Adenosine inhibits glutamatergic input to basal forebrain cholinergic neurons

Adenosine has been proposed as an endogenous homeostatic sleep factor that accumulates during waking and inhibits wake-active neurons to promote sleep. It has been specifically hypothesized that adenosine decreases wakefulness and promotes sleep recovery by directly inhibiting wake-active neurons of the basal forebrain (BF), particularly BF cholinergic neurons. We previously showed that adenosine directly inhibits BF cholinergic neurons. Here, we investigated 1) how adenosine modulates glutamatergic input to BF cholinergic neurons and 2) how adenosine uptake and adenosine metabolism are involved in regulating extracellular levels of adenosine. Our experiments were conducted using whole cell patch-clamp recordings in mouse brain slices. We found that in BF cholinergic neurons, adenosine reduced the amplitude of AMPA-mediated evoked glutamatergic excitatory postsynaptic currents (EPSCs) and decreased the frequency of spontaneous and miniature EPSCs through presynaptic A(1) receptors. Thus we have demonstrated that in addition to directly inhibiting BF cholinergic neurons, adenosine depresses excitatory inputs to these neurons. It is therefore possible that both direct and indirect inhibition may synergistically contribute to the sleep-promoting effects of adenosine in the BF. We also found that blocking the influx of adenosine through the equilibrative nucleoside transporters or inhibiting adenosine kinase and adenosine deaminase increased endogenous adenosine inhibitory tone, suggesting a possible mechanism through which adenosine extracellular levels in the basal forebrain are regulated.     

Activity-dependent release of adenosine inhibits the glutamatergic synaptic transmission and plasticity in the hypothalamic hypocretin/orexin neurons

The hypocretin (orexin) neurons in the lateral hypothalamus play a crucial role in the promotion of arousal. Adenosine, an endogenous sleep-promoting factor, modulates both neuronal excitatory and synaptic transmission in the CNS. In this study, the involvement of endogenous adenosine in the regulation of excitatory glutamatergic synaptic transmission to hypocretin neurons was investigated in the hypothalamic slices from transgenic mice by using different frequencies of stimulation. A train of low-frequency stimulation (0.033, 1 Hz) had no effect on the amplitude of evoked excitatory postsynaptic currents (evEPSCs) in hypocretin neurons. Blockade of adenosine A1 receptors with selective A1 receptor antagonist 8-cyclopentyltheophylline (CPT), the amplitude of evEPSCs did not change during 0.033 and 1 Hz stimuli. When the frequency of stimulation was increased upto 2 Hz, a time-dependent depression of amplitude was recorded in hypocretin neurons. Administration of CPT caused no significant change in depressed synaptic response induced by 2 Hz stimulus. While depression induced by 10 and 100 Hz stimuli was partially inhibited by the CPT but not by the selective A2 receptor antagonist 3,7-dimethyl-1-(2-propynyl)xanthine. Further findings have demonstrated that high-frequency stimulation could induce long-term potentiation (LTP) of glutamatergic synaptic transmission to hypocretin neurons in acute hypothalamic slices. The experiments with CPT suggested that A1 receptor antagonist could facilitate the induction of LTP, indicating that endogenous adenosine, acting through A1 receptors, may suppress the induction of LTP of excitatory synaptic transmission to hypocretin neurons. These results suggest that in the hypothalamus, endogenous adenosine will be released into extracellular space in an activity-dependent manner inhibiting both basal excitatory synaptic transmission and LTP in hypocretin neurons via A1 receptors. Our data provide further support for the notion that hypocretin neurons in the lateral hypothalamus may be another important target involved in the endogenous adenosine modulating the sleep and wakefulness cycle in the mammalian CNS.     

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.     

What keeps us awake: the neuropharmacology of stimulants and wakefulness-promoting medications

Numerous studies dissecting the basic mechanisms that control sleep regulation have led to considerable improvement in our knowledge of sleep disorders. It is now well accepted that transitions between sleep and wakefulness are regulated by complex neurobiologic mechanisms, which, ultimately, can be delineated as oscillations between two opponent processes, one promoting sleep and the other promoting wakefulness. The role of several neurotransmitter or neuromodulator systems, including noradrenergic, serotonergic, cholinergic, adenosinergic, and histaminergic systems and, more recently, the hypocretin/orexin and dopamine systems, has been clearly established. Amphetamine-like stimulants are known to increase wakefulness by blocking dopamine reuptake, by stimulating dopamine release, or by both mechanisms. Modafinil may increase wakefulness through activation of noradrenergic and dopaminergic systems, possibly through interaction with the hypocretin/orexin system. Caffeine inhibits adenosinergic receptors, which in turn can produce activation via interaction with GABAergic and dopaminergic neurotransmission. Nicotine enhances acetylcholine neurotransmission in the basal forebrain and dopamine release. Understanding the exact role of the hypocretin/orexin and dopamine systems in the physiology and pharmacology of sleep-wake regulation may reveal new insights into current and future wakefulness-promoting drugs.     

Adenosine inhibits basal forebrain cholinergic and noncholinergic neurons in vitro

Adenosine has been proposed as a homeostatic "sleep factor" that promotes the transition from waking to sleep by affecting several sleep-wake regulatory systems. In the basal forebrain, adenosine accumulates during wakefulness and, when locally applied, suppresses neuronal activity and promotes sleep. However, the neuronal phenotype mediating these effects is unknown. We used whole-cell patch-clamp recordings in in vitro rat brain slices to investigate the effect of adenosine on identified cholinergic and noncholinergic neurons of the magnocellular preoptic nucleus and substantia innominata. Adenosine (0.5-100 microM) reduced the magnocellular preoptic nucleus and substantia innominata cholinergic neuronal firing rate by activating an inwardly rectifying potassium current that reversed at -82 mV and was blocked by barium (100 microM). Application of the A1 receptor antagonist 8-cyclo-pentyl-theophylline (200 nM) blocked the effects of adenosine. Adenosine was also tested on two groups of electrophysiologically distinct noncholinergic magnocellular preoptic nucleus and substantia innominata neurons. In the first group adenosine, via activation of postsynaptic A1 receptors, reduced spontaneous firing via inhibition of the hyperpolarization-activated cation current. Blocking the H-current with ZD7288 (20 microM) abolished adenosine effects on these neurons. The second group was not affected by adenosine. These results demonstrate that, in the magnocellular preoptic nucleus and substantia innominata region of the basal forebrain, adenosine inhibits both cholinergic neurons and a subset of noncholinergic neurons. Both of these effects occur via postsynaptic A1 receptors, but are mediated downstream by two separate mechanisms.     

Orexin peptides enhance median preoptic nucleus neuronal excitability via postsynaptic membrane depolarization and enhancement of glutamatergic afferents

Subpopulations of neurons in the median preoptic nucleus (MnPO) located within the lamina terminalis contribute to thermoregulatory, cardiovascular and hydromineral homeostasis, and sleep-promotion. MnPO is innervated by lateral hypothalamic neurons that synthesize and secrete the arousal-promoting and excitatory orexin (hypocretin) neuropeptides. To evaluate the hypothesis that orexins modulate the excitability of MnPO neurons, we used patch-clamp recording techniques applied in rat brain slice preparations to assess the effects of exogenously applied orexin A and orexin B peptides on their intrinsic and synaptic properties. Whole cell recordings under current-clamp mode revealed that 11/15 tested MnPO neurons responded similarly to either orexin A or B (500-1000 nM) with a slowly rising, prolonged (10-15 min) and reversible membrane depolarization. Under voltage-clamp mode, orexin applications induced a tetrodotoxin-resistant inward current of -7.2+/-1.6 pA, indicating a direct (postsynaptic) activation, with a time course similar to the observed membrane depolarization. The orexin-induced responses in 4/7 neurons were associated with a significant decrease in membrane conductance and the net orexin-induced current that reversed at -99+/-5 mV, suggesting closure of potassium channels. Orexins did not attenuate the properties of excitatory (n=4) or inhibitory (n=7) postsynaptic currents evoked by subfornical organ stimulation. By contrast, orexins applications induce a significant increase in both frequency and amplitude of spontaneous glutamatergic postsynaptic currents (5/7 cells) but had no influence on spontaneous GABAergic currents (6/6 cells). Thus, in addition to a direct postsynaptic receptor-mediated excitation, orexins can also increase the excitability of MnPO neurons via increasing their excitatory inputs, presumably through an orexin receptor-mediated excitation of local glutamatergic neurons whose axons project to MnPO neurons.     

Orexins/hypocretins excite basal forebrain cholinergic neurones.

The orexins (orexin A and B, also known as hypocretin 1 and 2) are two recently identified neuropeptides (de Lecea et al., 1998; Sakurai et al., 1998) which are importantly implicated in the control of wakefulness (for reviews see Hungs and Mignot, 2001; van den Pol, 2000; Willie et al., 2001 ). Indeed, alteration in these peptides' precursor, their receptors or the hypothalamic neurones that produce them leads to the sleep disorder narcolepsy (Chemelli et al., 1999; Lin et al., 1999; Peyron et al., 2000; Thannickal et al., 2000). The mechanisms by which the orexins modulate wakefulness, however, are still unclear. Their presence in fibres coursing from the hypothalamus (Peyron et al., 1998) up to the preoptic area (POA) and basal forebrain (BF) suggests that they might influence the important sleep and waking neural systems situated there (Jones, 2000). The present study, performed in rat brain slices, demonstrates, however, that the orexins have no effect on the GABA sleep-promoting neurones of the POA, whereas they have a strong and direct excitatory effect on the cholinergic neurones of the contiguous BF. In addition, by comparing the effects of orexin A and B we demonstrate here that orexins' action depends upon orexin type 2 receptors (OX(2)), which are those lacking in narcoleptic dogs (Lin et al., 1999). These results suggest that the orexins excite cholinergic neurones that release acetylcholine in the cerebral cortex and thereby contribute to the cortical activation associated with wakefulness.     

Selective enhancement of excitatory synaptic activity in the rat nucleus tractus solitarius by hypocretin 2

Hypocretin 2 (orexin B) is a hypothalamic neuropeptide thought to be involved in regulating energy homeostasis, autonomic function, arousal, and sensory processing. Neural circuits in the caudal nucleus tractus solitarius (NTS) integrate viscerosensory inputs, and are therefore implicated in aspects of all these functions. We tested the hypothesis that hypocretin 2 modulates fast synaptic activity in caudal NTS areas that are generally associated with visceral sensation from cardiorespiratory and gastrointestinal systems. Hypocretin 2-immunoreactive fibers were observed throughout the caudal NTS. In whole-cell recordings from neurons in acute slices, hypocretin 2 depolarized 48% and hyperpolarized 10% of caudal NTS neurons, effects that were not observed when Cs(+) was used as the primary cation carrier. Hypocretin 2 also increased the amplitude of tractus solitarius-evoked excitatory postsynaptic currents (EPSCs) in 36% of neurons and significantly enhanced the frequency of spontaneous EPSCs in most (59%) neurons. Spontaneous inhibitory postsynaptic currents (IPSCs) were relatively unaffected by the peptide. The increase in EPSC frequency persisted in the presence of tetrodotoxin, suggesting a role for the peptide in regulating glutamate release in the NTS by acting at presynaptic terminals.These data suggest that hypocretin 2 modulates excitatory, but not inhibitory, synapses in caudal NTS neurons, including viscerosensory inputs. The selective nature of the effect supports the hypothesis that hypocretin 2 plays a role in modulating autonomic sensory signaling in the NTS.     

The endogenous somnogen adenosine excites a subset of sleep-promoting neurons via A2A receptors in the ventrolateral preoptic nucleus

Recent research has shown that neurons in the ventrolateral preoptic nucleus are crucial for sleep by inhibiting wake-promoting systems, but the process that triggers their activation at sleep onset remains to be established. Since evidence indicates that sleep induced by adenosine, an endogenous sleep-promoting substance, requires activation of brain A(2A) receptors, we examined the hypothesis that adenosine could activate ventrolateral preoptic nucleus sleep neurons via A(2A) adenosine receptors in rat brain slices. Following on from our initial in vitro identification of these neurons as uniformly inhibited by noradrenaline and acetylcholine arousal transmitters, we established that the ventrolateral preoptic nucleus comprises two intermingled subtypes of sleep neurons, differing in their firing responses to serotonin, inducing either an inhibition (Type-1 cells) or an excitation (Type-2 cells). Since both cell types contained galanin and expressed glutamic acid decarboxylase-65/67 mRNAs, they potentially correspond to the sleep promoting neurons inhibiting arousal systems. Our pharmacological investigations using A(1) and A(2A) adenosine receptors agonists and antagonists further revealed that only Type-2 neurons were excited by adenosine via a postsynaptic activation of A(2A) adenosine receptors. Hence, the present study is the first demonstration of a direct activation of the sleep neurons by adenosine. Our results further support the cellular and functional heterogeneity of the sleep neurons, which could enable their differential contribution to the regulation of sleep. Adenosine and serotonin progressively accumulate during arousal. We propose that Type-2 neurons, which respond to these homeostatic signals by increasing their firing are involved in sleep induction. In contrast, Type-1 neurons would likely play a role in the consolidation of sleep.     

Adenosine A1 receptors decrease thalamic excitation of inhibitory and excitatory neurons in the barrel cortex

Caffeine is consumed worldwide to enhance wakefulness, but the cellular mechanisms are poorly understood. Caffeine blocks adenosine receptors suggesting that adenosine decreases cortical arousal. Given the widespread innervation of the cerebral cortex by thalamic fibers, adenosine receptors on thalamocortical terminals could provide an efficient method of limiting thalamic activation of the cortex. Using a mouse thalamocortical slice preparation and whole-cell patch clamp recordings, we examined whether thalamocortical terminals are modulated by adenosine receptors. Bath application of adenosine decreased excitatory postsynaptic currents elicited by stimulation of the ventrobasal thalamus. Thalamocortical synapses onto inhibitory and excitatory neurons were equally affected by adenosine. Adenosine also increased the paired pulse ratio and the coefficient of variation of the excitatory postsynaptic currents, suggesting that adenosine decreased glutamate release. The inhibition produced by adenosine was reversed by a selective antagonist of adenosine A1 receptors (8-cyclopentyltheophylline) and mimicked by a selective A1 receptor agonist (N6-cyclopentyladenosine). Our results indicate that thalamocortical excitation is regulated by presynaptic adenosine A1 receptors and provide a mechanism by which increased adenosine levels can directly reduce cortical excitability.     

Presynaptic inhibition of synaptic transmission by adenosine in rat subthalamic nucleus in vitro

Whole-cell patch clamp recordings were made from the subthalamic nucleus in rat brain slice preparations to examine the effect of adenosine on inhibitory and excitatory synaptic transmission. Adenosine reversibly inhibited both GABA-mediated inhibitory and glutamate-mediated excitatory postsynaptic currents. Adenosine at 100 microM reduced the amplitude of inhibitory and excitatory postsynaptic currents by 42+/-5% and 34+/-6%, respectively. Reductions in the amplitude of both inhibitory and excitatory postsynaptic currents were accompanied by increases in paired-pulse ratios. In addition, adenosine decreased the frequency of spontaneous miniature excitatory postsynaptic currents but had no effect on their amplitude. These results are consistent with a presynaptic site of action. The adenosine A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine completely reversed the adenosine-induced attenuation of inhibitory and excitatory postsynaptic currents, but 8-cyclopentyl-1,3-dipropylxanthine alone had no effect on synaptic currents evoked at 0.1 Hz. However, 8-cyclopentyl-1,3-dipropylxanthine inhibited a time-dependent depression of excitatory postsynaptic currents that was normally observed in response to a 5 Hz train of stimuli, suggesting that endogenous adenosine could be released during higher frequencies of stimulation. These results suggest that adenosine inhibits synaptic release of GABA and glutamate by stimulation of presynaptic A(1) receptors in the subthalamic nucleus.     

Hypocretin-2 (orexin-B) modulation of superficial dorsal horn activity in rat

The hypothalamic peptides hypocretin-1 (orexin A) and hypocretin-2 (Hcrt-2; orexin B) are important in modulating behaviours demanding arousal, including sleep and appetite. Fibres containing hypocretin project from the hypothalamus to the superficial dorsal horn (SDH) of the spinal cord (laminae I and II); however, the effects produced by hypocretins on SDH neurones are unknown. To study the action of Hcrt-2 on individual SDH neurones, tight-seal, whole-cell recordings were made with biocytin-filled electrodes from rat lumbar spinal cord slices. In 19 of 63 neurones, Hcrt-2 (30 n m to 1 μ m ) evoked an inward (excitatory) current accompanied by an increase in baseline noise. The inward current and noise were unaffected by TTX but were blocked by the P_2X purinergic receptor antagonist suramin (300–500 μ m ). Hcrt-2 (30 n m to 1 μ m ) increased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) in the majority of neurones. The sIPSC increase was blocked by strychnine (1 μ m ) and by TTX (1 μ m ), suggesting that the increased sIPSC frequency was glycine and action potential dependent. Hcrt-2 increased the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) in a few neurones but had no effect on dorsal root-evoked EPSCs in these or in other neurones. Neurones located in outer lamina II, particularly radial and vertical cells, were most likely to respond to Hcrt-2. We conclude that Hcrt-2 has excitatory effects on certain SDH neurones, some of which exert inhibitory influences on other cells of the region, consistent with the perspective that hypocretin has a role in orchestrating reactions related to arousal, including nociception, pain and temperature sense.     

Metabotropic glutamate and muscarinic cholinergic receptor-mediated preferential inhibition of N-methyl-D-aspartate component of transmissions in rat ventral tegmental area

Presynaptic inhibition is one of the major control mechanisms in the CNS. Our laboratory recently reported that presynaptic GABA(B) and adenosine A(1) receptors mediate a preferential inhibition on N-methyl-D-aspartate receptor-mediated excitatory postsynaptic currents recorded in rat midbrain dopamine neurons. Here we extended these findings to metabotropic glutamate and muscarinic cholinergic receptors. Intracellular voltage clamp recordings were made from dopamine neurons in rat ventral tegmental area in slice preparations. (+/-)-1-Aminocyclopentane-trans-1,3-dicarboxylic acid (agonist for groups I and II metabotropic glutamate receptors) and L(+)-2-amino-4-phosphonobutyric acid (L-AP4; agonist for group III metabotropic glutamate receptors) were significantly more potent for inhibiting N-methyl-D-aspartate receptor-mediated excitatory postsynaptic currents, as compared with inhibition of excitatory postsynaptic currents mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors. Such preferential inhibition of the N-methyl-D-aspartate component was also observed for muscarine (agonist for muscarinic cholinergic receptors). Inhibitory effects of (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid, L-AP4, and muscarine were blocked reversibly by their respective antagonists [(RS)-alpha-methyl-4-carboxyphenylglycine, (RS)-alpha-methyl-4-phosphonophenylglycine, and 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide]. In addition, all three agonists increased the ratio of excitatory postsynaptic currents in paired-pulse studies and did not reduce currents induced by exogenous N-methyl-D-aspartate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid. Interestingly, the glutamate release stimulator 4-aminopyridine (30 microM) and the glutamate uptake inhibitor L-anti-endo-3,4-methanopyrrolidine dicarboxylate (300 microM) preferentially increased the amplitude of N-methyl-D-aspartate excitatory postsynaptic currents.Thus, agonists for metabotropic glutamate and muscarinic cholinergic receptors act presynaptically to cause a preferential reduction in the N-methyl-D-aspartate component of excitatory synaptic transmissions. Together with the evidence for GABA(B) and adenosine A(1) receptor-mediated preferential inhibition of the N-methyl-D-aspartate component, the present results suggest that limiting glutamate spillover onto postsynaptic N-methyl-D-aspartate receptors may be a general rule for presynaptic modulation in midbrain dopamine neurons.     

Delayed orexin signaling consolidates wakefulness and sleep: physiology and modeling

Orexin-producing neurons are clearly essential for the regulation of wakefulness and sleep because loss of these cells produces narcolepsy. However, little is understood about how these neurons dynamically interact with other wake- and sleep-regulatory nuclei to control behavioral states. Using survival analysis of wake bouts in wild-type and orexin knockout mice, we found that orexins are necessary for the maintenance of long bouts of wakefulness, but orexin deficiency has little impact on wake bouts <1 min. Since orexin neurons often begin firing several seconds before the onset of waking, this suggests a surprisingly delayed onset (>1 min) of functional effects. This delay has important implications for understanding the control of wakefulness and sleep because increasing evidence suggests that different mechanisms are involved in the production of brief and sustained wake bouts. We incorporated these findings into a mathematical model of the mouse sleep/wake network. Orexins excite monoaminergic neurons and we hypothesize that orexins increase the monoaminergic inhibition of sleep-promoting neurons in the ventrolateral preoptic nucleus. We modeled orexin effects as a time-dependent increase in the strength of inhibition from wake- to sleep-promoting populations and the resulting simulated behavior accurately reflects the fragmented sleep/wake behavior of narcolepsy and leads to several predictions. By integrating neurophysiology of the sleep/wake network with emergent properties of behavioral data, this model provides a novel framework for investigating network dynamics and mechanisms associated with normal and pathologic sleep/wake behavior.     

Cholinergic-mediated response enhancement in barrel cortex layer V pyramidal neurons

Neocortical cholinergic activity plays a fundamental role in sensory processing and cognitive functions, but the underlying cellular mechanisms are largely unknown. We analyzed the effects of acetylcholine (ACh) on synaptic transmission and cell excitability in rat "barrel cortex" layer V (L5) pyramidal neurons in vitro. ACh through nicotinic and M1 muscarinic receptors enhanced excitatory postsynaptic currents and through nicotinic and M2 muscarinic receptors reduced inhibitory postsynaptic currents. These effects increased excitability and contributed to the generation of Ca(2+) spikes and bursts of action potentials (APs) when inputs in basal dendrites were stimulated. Ca(2+) spikes were mediated by activation of NMDA receptors (NMDARs) and L-type voltage-gated Ca(2+) channels. Additionally, we demonstrate in vivo that basal forebrain stimulation induced an atropine-sensitive increase of L5 AP responses evoked by vibrissa deflection, an effect mainly due to the enhancement of an NMDAR component. Therefore, ACh modified the excitatory/inhibitory balance and switched L5 pyramidal neurons to a bursting mode that caused a potent and sustained response enhancement with possible fundamental consequences for the function of the barrel cortex.     

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.     

Sleep neurobiology from a clinical perspective

Many neurochemical systems interact to generate wakefulness and sleep. Wakefulness is promoted by neurons in the pons, midbrain, and posterior hypothalamus that produce acetylcholine, norepinephrine, dopamine, serotonin, histamine, and orexin/hypocretin. Most of these ascending arousal systems diffusely activate the cortex and other forebrain targets. NREM sleep is mainly driven by neurons in the preoptic area that inhibit the ascending arousal systems, while REM sleep is regulated primarily by neurons in the pons, with additional influence arising in the hypothalamus. Mutual inhibition between these wake- and sleep-regulating regions likely helps generate full wakefulness and sleep with rapid transitions between states. This up-to-date review of these systems should allow clinicians and researchers to better understand the effects of drugs, lesions, and neurologic disease on sleep and wakefulness.     

Adenosine preferentially suppresses serotonin2A receptor-enhanced excitatory postsynaptic currents in layer V neurons of the rat medial prefrontal cortex

Serotonin induces 'spontaneous' (non-electrically evoked) excitatory postsynaptic currents in layer V pyramidal neurons in the prefrontal cortex. This is likely due to a serotonin2A receptor-mediated focal release of glutamate onto apical dendrites. In addition, activation of the serotonin2A receptor selectively enhances late components of electrically evoked excitatory postsynaptic currents. In this study, using in vitro intracellular and whole-cell recording in rat brain slices, we examined the role of adenosine in modulating serotonin2A-enhanced 'spontaneous' and electrically evoked excitatory postsynaptic currents in layer V pyramidal neurons in the medial prefrontal cortex. Adenosine and N6-cyclopentyladenosine, an A1 adenosine agonist, markedly suppressed the serotonin2A-induced ('spontaneous') excitatory postsynaptic currents. However, adenosine had no effect on spontaneous miniature (tetrodotoxin-insensitive) postsynaptic potentials. Adenosine also blocked the late excitatory postsynaptic currents induced by the serotonin2A/2C agonist R(-)-2,5-dimethoxy-4-iodoamphetamine hydrochloride. Surprisingly, in contrast to other regions, adenosine had a relatively small effect on electrically evoked fast excitatory postsynaptic currents.These findings represent a novel demonstration of adenosine's ability to preferentially modulate serotonin2A-mediated synaptic events in the medial prefrontal cortex. As the serotonin2A receptor is closely linked with the effects of atypical antipsychotics and hallucinogens, further understanding of the modulators of this receptor such as adenosine may provide useful therapeutic applications.     

Excitatory action of hypocretin/orexin on neurons of the central medial amygdala

The neurons of the lateral hypothalamus that contain hypocretin/orexin (hcrt/orx) are thought to promote arousal through the excitatory action they exert on the multiple areas to which they project within the CNS. We show here that the hcrt/orx peptides can also exert a strong action on the amygdala, a structure known for its implication in emotional aspects of behavior. Indeed, the hcrt/orx peptides, applied in acute rat brain slices, excite a specific class of "low threshold burst" neurons in the central medial (CeM) nucleus which is considered as a major output of the amygdala. These excitatory effects are postsynaptic, mediated by Hcrt2/OX2 receptors and result from the closure of a potassium conductance. They occur on a class of neurons that are also excited by vasopressin acting through V1a receptors. These results suggest that the hcrt/orx system can act through the amygdala to augment arousal and evoke the autonomic and behavioral responses associated with fear, stress or emotion.     

Transient enhancement of inhibitory synaptic transmission in hippocampal CA1 pyramidal neurons after cerebral ischemia

Pyramidal neurons in hippocampal CA1 regions are highly sensitive to cerebral ischemia. Alterations of excitatory and inhibitory synaptic transmission may contribute to the ischemia-induced neuronal degeneration. However, little is known about the changes of GABAergic synaptic transmission in the hippocampus following reperfusion. We examined the GABA_A receptor-mediated inhibitory postsynaptic currents (IPSCs) in CA1 pyramidal neurons 12 and 24 h after transient forebrain ischemia in rats. The amplitudes of evoked inhibitory postsynaptic currents (eIPSCs) were increased significantly 12 h after ischemia and returned to control levels 24 h following reperfusion. The potentiation of eIPSCs was accompanied by an increase of miniature inhibitory postsynaptic current (mIPSC) amplitude, and an enhanced response to exogenous application of GABA, indicating the involvement of postsynaptic mechanisms. Furthermore, there was no obvious change of the paired-pulse ratio (PPR) of eIPSCs and the frequency of mIPSCs, suggesting that the potentiation of eIPSCs might not be due to the increased presynaptic release. Blockade of adenosine A1 receptors led to a decrease of eIPSCs amplitude in post-ischemic neurons but not in control neurons, without affecting the frequency of mIPSCs and the PPR of eIPSCs. Thus, tonic activation of adenosine A1 receptors might, at least in part, contribute to the enhancement of inhibitory synaptic transmission in CA1 neurons after forebrain ischemia. The transient enhancement of inhibitory neurotransmission might temporarily protect CA1 pyramidal neurons, and delay the process of neuronal death after cerebral ischemia.