putamen (CP). In fact, the highest dose produced a small (20%) decrease in DA levels in the NAc. Extracellular levels of the 5-HT metabolite 5HIAA were consistently decreased at all doses of fluoxetine in both structures. Since SSRIs generally require several weeks of treatment to be effective clinically, a second experiment examined the effect of chronic administration of fluoxetine. Chronic (21 day) daily treatment with 5 mg/kg had no effect on NAc basal levels of DA, DA metabolites, or 5HIAA, relative to a saline-treated control group. Finally, pretreatment with fluoxetine appeared to slightly enhance the elevation of NAc DA induced by an injection of cocaine (10 mg/kg, i.p.), an effect that was not quite significant (P < .06). In conclusion, the 5-HT-induced facilitation of NAc DA neurotransmission described in the literature may not be relevant to the therapeutic action of fluoxetine.