Regulation Of Behavioral State Research Articles

Brainstem cholinergic modulation of muscle tone in infant rats

In week-old rats, lesions of the dorsolateral pontine tegmentum (DLPT) and nucleus pontis oralis (PnO) have opposing effects on nuchal muscle tone. Specifically, pups with DLPT lesions exhibit prolonged bouts of nuchal muscle atonia (indicative of sleep) and pups with PnO lesions exhibit prolonged bouts of high nuchal muscle tone (indicative of wakefulness). Here we test the hypothesis that nuchal muscle tone is modulated, at least in part, by cholinergically mediated interactions between these two regions. First, in unanesthetized pups, we found that chemical infusion of the cholinergic agonist carbachol (22 mm, 0.1 microL) within the DLPT produced high muscle tone. Next, chemical lesions of the PnO were used to produce a chronic state of high nuchal muscle tone, at which time the cholinergic antagonist scopolamine (10 mm, 0.1 microL) was infused into the DLPT. Scopolamine effectively decreased nuchal muscle tone, thus suggesting that lesions of the PnO increase muscle tone via cholinergic activation of the DLPT. Using 2-deoxyglucose autoradiography, metabolic activation throughout the DLPT was observed after PnO lesions. Finally, consistent with the hypothesis that PnO inactivation produces high muscle tone, infusion of the sodium channel blocker lidocaine (2%) into the PnO of unanesthetized pups produced rapid increases in muscle tone. We conclude that, even early in infancy, the DLPT is critically involved in the regulation of muscle tone and behavioral state, and that its activity is modulated by a cholinergic mechanism that is directly or indirectly controlled by the PnO.

Circadian rhythms of TRH-like peptide levels in rat brain

This is the first report of diurnal variations in the levels of thyrotropin-releasing hormone-like peptides (pGlu-X-Pro-NH 2, where “X” can be any amino acid residue) in brain regions involved in mood regulation. These peptides have neuroprotective and antidepressant-like properties that may help stabilize chronobiologic systems that are often abnormal in neuropsychiatric disease. We hypothesized that diurnal fluctuations in the levels of these neuropeptides are components of the chronobiologic regulation of autonomic, behavioral and emotional states. Optimal use of these potentially therapeutic agents will benefit from an understanding of their response to, and effect on, normal vegetative, activity and sleep patterns, and the corresponding disordered patterns of mental illness. For these reasons, 16 male, 200 g, Sprague–Dawley rats were maintained for 4 weeks in a stable 12 h lights on, 12 h lights off photoperiod. Levels of TRH and TRH-like peptides were measured at 3.0 h, 10.5 h, 13.5 h and 21.0 h, where the subjective midnight was 0.0 h, by a combination of HPLC and RIA. Highly significant changes in TRH-like peptide levels were observed in the striatum, posterior cingulate, cerebellum, pyriform cortex, nucleus accumbens and medulla oblongata. TRH-like peptide levels, in general, were highly correlated with changes in TRH concentration, within and between brain regions, and may be colocalized in large glutamatergic neurons innervating the rat limbic system. We conclude that TRH-like peptides may be important components of chronobiologic systems involved in maintaining autonomic, behavioral and mood equilibria.

Organization of noradrenergic efferents to arousal‐related basal forebrain structures

Norepinephrine acts within select basal forebrain regions to modulate behavioral state and/or state-dependent processes, including the general regions encompassing the medial septal area, the medial preoptic area, and the substantia innominata. The present study examined the origin and organization of noradrenergic efferents to these basal forebrain regions by using combined immunohistochemical identification of noradrenergic neurons with retrograde tracing. Results indicate that the locus coeruleus provides the majority of noradrenergic input to these regions. Lesser, although at times substantial, contributions from the A1/C1 and A2/C2 adrenergic cell groups were also observed, particularly in the case of the medial preoptic region. Given the prominent state-modulating actions of the locus coeruleus, additional studies examined: 1) lateralization of locus coeruleus efferents to these regions; 2) the topographical organization of basal forebrain-projecting locus coeruleus neurons; and 3) the degree of collateralization of individual locus coeruleus neurons across these regions. Approximately 80-85% of locus coeruleus efferents to these regions project ipsilaterally. In general, basal forebrain-projecting neurons were distributed throughout the entire dorsoventral and rostrocaudal extent of the locus coeruleus. Additionally, a large proportion of locus coeruleus neurons project simultaneously to these basal forebrain terminal fields. Combined, these observations indicate coordinated actions of locus coeruleus neurons across these basal forebrain regions implicated in the regulation of behavioral state and/or state-dependent processes.

Movement-related correlates of single cell activity in the interpeduncular nucleus and habenula of the rat during a pellet-chasing task

The habenula and interpeduncular nucleus (IPN) are part of a dorsal diencephalic conduction system which receives input from cholinergic, striatal, and hypothalamic areas, and sends output to several, disparate midbrain regions. These output regions include the dorsal tegmental nucleus, which is part of a navigation-related system that provides a signal for directional heading. The habenula and IPN also project to the dorsal and medial Raphe nuclei, thought to be involved in mood and behavioral state regulation. Here, cells in both the habenula and IPN were recorded in freely moving rats while they foraged for food pellets. There were four major findings. First, many of the cells tended to fire in sporadic bouts of relatively high versus low rates, and this may be related to intrinsic cell properties discovered during in vitro studies. Second, although these regions are connected to the direction signaling circuit, they do not, themselves demonstrate a directional signal. Third, about 10% of the cells in the lateral habenula showed a strong correlation between rate and angular head motion. This may constitute an important, requisite input to the above-mentioned head direction circuit. Finally, many of the cells in each region showed a temporally coarse correlation with running speed, so that bouts of high frequency firing coincided with episodes of higher behavioral activation. This last finding may be related to work which shows an influence of the habenula on locomotor activity, and in relation to the protective effects of exercise in relation to stress, as mediated by the Raphe nuclei.

Injection of glutamate into the pedunculopontine tegmental nuclei of anesthetized rat causes respiratory dysrhythmia and alters EEG and EMG power

The pedunculopontine tegmental nucleus (PPT) has been shown to have important functions relevant to the regulation of behavioral states and various motor control systems, including breathing control. Our previous work has shown that the activation of neurons within the PPT, a structure that is typically active during rapid eye movement (REM) sleep, can produce respiratory disturbances in freely moving and anesthetized rats. The aim of this study was to test the hypothesis that respiratory modulation by the PPT in anesthetized rats can be evoked in the absence of other signs of an REM-sleep-like state. We characterized electroencephalogram (EEG) and electromyogram (EMG) changes during respiratory disturbances induced by glutamatergic stimulation of the PPT in spontaneously breathing, adult male Sprague-Dawley rats anesthetized with a ketamine/xylazine combination or with nembutal. Respiratory movements were monitored by a piezoelectric strain gauge. Two-barrel glass pipettes were used to pressure inject glutamate, to probe for respiratory effective sites within the PPT, and to inject oil red dye at the end of the experiments for histological verification of the injection sites. The EEGs were recorded from the sensorimotor cortex, hippocampus, and from the pons contralateral from the injection site. The EMGs were recorded from the genioglossus muscle. The initial response to glutamate injection into the respiratory modulating region of the PPT was always a respiratory pattern disturbance. Subsequent activation of EMG and EEG often occurred in ketamine/xylazine-anesthetized rats, but REM-sleep-like patterns were not observed. Respiratory pattern and EMG power changes in nembutal-anesthetized rats were similar, but EEG activation was never observed. Thus, we conclude that respiratory suppression produced by the local activation of PPT neurons may not necessarily be accompanied by an REM-sleep-like cortical state in this anesthetized model.

Organization of hypocretin/orexin efferents to locus coeruleus and basal forebrain arousal‐related structures

Hypocretin/orexin neurons give rise to an extensive projection system, portions of which innervate multiple regions associated with the regulation of behavioral state. These regions include the locus coeruleus, medial septal area, medial preoptic area, and substantia innominata. Evidence indicates that hypocretin modulates behavioral state via actions within each of these terminal fields. To understand better the circuitry underlying hypocretin-dependent modulation of behavioral state, the present study characterized the degree to which there exists: 1) lateralization of hypocretin efferents to basal forebrain and brainstem arousal-related regions, 2) topographic organization of basal forebrain- and brainstem-projecting hypocretin neurons, and 3) collateralization of individual hypocretin neurons to these arousal-related terminal fields. These studies utilized combined immunohistochemical identification of hypocretin neurons with single or double retrograde tracing from the locus coeruleus, medial preoptic area, medial septal area, and substantia innominata. Results indicate that approximately 80% of hypocretin efferents to basal forebrain regions project ipsilaterally, whereas projections to the locus coeruleus are more bilateral (65%). There was a slight preference for basal forebrain-projecting hypocretin neurons to be distributed within the medial half of the hypocretin cell group. In contrast, hypocretin neurons projecting to the locus coeruleus were located primarily within the dorsal half of the hypocretin cell group. Finally, a large proportion of hypocretin neurons appear to project simultaneously to at least two of the examined terminal fields. These latter observations suggest coordinated actions of hypocretin across multiple arousal-related regions.

Zaprinast stimulates extracellular adenosine accumulation in rat pontine slices

Adenosine appears to be an endogenous somnogen. The lateral dorsal tegmental/pedunculopontine nucleus (LDT/PPT) located in the mesopontine tegmentum is important in the regulation of arousal. Neurons in this nucleus are strongly hyperpolarized by adenosine and express neuronal nitric oxide synthase. Zaprinast is a cyclic nucleotide phosphodiesterase inhibitor, and has been shown in the hippocampal slice to inhibit the field excitatory postsynaptic potential. This action could be blocked by an adenosine receptor antagonist, and therefore is presumably due to adenosine release stimulated by zaprinast. In the present study we tested the effect of zaprinast on extracellular adenosine accumulation in pontine slices containing the LDT. Zaprinast at 10 μM evoked an increase in extracellular adenosine concentration. This effect was blocked by impermeant inhibitors of 5′-nucleotidase, indicating that the extracellular adenosine was derived from extracellular AMP. However, inhibitors of cAMP degradation had little or no effect on zaprinast-evoked adenosine accumulation, suggesting that extracellular cAMP was not the source. Removal of extracellular calcium inhibited the effect of zaprinast. These results demonstrate that a pathway exists by which zaprinast stimulates extracellular adenosine accumulation, and the presence of this pathway in the pontine slice suggests the possibility that it may be relevant for the regulation of behavioral state.

A serotonergic (5-HT 2) receptor mechanism in the laterodorsal tegmental nucleus participates in regulating the pattern of rapid-eye-movement sleep occurrence in the rat

Serotonin [5-hydroxytryptamine (5-HT)] plays an inhibitory role in rapid-eye-movement (REM) sleep although the exact mechanism(s) and site(s) of action are not known. It is commonly assumed that 5-HT exerts its influence on REM sleep via input from the dorsal raphe nucleus (DRN) directly onto cholinergic neurons involved in the generation of REM sleep. 5-HT 2 receptor sites have been found on cholinergic neurons in the laterodorsal tegmental nucleus (LDT) and pedunculopontine tegmental nucleus (PPT). We locally microinjected the 5-HT 2 agonist DOI ((±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCl) and the 5-HT 2 antagonist, ketanserin, in LDT in rats to determine whether these receptor sites are involved in the regulation of behavioral states. DOI and ketanserin primarily affected REM sleep, by significantly decreasing or increasing, respectively, the number, but not the duration, of REM sleep episodes. DOI specifically decreased the occurrence of clusters of REM sleep episodes appearing at intervals less than or equal to 3 min (sequential episodes) without affecting single episodes separated by more than 3 min. An opposite effect of ketanserin on REM sleep clusters, although not statistically significant, was observed.

Age-related changes in histamine receptor mRNA levels in the mouse brain

Several lines of evidence indicate that the histaminergic (HA) system is important for wakefulness and behavioral state regulation. We investigated the hypothesis that age-related changes in HA system occur which may be related to decreased alertness in aging. Although histidine decarboxylase mRNA levels did not change with age in C57BL/6 mice, significant differences were found in histamine H1 receptor ( H 1R ), histamine H2 receptor ( H 2R ), and histamine H3 receptor ( H 3R ) mRNA levels in several brain regions. The most widespread changes were observed in H 1R mRNA, which were significantly lower (27–38%) in the cortex, hypothalamus, hippocampus and medulla of 24-month-old mice relative to 3-month-old animals. Age-related changes in H 2R mRNA levels were restricted to the pons and cerebellum and decreased H 3R mRNA was found only in the medulla. In conjunction with the age-related decrease in hypocretin receptor 2 mRNA levels we have previously reported, decreased HA receptor mRNA levels may contribute to diminished alertness, sleep continuity, and diurnal rhythms of sleep and wakefulness in the aged.

Colocalization of γ-aminobutyric acid and acetylcholine in neurons in the laterodorsal and pedunculopontine tegmental nuclei in the cat: a light and electron microscopic study

Cholinergic and γ-aminobutyric acid (GABA) mechanisms in the dorsolateral pontomesencephalic tegmentum have been implicated in the control of active (REM) sleep and wakefulness. To determine the relationships between neurons that contain these neurotransmitters in this region of the brainstem in adult cats, combined light and electron microscopic immunocytochemical procedures were employed. Light microscopic analyses revealed that choline acetyltransferase (ChAT) and GABA immunoreactive neurons were distributed throughout the laterodorsal and pedunculopontine tegmental nuclei (LDT and PPT). Surprisingly, approximately 50% of the ChAT immunoreactive neurons in these nuclei also contained GABA. Using electron microscopic pre-embedding immunocytochemistry, GABA immunoreactivity was observed in somas, dendrites and axon terminals in both the LDT and PPT. Most of the GABA immunoreactive terminals formed symmetrical synapses with non-immunolabeled dendrites. Electron microscopic double-immunolabeling techniques revealed that ChAT and GABA were colocalized in axon terminals in the LDT/PPT. Approximately 30% of the ChAT immunoreactive terminals were also GABA immunoreactive, whereas only 6–8% of the GABA immunoreactive terminals were ChAT immunoreactive. Most of the ChAT/GABA immunoreactive terminals formed symmetrical synapses with non-immunolabeled dendrites; however, ChAT/GABA immunoreactive terminals were also observed that contacted ChAT immunoreactive dendrites. With respect to ChAT immunoreactive postsynaptic profiles, approximately 40% of the somas and 50% of the dendrites received synaptic contact from GABA immunoreactive terminals in both the LDT and PPT. These findings (a) indicate that there are fundamental interactions between cholinergic and GABAergic neurons within the LDT/PPT that play an important role in the control of active sleep and wakefulness and (b) provide an anatomical basis for the intriguing possibility that a mechanism of acetylcholine and GABA co-release from the terminals of LDT/PPT neurons is involved in the regulation of behavioral states.

Wake-Promoting Actions of Dopamine D1 and D2 Receptor Stimulation

Multiple ascending neurotransmitter systems participate in the regulation of behavioral state. For example, noradrenergic, cholinergic, and serotonergic systems increase EEG and, in some cases, behavioral indices of arousal. The extent to which dopaminergic systems exert a similar activating influence on behavioral state remains unclear. The current studies examined the wake-promoting actions of centrally administered D1 and D2 receptor agonists. In these studies, intracerebroventricular infusions of a D1 (SKF-82958; 2.5 and 25 nmol) or D2 (quinpirole; 40 and 140 nmol)-agonist were made into sleeping animals. The effects of these infusions on electroencephalogram/electromyographic indices of sleep-wake state and behavior were examined. D1 agonist administration dose dependently increased time spent awake and suppressed rapid eye movement and slow-wave sleep in the 2 h immediately after infusion. D1 agonist administration also elicited modest increases in measures of locomotion and time spent grooming and eating. D2 agonist administration had similar wake-promoting actions, accompanied by modest effects on drinking and locomotion. Interestingly, D2 agonist administration also significantly increased time spent chewing on inedible material, an arousal/stress-related behavior. Overall, these results demonstrate that dopamine contributes to the alert waking state via actions of D1 and D2 receptors. Additionally or alternatively, these results further suggest a potential involvement of dopamine receptors in the induction of high-arousal states, including stress.

Influence of inhibitory and excitatory inputs on serotonin efflux differs in the dorsal and median raphe nuclei

The dorsal (DRN) and median raphe nuclei (MRN) are two major sources of serotonergic projections to forebrain that are involved in regulation of behavioral state and motor activity, and implicated in affective disorders such as depression and schizophrenia. To investigate afferent influences on serotonergic neurons, this study compared the role of endogenous GABA and glutamate in the DRN and MRN using microdialysis and measurement of locomotor activity in freely behaving rats. Local infusion of the GABA A receptor antagonist bicuculline increased serotonin (5-HT) efflux in the DRN but not the MRN. In contrast, infusion of glutamate receptor antagonists produced larger decreases in 5-HT efflux in the MRN compared with the DRN. Moreover, glutamate receptor antagonists attenuated the increase in 5-HT efflux produced by GABA receptor blockade in the DRN. Thus, the disinhibitory effect of GABA blockers could be ascribed in part to an enhanced influence of glutamate. Measurements of locomotor activity indicate that changes in 5-HT were not simply correlated with behavioral activity induced by drug infusion. In summary, the role of inhibitory and excitatory afferents was strikingly different in the DRN and MRN. GABA afferents were the predominant tonic influence on serotonergic neurons in the DRN. In contrast, glutamatergic but not GABAergic afferents had a strong tonic influence on serotonergic neurons in the MRN.

Regulation of Body Size and Behavioral State of C. elegans by Sensory Perception and the EGL-4 cGMP-Dependent Protein Kinase

The growth and behavior of higher organisms depend on the accurate perception and integration of sensory stimuli by the nervous system. We show that defects in sensory perception in C. elegans result in abnormalities in the growth of the animal and in the expression of alternative behavioral states. Our analysis suggests that sensory neurons modulate neural or neuroendocrine functions, regulating both bodily growth and behavioral state. We identify genes likely to be required for these functions downstream of sensory inputs. Here, we characterize one of these genes as egl-4, which we show encodes a cGMP-dependent protein kinase. We demonstrate that this cGMP-dependent kinase functions in neurons of C. elegans to regulate multiple developmental and behavioral processes including the orchestrated growth of the animal and the expression of particular behavioral states.

Pain and opioids in preterm and newborns

Despite the fact that neonates and infants are not capable of expressing their subjective pain sensations, it has become clear that they do perceive nociception, as pain correlates to hormonal, metabolic, immune, and cardiovascular changes. New findings support the notion that repetitive painful stimuli result in long term psycho-physiological effects with ensuing decreased attentiveness and orientation, poor regulation of behavioral state and motor processes, increase in irritability as well as an altered pattern of feeding and sleeping. These sequelae of repetitive painful experiences with an increase in sensitization of sensory afferent input supports the view of a sufficient analgesia during all kinds of painful procedures in the preterm and neonate. In order to sufficiently diminish nociceptive afferent input during surgery opioids are the drugs of choice aside from local anesthetics. However, the use of opioids in neonates and especially preterm infants must be considered in the light of certain pharmacokinetic and pharmacodynamic differences when compared to adults: 1. There is a longer elimination rate, which may result in post-operative overhang of respiratory depression, especially when opioids are given repetitively, resulting in an accumulation and an increased duration of action. 2. There is a reduced hepatic enzyme activity, which ultimately affects clearance rate. 3. The blood-brain-barrier is not fully developed in the preterm, which results in more access of opioids to binding sites in the CNS. 4. Differentiation of opioid-binding sites into mu, delta, and kappa has not reached its peak; thus, higher doses relative to body weight are needed to establish a sufficient deep plane of analgesia. 5. Caudal parts of the CNS, especially the pons-medullar region exhibit an earlier expression of receptors than the rostral parts. Sequelae of such differences are a more pronounced respiratory depression, often due to muscular rigidity, and bradycardia after which a full analgesic effect takes place. Despite such potential drawbacks, opioids are still the best choice as they sufficiently block nociceptive afferent input and when compared to other anesthetics, they show the least cardiovascular changes. One, however, has to bear in mind that dosing is done according to effect and not to body weight while potential side effects are most prominent in the preterm infant.

Wake-promoting and sleep-suppressing actions of hypocretin (orexin): basal forebrain sites of action

The hypocretins (orexins) are a newly identified peptide family comprised of two peptides, hypocretin-1 and hypocretin-2. Recent observations suggest an involvement of these peptides in the regulation of behavioral state. For example, these peptides are found in a variety of brain regions associated with the regulation of forebrain neuronal and behavioral activity states. Furthermore, when infused into the lateral ventricles in awake animals, hypocretin-1 elicits increased duration of waking beyond that observed in vehicle-treated animals. Previous studies have been limited to an examination of the sleep–wake effects of hypocretin-1 in awake animals. Currently, the sleep–wake effects of hypocretin-2 and the extent to which hypocretins can initiate waking in the sleeping animal remain unclear. To better characterize the wake-promoting actions of the hypocretins, the current studies examined the sleep–wake effects of varying doses (0.007, 0.07 and 0.7 nmol) of hypocretin-1 and hypocretin-2 when administered into sleeping rats (e.g. remote-controlled infusions). Infusions of hypocretin-1 and hypocretin-2 into the lateral ventricles elicited a short latency (0.7 nmol hypocretin-1; 93±30 s from the start of the 120-s infusion) increase in electroencephalographic, electromyographic, and behavioral indices of waking. These infusions also produced substantial decreases in slow-wave and rapid-eye movement sleep. Hypocretin-1 was more potent than hypocretin-2 in these actions. Interestingly, hypocretin-1 infused into the fourth ventricle elicited less robust waking which occurred with a longer latency than infusions into the lateral ventricles. These latter observations suggest a forebrain site of action participates in hypocretin-1-induced waking. Within the forebrain, a variety of basal forebrain structures, including the medial preoptic area, the medial septal area and the substantia innominata, receive a moderate hypocretin innervation. Therefore, additional studies examined the sleep–wake effects of bilateral hypocretin-1 infusions into these basal forebrain structures. Robust increases in waking were observed following infusions into, but not outside, the medial septal area, the medial preoptic area and the substantia innominata. These results indicate a potentially prominent role of hypocretins in sleep–wake regulation via actions within certain basal forebrain structures and are consistent with studies indicating a prominent role of hypocretins in sleep/arousal disorders.

Unravelling the neural circuitry for circadian regulation of sleep–wake cycles

A requirement for the fruitful analysis of a neurobiological process is the identification of neural circuits that are crucial for effecting the process. Circadian rhythms constitute one ubiquitous, but mechanistically complex, process that is important for regulating a variety of behavioral states, including sleep and waking. The circadian rhythmicity of sleep–wake cycles might be regulated by trans-synaptic signalling between neurons located in hypothalamic nuclei and in the noradrenergic locus coeruleus (LC). LC neurons fire briskly during waking, and they are less active during slow-wave sleep. However, it is not known whether the LC system is importantly involved in the circadian regulation of arousal. Circadian regulation of the sleep–wake cycle probably involves the suprachiasmatic nucleus (SCN) of the hypothalamus; lesions of the SCN eliminate the circadian rhythmicity of the sleep–wake cycle. This observation, and others, led to the hypothesis that connections between the SCN and brain structures that modulate arousal might underlie the circadian regulation of sleep–wake cycles. Direct demonstration of functional projections from the SCN to brain centers that regulate arousal, such as the LC, is vital for revealing how the SCN and LC regulate arousal.A study by Aston-Jones et al 1xA neural circuit for circadian regulation of arousal. Aston-Jones, G. et al. Nat. Neurosci. 2001; 4: 732–738Crossref | PubMed | Scopus (352)See all

A neural circuit for circadian regulation of arousal.

An unknown aspect of behavioral state regulation is how the circadian oscillator of the suprachiasmatic nucleus (SCN) regulates sleep and waking. In this report, we describe the necessary elements for a circuit that provides circadian regulation of arousal. Trans-synaptic retrograde tracing revealed a prominent indirect projection from the SCN to the noradrenergic nucleus locus coeruleus (LC), a brain arousal system. Double-labeling experiments revealed several possible links between the SCN and the LC, including the dorsomedial (DMH) and paraventricular hypothalamic nuclei (PVN), as well as medial and ventrolateral pre-optic areas. Lesion studies confirmed that the DMH is a substantial relay in this circuit. Next, neurophysiology experiments revealed circadian variations in LC impulse activity. Lesions of the DMH eliminated these circadian changes in LC activity, confirming the functionality of the SCN-DMH-LC circuit. These results reveal mechanisms for regulation of circadian and sleep-waking functions.

Insights from functional neuroimaging studies of behavioral state regulation in healthy and depressed subjects

New data are presented showing excellent replicability and test-retest reliability of REM sleep findings from functional brain imaging studies in healthy subjects on which newer brain-based models of human dreaming have been constructed. Preliminary region-of-interest findings related to bottom-up versus dissociable brain systems mediating REM sleep and dreaming are also presented.[Hobson et al.; Solms]>

Nitric Oxide-Stimulated Increase in Extracellular Adenosine Accumulation in Rat Forebrain Neurons in Culture Is Associated with ATP Hydrolysis and Inhibition of Adenosine Kinase Activity

Adenosine is a putative endogenous sleep-inducing substance, and nitric oxide has been implicated in arousal and sleep mechanisms. We found that various nitric oxide donors, including diethylamine NONOate (DEA/NO), stimulated large increases in extracellular adenosine in nearly pure cultures of forebrain neurons. The effect of DEA/NO could be blocked by 2-phenyl-4,4,5, 5-tetramethyl-imidazoline-1-oxyl-oxide and could not be mimicked by degraded solutions of DEA/NO or by DEA itself; therefore, it was caused by nitric oxide release on hydrolysis of the parent compound. The accumulation of adenosine was not blocked by probenecid or GMP, suggesting that neither extracellular cAMP nor extracellular AMP was the source, and that adenosine was therefore the most likely species transported across the plasma membrane. To pursue this further, we tested the effect of DEA/NO on cellular ATP and found a significant fall in ATP associated with exposure to nitric oxide. In addition, exposure to DEA/NO nearly completely inhibited adenosine kinase activity. It has been found previously that adenosine kinase is inhibited by its substrate, adenosine. We found that exposure to nitric oxide increased intracellular adenosine to 125 +/- 18% of control values (p < 0.01), consistent with the possibility that in our system the inhibition of adenosine kinase is related to an increase in intracellular adenosine, and that the effect of nitric oxide on extracellular adenosine is significantly potentiated by substrate inhibition of adenosine kinase. Furthermore, nitric oxide-stimulated adenosine accumulation may be important in the regulation of behavioral state.

Amphetamine acts within the medial basal forebrain to initiate and maintain alert waking

Amphetamine-like stimulants exert well-known arousal-enhancing actions. Surprisingly, little is known concerning the neuroanatomical substrates through which these drugs enhance arousal. Previous work implicates a number of basal forebrain structures in the regulation of behavioral state. The current studies examined the effects of amphetamine infusions made directly within basal forebrain sites on behavioral, electroencephalographic, and electromyographic indices of arousal in anesthetized and unanesthetized rat. In the anesthetized rat, amphetamine elicited prolonged epochs of bilateral electroencephalographic activation when infused unilaterally (3.75 μg/150 nl) into an extended region of the medial basal forebrain, demarcated anteriorally by the anterior portion of the medial septal area (which includes posterior accumbens shell) and posteriorally by the posterior aspect of the preoptic area of the hypothalamus. In the unanesthetized (undisturbed, resting) rat, amphetamine infusions into this region elicited prolonged epochs of alert waking, which at the lowest dose (3.75 μg), qualitatively resembled normal waking. Infusions placed lateral (including within the substantia innominata), anterior (including within the core subregion of the nucleus accumbens), posterior, or dorsal to these structures, as well as directly within the lateral ventricles did not alter electroencephalographic or behavioral measures. These results indicate that a region of the medial basal forebrain, extending from the anterior medial septum/accumbens shell to the posterior preoptic area, is a site within which amphetamine-like stimulants act to enhance behavioral and electroencephalographic measures of arousal.