Sleep-wakefulness in alcohol preferring and non-preferring rats following binge alcohol administration.

The alcohol-preferring (P) rat is a valid animal model of alcoholism. However, the effect of alcohol on sleep in P or alcohol non-preferring (NP) rats is unknown. Since alcohol consumption has tremendous impact on sleep, the present study compared the effects of binge alcohol administration on sleep-wakefulness in P and NP rats. Using standard surgical procedures, the P and NP rats were bilaterally implanted with sleep recording electrodes. Following post-operative recovery and habituation, pre-ethanol (baseline) sleep-wakefulness was electrographically recorded for 48 h. Subsequently, ethanol was administered beginning with a priming dose of 5 g/Kg followed by two doses of 2 g/Kg every 8 h on the first day and three doses of 3 g/Kg/8 h on the second day. On the following day (post-ethanol), undisturbed sleep-wakefulness was electrographically recorded for 24 h. Our initial results suggest that, during baseline conditions, the time spent in each of the three behavioral states: wakefulness, non-rapid eye movement (NREM) sleep and REM sleep, was comparable between P and NP rats. However, the P rats were more susceptible to changes in sleep-wakefulness following 2 days of binge ethanol treatment. As compared to NP rats, the P rats displayed insomnia like symptoms including a significant reduction in the amount of time spent in NREM sleep coupled with a significant increase in wakefulness on post-ethanol day. Subsequent analysis revealed that binge ethanol induced increased wakefulness and reduced NREM sleep in P rats occurred mainly in the dark period. This is the first study that: (1) demonstrates spontaneous sleep-wake profile in P and NP rats, and (2) compares the effects of binge ethanol treatment on sleep in P and NP rats. Our results suggest that, as compared to NP rats, the P rats were more susceptible to sleep disruptions after binge ethanol treatment. In addition, the P rats exhibited insomnia-like symptoms observed during abstinence from alcohol in human subjects.

Effect of microdialysis perfusion of 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridine-3-ol in the perifornical hypothalamus on sleep-wakefulness: role of delta-subunit containing extrasynaptic GABAA receptors.

Gaboxadol or 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridine-3-ol (THIP) is a selective agonist for the delta-subunit containing extrasynaptic GABA(A) receptors that will soon enter the U.S. market as a sleep aid [Winsky-Sommerer R, Vyazovskiy VV, Homanics GE, Tobler I (2007) The EEG effects of THIP (gaboxadol) on sleep and waking are mediated by the GABA(A)delta-subunit-containing receptors. Eur J Neurosci 25:1893-1899]. Numerous studies have shown that systemic administration of THIP reduces wakefulness and increases sleep both in humans and rats [Lancel M, Langebartels A (2000) Gamma-aminobutyric acid(A) (GABA(A)) agonist 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol persistently increases sleep maintenance and intensity during chronic administration to rats. J Pharmacol Exp Ther 293:1084-1090; Walsh JK, Deacon S, Dijk DJ, Lundahl J (2007) The selective extrasynaptic GABAA agonist, gaboxadol, improves traditional hypnotic efficacy measures and enhances slow wave activity in a model of transient insomnia. Sleep 30:593-602]. However, it is yet unclear where in the brain THIP acts to promote sleep. Since the perifornical lateral hypothalamus (PFH) contains orexin neurons and orexin neurons are critical for maintenance of arousal [McCarley RW (2007) Neurobiology of rapid eye movement (REM) and NREM sleep. Sleep Med 8:302-330], we hypothesized that THIP may act on PFH neurons to promote sleep. To test our hypothesis, we used reverse microdialysis to perfuse THIP unilaterally into the PFH and studied its effects on sleep-wakefulness during the light period in freely behaving rats. Microdialysis perfusion of THIP (100 microM) into the PFH produced a significant reduction in wakefulness with a concomitant increase in non-rapid eye movement or slow wave sleep as compared with artificial cerebrospinal fluid perfusion. REM sleep was unaffected. This is the first study implicating the delta-subunit containing extrasynaptic GABA(A) receptors in PFH in control of sleep-wakefulness in freely behaving rats.

Adenosine and the homeostatic control of sleep: effects of A1 receptor blockade in the perifornical lateral hypothalamus on sleep-wakefulness.

The orexinergic neurons of the lateral hypothalamus (LH) are critical for wakefulness [McCarley RW (2007) Neurobiology of REM and NREM sleep. Sleep Med 8:302-330]. Recent evidence suggests that adenosine (AD), a homeostatic sleep factor, may act via A1 receptor (A1R) to control orexinergic activity and regulate sleep-wakefulness [Thakkar MM, Winston S, McCarley RW (2002) Orexin neurons of the hypothalamus express adenosine A1 receptors. Brain Res 944:190-194; Liu ZW, Gao XB (2006) Adenosine inhibits activity of hypocretin/orexin neurons via A1 receptor in the lateral hypothalamus: a possible sleep-promoting effect. J Neurophysiol]. To evaluate the role of AD in the orexinergic LH and its influences on sleep-wakefulness, we designed two experiments in freely behaving rats: First, we bilaterally microinjected 1,3-dipropyl-8-phenylxanthine (DPX) (1.5 pmol and 15 pmol), a selective A1R antagonist into the LH during the light cycle and examined its effect on spontaneous sleep-wakefulness. Second, we performed 6 h of sleep deprivation. Thirty minutes before the animals were allowed to enter recovery sleep, 15 pmol of DPX was bilaterally microinjected into the LH and its effects on recovery sleep were monitored. Microinjection of DPX into the orexinergic LH produced a significant increase in wakefulness with a concomitant reduction in sleep, both during spontaneous bouts of sleep-wakefulness and during recovery sleep. Local administration of DPX into the LH produced a significant increase in the latency to non-REM sleep during recovery sleep. However, total slow wave (delta) activity during non-REM sleep phase of recovery sleep remained unaffected after DPX treatment. This is the first study that implicates endogenous adenosine to have a functional role in controlling orexinergic tone and influencing the homeostatic regulation of sleep-wakefulness.

Nociceptin/orphanin FQ decreases serotonin efflux in the rat brain but in contrast to a kappa-opioid has no antagonistic effect on mu-opioid-induced increases in serotonin efflux.

Similar to kappa-opioids, nociceptin/orphanin FQ (OFQ) exerts anti-mu-opioid actions. This may involve interactions within the circuitry controlling 5-HT neurons in the dorsal raphe nucleus (DRN) that project to the nucleus accumbens (NAcc). To test this hypothesis, we compared the effects of OFQ and kappa-opioids on 5-HT efflux in the CNS of freely behaving rats. First, OFQ (30-300 microM) infused into the DRN for 120 min dose-dependently decreased 5-HT efflux in the DRN. The opioid receptor-like 1 (ORL-1) antagonist [Nphe(1)]nociceptin(1-13)NH(2) blocked this effect. Using dual-probe microdialysis we observed that OFQ (300 microM) infused into the DRN for 120 min produced parallel decreases in 5-HT efflux in the DRN and NAcc, suggesting that ORL-1 receptors in the DRN inhibit serotonergic neurons projecting to the NAcc. Also, 5-HT efflux in the NAcc was dose-dependently decreased during OFQ (30-300 microM) infusion into the NAcc. This suggests that OFQ can reduce 5-HT efflux in the NAcc both by inhibiting serotonergic neurons in the DRN and by stimulating ORL-1 receptors in the NAcc. Similar to OFQ, the kappa-opioids U-50,488 (300 microM) and dynorphin A(1-13) (300 microM) infused into the DRN for 120 min decreased 5-HT efflux in the DRN. This effect was blocked only by the kappa-opioid receptor antagonist nor-BNI. Lastly, we compared the ability of OFQ and U-50,488 to block mu-opioid-induced increases in 5-HT. The kappa-opioid U-50,488 (1000 microM) attenuated the increase in 5-HT induced by the mu-opioid agonist endomorphin-1 (300 microM) in the DRN. In contrast, OFQ (300-1000 microM) did not alter mu-opioid-induced increases in 5-HT efflux. In summary, kappa-opioids and OFQ both decreased 5-HT efflux in the CNS. However, in contrast to kappa-opioids, which reversed mu-opioid-induced increases in 5-HT efflux, the anti-mu-opioid effects of OFQ apparently do not involve changes in 5-HT transmission under our experimental conditions.

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.

Electrophysiological characterization of neurons in the dorsolateral pontine rapid-eye-movement sleep induction zone of the rat: Intrinsic membrane properties and responses to carbachol and orexins.

Pharmacological, lesion and single-unit recording techniques in several animal species have identified a region of the pontine reticular formation (subcoeruleus, SubC) just ventral to the locus coeruleus as critically involved in the generation of rapid-eye-movement (REM) sleep. However, the intrinsic membrane properties and responses of SubC neurons to neurotransmitters important in REM sleep control, such as acetylcholine and orexins/hypocretins, have not previously been examined in any animal species and thus were targeted in this study. We obtained whole-cell patch-clamp recordings from visually identified SubC neurons in rat brain slices in vitro. Two groups of large neurons (mean diameter 30 and 27 mum) were tentatively identified as cholinergic (rostral SubC) and noradrenergic (caudal SubC) neurons. SubC reticular neurons (non-cholinergic, non-noradrenergic) showed a medium-sized depolarizing sag during hyperpolarizing current pulses and often had a rebound depolarization (low-threshold spike, LTS). During depolarizing current pulses they exhibited little adaptation and fired maximally at 30-90 Hz. Those SubC reticular neurons excited by carbachol (n=27) fired spontaneously at 6 Hz, often exhibited a moderately sized LTS, and varied widely in size (17-42 mum). Carbachol-inhibited SubC reticular neurons were medium-sized (15-25 mum) and constituted two groups. The larger group (n=22) was silent at rest and possessed a prominent LTS and associated one to four action potentials. The second, smaller group (n=8) had a delayed return to baseline at the offset of hyperpolarizing pulses. Orexins excited both carbachol excited and carbachol inhibited SubC reticular neurons. SubC reticular neurons had intrinsic membrane properties and responses to carbachol similar to those described for other reticular neurons but a larger number of carbachol inhibited neurons were found (>50%), the majority of which demonstrated a prominent LTS and may correspond to pontine-geniculate-occipital burst neurons. Some or all carbachol-excited neurons are presumably REM-on neurons.

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.

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'.

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.

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.