M.M. Thakkar

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.

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.

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.

Hippocampal synaptic plasticity and spatial learning are impaired in a rat model of sleep fragmentation

Sleep fragmentation, a symptom in many clinical disorders, leads to cognitive impairments. To investigate the mechanisms by which sleep fragmentation results in memory impairments, rats were awakened once every 2u2003min via 30u2003s of slow movement on an automated treadmill. Within 1u2003h of this sleep interruption (SI) schedule, rats began to sleep in the 90‐s periods without treadmill movement. Total non‐rapid eye movement sleep (NREM) sleep time did not change over the 24u2003h of SI, although there was a significant decline in rapid eye movement sleep (REM) sleep and a corresponding increase in time spent awake. In the SI group, the mean duration of sleep episodes decreased and delta activity during periods of wake increased. Control rats either lived in the treadmill without movement (cage controls, CC), or had 10u2003‐min periods of movement followed by 30u2003min of non‐movement allowing deep/continuous sleep (exercise controls, EC). EC did not differ from baseline in the total time spent in each vigilance state. Hippocampal long‐term potentiation (LTP), a long‐lasting change in synaptic efficacy thought to underlie declarative memory formation, was absent in rats exposed to 24 and 72u2003h SI. In contrast, LTP was normal in EC rats. However, long‐term depression and paired‐pulse facilitation were unaltered by 24u2003h SI. Twenty‐four hour SI also impaired acquisition of spatial learning in the hippocampus‐dependent water maze test. Twenty‐four hour SI elevated plasma corticosterone (CORT) to levels previously shown to enhance LTP (125u2003ng/mL). The results suggest that sleep fragmentation negatively impacts spatial learning. Loss of N‐methyl‐d‐aspartate (NMDA) receptor‐dependent LTP in the hippocampal CA1 region may be one mechanism involved in this deficit.

Chronic low-amplitude electrical stimulation of the laterodorsal tegmental nucleus of freely moving cats increases REM sleep.

While cholinergic stimulation of the PRF evokes a REM-like state, electrical stimulation of LDT/PPT neurons has not been used to test the hypothesis of mesopontine cholinergic control of REM sleep. Adult cats were implanted for electrographic recording and with bipolar unilateral stimulating electrodes, either in the LDT or within the PRF (stimulation control). Baseline recordings of the normal sleep-wake cycle were carried out for 5 h. On the next day, continuous stimulation of the LDT or mPRF was carried out during the same time period (0.5 ms pulses, 1 microA, 8 Hz) and with post-stimulation recording for 3 h. A second baseline recording day followed with same protocol as the first baseline day. This 3-day sequence, separated by 3 days, was repeated three times in each of the three LDT and the three medial PRF cats. Five hours of chronic low-amplitude stimulation of the LDT induced a highly significant increase in total REM and in the duration of REM sleep bouts. Stimulation of the mPRF did not affect any of the behavioral states. This study, the first to our knowledge to use low-amplitude stimulation of LDT in freely moving cats, indicates the importance of mesopontine cholinergic neurons in REM sleep.

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.

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.

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.

Characterization of GABAergic neurons in rapid-eye-movement sleep controlling regions of the brainstem reticular formation in GAD67–green fluorescent protein knock-in mice

Recent experiments suggest that brainstem GABAergic neurons may control rapid‐eye‐movement (REM) sleep. However, understanding their pharmacology/physiology has been hindered by difficulty in identification. Here we report that mice expressing green fluorescent protein (GFP) under the control of the GAD67 promoter (GAD67‐GFP knock‐in mice) exhibit numerous GFP‐positive neurons in the central gray and reticular formation, allowing on‐line identification inu2003vitro. Small (10–15u2003µm) or medium‐sized (15–25u2003µm) GFP‐positive perikarya surrounded larger serotonergic, noradrenergic, cholinergic and reticular neurons, and >u200a96% of neurons were double‐labeled for GFP and GABA, confirming that GFP‐positive neurons are GABAergic. Whole‐cell recordings in brainstem regions important for promoting REM sleep [subcoeruleus (SubC) or pontine nucleus oralis (PnO) regions] revealed that GFP‐positive neurons were spontaneously active at 3–12u2003Hz, fired tonically, and possessed a medium‐sized depolarizing sag during hyperpolarizing steps. Many neurons also exhibited a small, low‐threshold calcium spike. GFP‐positive neurons were tested with pharmacological agents known to promote (carbachol) or inhibit (orexinu2003A) REM sleep. SubC GFP‐positive neurons were excited by the cholinergic agonist carbachol, whereas those in the PnO were either inhibited or excited. GFP‐positive neurons in both areas were excited by orexins/hypocretins. These data are congruent with the hypothesis that carbachol‐inhibited GABAergic PnO neurons project to, and inhibit, REM‐on SubC reticular neurons during waking, whereas carbachol‐excited SubC and PnO GABAergic neurons are involved in silencing locus coeruleus and dorsal raphe aminergic neurons during REM sleep. Orexinergic suppression of REM during waking is probably mediated in part via excitation of acetylcholine‐inhibited GABAergic neurons.

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 A1-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 A1 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 A1 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 A1 receptor binding and the levels of its mRNA. We observed that A1 receptor mRNA levels increase after 6 h of sleep deprivation. One of the transcription factors that showed increased DNA-binding activity was nuclear factor κB (NF-κB) and may regulate the expression of A1 mRNA. We observed, using a gel shift assay, that the DNA-binding activity of NF-κB increased following 3 h of sleep deprivation. This was further supported by the increased appearance of NF-κB protein in the nuclear extracts and the consequent disappearance of cytoplasmic protein inhibitor κB (I-κB). Together our results reviewed in this report suggest that the somnogenic effects of adenosine in the BF area may be mediated by the A1 subtype of adenosine receptor, and its expression might be regulated by induction in the NF-κB protein as its transcription factor. This positive feedback might mediate some of long-duration effects of sleep deprivation, including ‘sleep debt’.