Rodrigo A. España

Afferents to the Orexin Neurons of the Rat Brain

Emotions, stress, hunger, and circadian rhythms all promote wakefulness and behavioral arousal. Little is known about the pathways mediating these influences, but the orexin‐producing neurons of the hypothalamus may play an essential role. These cells heavily innervate many wake‐promoting brain regions, and mice lacking the orexin neurons have narcolepsy and fail to rouse in response to hunger (Yamanaka et al. [ 2003 ] Neuron 38:701–713). To identify the afferents to the orexin neurons, we first injected a retrograde tracer into the orexin neuron field of rats. Retrogradely labeled neurons were abundant in the allocortex, claustrum, lateral septum, bed nucleus of the stria terminalis, and in many hypothalamic regions including the preoptic area, dorsomedial nucleus, lateral hypothalamus, and posterior hypothalamus. Retrograde labeling in the brainstem was generally more modest, but labeling was strong in the periaqueductal gray matter, dorsal raphe nucleus, and lateral parabrachial nucleus. Injection of an anterograde tracer confirmed that most of these regions directly innervate the orexin neurons, with some of the heaviest input coming from the lateral septum, preoptic area, and posterior hypothalamus. In addition, hypothalamic regions preferentially innervate orexin neurons in the medial and perifornical parts of the field, but most projections from the brainstem target the lateral part of the field. Inputs from the suprachiasmatic nucleus are mainly relayed via the subparaventricular zone and dorsomedial nucleus. These observations suggest that the orexin neurons may integrate a variety of interoceptive and homeostatic signals to increase behavioral arousal in response to hunger, stress, circadian signals, and autonomic challenges. J. Comp. Neurol. 494:845–861, 2006.

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.

Demon voltammetry and analysis software: Analysis of cocaine-induced alterations in dopamine signaling using multiple kinetic measures

The fast sampling rates of fast scan cyclic voltammetry make it a favorable method for measuring changes in brain monoamine release and uptake kinetics in slice, anesthetized, and freely moving preparations. The most common analysis technique for evaluating changes in dopamine signaling uses well-established Michaelis-Menten kinetic methods that can accurately model dopamine release and uptake parameters across multiple experimental conditions. Nevertheless, over the years, many researchers have turned to other measures to estimate changes in dopamine release and uptake, yet to our knowledge no systematic comparison amongst these measures has been conducted. To address this lack of uniformity in kinetic analyses, we have created the Demon Voltammetry and Analysis software suite, which is freely available to academic and non-profit institutions. Here we present an explanation of the Demon Voltammetry acquisition and analysis features, and demonstrate its utility for acquiring voltammetric data under in vitro, in vivo anesthetized, and freely moving conditions. Additionally, the software was used to compare the sensitivity of multiple kinetic measures of release and uptake to cocaine-induced changes in electrically evoked dopamine efflux in nucleus accumbens core slices. Specifically, we examined and compared tau, full width at half height, half-life, T₂₀, T₈₀, slope, peak height, calibrated peak dopamine concentration, and area under the curve to the well-characterized Michaelis-Menten parameters, dopamine per pulse, maximal uptake rate, and apparent affinity. Based on observed results we recommend tau for measuring dopamine uptake and calibrated peak dopamine concentration for measuring dopamine release.

The hypocretin–orexin system regulates cocaine self-administration via actions on the mesolimbic dopamine system

Recent evidence suggests that the hypocretin–orexin system participates in the regulation of reinforcement processes. The current studies examined the extent to which hypocretin neurotransmission regulates behavioral and neurochemical responses to cocaine, and behavioral responses to food reinforcement. These studies used a combination of fixed ratio, discrete trials, progressive ratio and threshold self‐administration procedures to assess whether the hypocretin 1 receptor antagonist, SB‐334867, reduces cocaine self‐administration in rats. Progressive ratio sucrose self‐administration procedures were also used to assess the extent to which SB‐334867 reduces responding to a natural reinforcer in food‐restricted and food‐sated rats. Additionally, these studies used microdialysis and in vivo voltammetry in rats to examine whether SB‐334867 attenuates the effects of cocaine on dopamine signaling within the nucleus accumbens core. Furthermore, in vitro voltammetry was used to examine whether hypocretin knockout mice display attenuated dopamine responses to cocaine. Results indicate that when SB‐334867 was administered peripherally or within the ventral tegmental area, it reduced the motivation to self‐administer cocaine and attenuated cocaine‐induced enhancement of dopamine signaling. SB‐334867 also reduced the motivation to self‐administer sucrose in food‐sated but not food‐restricted rats. Finally, hypocretin knockout mice displayed altered baseline dopamine signaling and reduced dopamine responses to cocaine. Combined, these studies suggest that hypocretin neurotransmission participates in reinforcement processes, likely through modulation of the mesolimbic dopamine system. Additionally, the current observations suggest that the hypocretin system may provide a target for pharmacotherapies to treat cocaine addiction.

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.

Fos immunoreactivity in hypocretin-synthesizing and hypocretin-1 receptor-expressing neurons: effects of diurnal and nocturnal spontaneous waking, stress and hypocretin-1 administration.

Hypocretin/orexin modulates sleep-wake state via actions across multiple terminal fields. Within waking, hypocretin may also participate in high-arousal processes, including those associated with stress. The current studies examined the extent to which alterations in neuronal activity, as measured by Fos immunoreactivity, occur within both hypocretin-synthesizing and hypocretin-1 receptor-expressing neurons across varying behavioral state/environmental conditions associated with varying levels of waking and arousal. Double-label immunohistochemistry was used to visualize Fos and either prepro-hypocretin in the lateral hypothalamus or hypocretin-1 receptors in the locus coeruleus and select basal forebrain regions involved in the regulation of behavioral state/arousal. Animals were tested under the following conditions: 1). diurnal sleeping; 2). diurnal spontaneous waking; 3). nocturnal spontaneous waking; and 4). high-arousal waking (diurnal novelty-stress). Additionally, the effects of hypocretin-1 administration (0.07 and 0.7 nmol) on levels of Fos were examined within these two neuronal populations. Time spent awake, scored for the 90-min preceding perfusion, was largely comparable in diurnal spontaneous waking, nocturnal spontaneous waking and high-arousal waking. Nocturnal spontaneous waking and high-arousal waking, but not diurnal spontaneous waking, were associated with increased levels of Fos within hypocretin-synthesizing neurons, relative to diurnal sleeping. Within hypocretin-1 receptor-expressing neurons, only high-arousal waking was associated with increased levels of Fos. Hypocretin-1 administration dose-dependently increased levels of Fos within hypocretin-1 receptor-expressing neurons to levels comparable to, or exceeding, levels observed in high-arousal waking. Combined, these observations support the hypothesis that hypocretin neuronal activity varies across the circadian cycle. Additionally, these data suggest that waking per se may not be associated with increased hypocretin neurotransmission. In contrast, high-arousal states, including stress, appear to be associated with substantially higher rates of hypocretin neurotransmission. Finally, these studies provide further evidence indicating coordinated actions of hypocretin across a variety of arousal-related basal forebrain and brainstem regions in the behavioral state modulatory actions of this peptide system.

Noradrenergic Modulation of Wakefulness/Arousal

The locus coeruleus-noradrenergic system supplies norepinephrine throughout the central nervous system. State-dependent neuronal discharge activity of locus coeruleus noradrenergic neurons has long-suggested a role of this system in the induction of an alert waking state. Work over the past two decades provides unambiguous evidence that the locus coeruleus, and likely other noradrenergic nuclei, exert potent wake-promoting actions via an activation of noradrenergic β- and α₁-receptors located within multiple subcortical structures, including the general regions of the medial septal area, the medial preoptic area and, most recently, the lateral hypothalamus. Conversely, global blockade of β- and α₁-receptors or suppression of norepinephrine release results in profound sedation. The wake-promoting action of central noradrenergic neurotransmission has clinical implications for treatment of sleep/arousal disorders, such as insomnia and narcolepsy, and clinical conditions associated with excessive arousal, such as post-traumatic stress disorder.

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. J. Comp. Neurol. 481:160–178, 2005.

Hypocretin/Orexin in Arousal and Stress

Multiple lines of evidence indicate that hypocretin/orexin (HCRT) participates in the regulation of arousal and arousal-related process. For example, HCRT axons and receptors are found within a variety of arousal-related systems. Moreover, when administered centrally, HCRT exerts robust wake-promoting actions. Finally, a dysregulation of HCRT neurotransmission is associated with the sleep/arousal disorder, narcolepsy. Combined, these observations suggested that HCRT might be a key transmitter system in the regulation of waking. Nonetheless, subsequent evidence indicates that HCRT may not play a prominent role in the initiation of normal waking. Instead HCRT may participate in a variety of processes such as consolidation of waking and/or coupling metabolic state with behavioral state. Additionally, substantial evidence suggests a potential involvement of HCRT in high-arousal conditions, including stress. Thus, HCRT neurotransmission is closely linked to high-arousal conditions, including stress, and HCRT administration exerts a variety of stress-like physiological and behavioral effects that are superimposed on HCRT-induced increases in arousal. Combined, this evidence suggests the hypothesis that HCRT may participate in behavioral responding under high-arousal aversive conditions. Importantly, these actions of HCRT may not be limited to stress. Like stress, appetitive conditions are associated with elevated arousal levels and a stress-like activation of various physiological systems. These and other observations suggest that HCRT may, at least in part, exert affectively neutral actions that are important under high-arousal conditions associated with elevated motivation and/or need for action.

Hypocretin 1/orexin A in the ventral tegmental area enhances dopamine responses to cocaine and promotes cocaine self-administration

RationaleRecent evidence indicates that the hypocretin/orexin system participates in the regulation of reinforcement and addiction processes. For example, manipulations that decrease hypocretin neurotransmission result in disruptions of neurochemical and behavioral responses to cocaine.ObjectivesTo further assess the relationship between the hypocretin system and cocaine reinforcement, the current studies used microdialysis and in vivo voltammetry to examine the effects of hypocretin 1 on cocaine-induced enhancement of dopamine signaling in the nucleus accumbens core. Fixed ratio, discrete trials, and progressive ratio self-administration procedures were also used to assess whether hypocretin 1 promotes cocaine self-administration behavior.ResultsInfusions of hypocretin 1 into the ventral tegmental area increased the effects of cocaine on tonic and phasic dopamine signaling and increased the motivation to self-administer cocaine on the discrete trials and progressive ratio schedules.ConclusionsTogether with previous observations demonstrating that a hypocretin 1 receptor antagonist disrupts dopamine signaling and reduces self-administration of cocaine, the current observations further indicate that the hypocretin system participates in reinforcement processes likely through modulation of the mesolimbic dopamine system.